Natural variation in siRNA activity and the influence of
chemical and mutational modifications
Torgeir Holen
A thesis in partial fulfilment of the requirements for the degree of Doctor Philosophiae
Επισκεπτεον δε µαλλον και περι του ηγεµονος, ει κεντρον εχει η µη.
(But further enquiry is necessary in fact about the leader, as to whether he has a sting or not)
Aristotle, Historia Animalium1
1
The Biotechnology Centre of Oslo
&
Department Group of Clinical Medicine, Faculty of Medicine
University of Oslo
2
- Kolofonside- settes opp av Unipub Forlag
- ISBN 82-8072-077-4
3
Contents
0.5 Acknowledgements
0.6 A note on this thesis writing
0.7 List of papers
0.8 Abbreviations
1 An introduction to the RNA interference field
1.0 A short overview
1.1 The three central publications in RNAi
1.2 The immune system hypothesis of RNA interference
1.3 Biochemical investigations of dsRNA and siRNA
1.4 The current RNAi model and a challenger
1.5 Non-complimentary short RNA, stRNA, inhibit translation
1.6 Synthetic siRNA versions
1.7 Endogenously expressed short hairpin RNA (shRNA)
1.8 Using siRNA and shRNA against viruses
2 Discussion of results in thesis papers I, II & III
2.0 Results introduction - a summary of ribozyme experiments
2.1 RNA interference in mammalian cells using dsRNA
2.2 Positional effects in a series of siRNA
2.3 On the possibility of finding local targeting rules
2.4 Investigating local area around hTF167i
2.5. Endogenous hTF mRNA targets differ markedly in siRNA accessibility
2.6 The main hypothesis: Natural variation in siRNA activity
2.7 Tissue Factor protein and procoagulant activity also show position effects
2.8 Tolerance of siRNA for mutations
2.9 Tolerance of siRNA for chemical modifications
2.10 Competition from inactive siRNA can block active siRNA
2.11 Cleavage fragment production from siRNA might indicate RNAi exonuclease
2.12 Dose-response experiments
2.13 Propagation of silencing not detected in human cells
2.14 PSKH1 siRNAs had not much activity against endogenous mRNA
2.15 Antisense siRNA compared with double-stranded siRNA
2.16 Blocking 3' OH does block activity in antisense siRNA
4
0.5 Acknowledgements
Without the encouragement and stimulating company of Mohammed Amarzguioui and Gaute
Brede, this work would never have seen the dusky light of our misty country.
Also I would like to acknowledge the work of Merete T. Wiiger for her contributions to our
coagulation assays, and Dimitrios Mantzilas for his assistance with the MALDI-TOF. Eshrat
Babaie is acknowledged for her running of the nuclic acid synthesis lab, Henning Johansen
for his running of the sequencing lab and Liv Bjørland for her running of the chemical
solutions lab. And all the other people at the Biotechnology Centre that makes the wheels go
around. You are appreciated. A special thanks to Erlend Ragnhildstveit for presenting that
paper by Hammond et al at our Journal Club.
Finally I would like to thank professor Hans Prydz for hiring me when I came back from
America, helping me write a grant application and also financing me for four months after my
grant money ran out. Last, but not least, I would like to acknowledge his unique style of
leadership, giving us the freedom to fail or succeed, as all of us, so to speak, are the
blacksmiths of our own destiny.
Torgeir Holen,
Oslo, January 2002
0.6 A note on this thesis writing
For some reason writing up this thesis seems to me somewhat meaningless. If I had additional
scientific insight that could be justified by the data, I should have included it in the papers.
Then again, anybody who claims that life has a meaning is selling something, and the
University of Oslo is selling doctorates.
The organizing principles of this thesis text are firstly chronological, to convey the richness of
a scientific field and a PHD thesis work, undergoing, so to speak, gastrulation. Secondly,
topical, to discuss the results in the three thesis papers as a whole, and in relation to various
interesting aspects and inconsistensies of RNA interference in the published literature. And
finally, eclectic, as RNA silencing is already such a wide and rapidly expanding field that a
5
work like this cannot possibly do justice to all the exiting things that are happening.
The strain that these three lines of exposition put on the text is necessarily visible.
Consequently, the text have been expanded somewhat to make it more readable and endurable
for the very few people who will ever read it. I hope it is appreciated.
0.7 List of papers and unpublished data
I)
Holen, T., Amarzguioui, M., Wiiger, M.T., Babaie, E. and Prydz, H. (2002).
Positional effects of short interfering RNAs targeting the human coagulation
trigger Tissue Factor. Nucleic Acids Research, 30, 1757-1766.
II)
Amarzguioui, M., Holen, T., Babaie, E. and Prydz, H. (2003). Tolerance for
mutations and chemical modifications in a siRNA. Nucleic Acids Research, 31,
589-595.
III)
Holen, T., Amarzguioui, M., Babaie, E. and Prydz, H. (2003). Similarity of RNAi
antisense and siRNA behaviour indicate a common RNA interference pathway.
Nucleic Acids Research (in press).
IV)
In addition comes some unpublished figures and unpublished data.
6
0.8 Abbreviations
2'MOE
2'-O-(2-methoxy)ethyl
3'deoxy
3' deoxyribose on 3' terminal nucleotide
3' OH
3' hydroxyl
Ago-2
Argonaute 2
Arabidopsis
Arabidopsis thaliana
ATP
adenosine triphosphate
bp
basepair(s)
CAF
carpel factory
CAP
7-methylguanylate on 5' nt
C. elegans
Caenorhabditis elegans
Drosophila
Drosophila melanogaster
DNA
dexoyribonucleic acid
dsRNA
double-stranded RNA
E.coli
Escherichia coli
esiRNA
endoribonuclease prepared siRNA
Fig.
figure
FITC
fluorescein isothiocyanate
g
gravities (multiples of 9.8 m/s2)
GC
guanosine and cytosine
GFP
green fluorescent protein
h
hour(s)
H. sapiens
Homo sapiens
hTF
human Tissue Factor
HIV
human immunodeficiency virus
kDa
kilo Dalton
Luc
luciferase
MALDI-TOF matrix assisted laser desorption ionization - time of flight
microRNA
small RNA expressed from a micro gene
mRNA
messenger RNA
mTF
murine Tissue Factor
nM
nanomolar
N. crassa
Neurospora crassa
7
nt
nucleotide(s)
ODN
oligodeoxynucleotide, oligo
oligo
oligonucleotide
PAGE
polyacrylamide gel electrophoresis
PCR
polymerase chain reaction
pGL2
plasmid GL2 (luciferase)
PHD
doctor (of philosophy)
PSKH1
protein serine kinase H1
PTGS
post-transcriptional gene silencing
PKR
protein kinase R
rbz
ribozyme
RdRP
RNA-dependent RNA polymerase
RISC
RNA induced silencing complex
RNA
ribonucleic acid
RNAi
RNA interference
RNase
ribonuclease
RSV
Rous Sarcoma Virus
S2
Schneider cells
S100
centrifugal supernatant after 100 000 g
shRNA
short hairpin RNA
siRNA
short interfering RNA
SNP
single nucleotide polymorphism
snRNA
small nuclear RNA
ssRNA
single-stranded RNA
stRNA
short temporal RNA, a microRNA
TRE
tetracycline response element
TGS
transcriptional gene silencing
TT
two successive thymidine nucleotides
UTR
untranslated region
8
1 An introduction to the RNA interference field
1.0 A short overview
Five years after Andrew Fire did his ground-breaking discovery of RNA interference (RNAi),
by showing that double-stranded RNA (dsRNA) can efficiently interfere with the
corresponding gene,5 a few things have become clear:
-
RNA interference is an over a billion years old immune system against virus and
transposons
-
the mechanism of action is starting to be uncovered, as a range of proteins have been
identified by mutational screens in various species and biochemical in vitro assays
have been developed
-
RNAi can be manipulated by introduction of various forms of RNA to knock down
mRNA of interest, something which has implications for both research and clinical
applications
-
related microRNA that are known to regulate development in Caenorhabditis elegans
by inhibiting translation of target mRNA, have been found in humans, but their
function is unknown
-
chemical modifications to the triggering RNA agents are tolerated by the biological
machinery, thus opening for further improvement in techniques
1.1 The three central publications in RNAi
Plant biologists had been seeing the effects of RNA interference since the early 90ies, as
plants tend to resist transgenic manipulation by turning off the offending genes by either TGS
(Transcriptional Gene Silencing) or PTGS (Post-Transcriptional Gene Silencing).10 PTGS is
presumably RNA interference, and plant researchers Andrew J. Hamilton and David C.
Baulcombe contributed with the key observation that silenced genes tended to have strange,
short (~ 25 nucleotides) RNAs associated with them.6 This observation led to Thomas
Tuschl’s breakthrough into the hitherto inaccessible field of human genetics, by
demonstrating how these short interfering RNA (siRNA) bypass both other RNAi
requirements and the mammalian virus detection system that had blocked RNAi in
mammalian cells so far.7 These three publications, by Fire et al, Hamilton & Baulcombe and
Elbashir et al are, in my view, key to understanding development of the RNA interference
field.
9
1.2 The immune system hypothesis of RNA interference
I will initially concentrate on the hypothesis that RNA interference is a billion years old
immune system. How do we know this? Primarily we know this due to comparative biology
studies done in C. elegans along with studies on plant viruses. In two key publications Ketting
et al 8 and Tabara et al 9 identified genes in C. elegans (mut-2, mut-7, rde-2 and rde-3) that
when mutated gave rise to stimulation of transposon activity, while having reduced RNAi.
Especially mut-7 was interesting, as its inactivation gave rise to a rapidly mutating mutator
strain. As this gene has sequence similarity with proteins with 3'-5' exonuclease domains like
RNaseD and Werner syndrome protein, Ketting et al proposed a speculative model where an
exonuclease degrades mRNA guided by the double-stranded RNA.8 Tabara et al, however,
warned against excessive speculation and pointed out that several mutator strains have intact
RNAi, among them mut-6. In addition comes mutations that inactivate RNAi, rde-1 and rde-4,
that do not mobilize transposons. All this suggest additional complexity.9
Another group of genes (mes-2, mes-3, mes-4 and mes-6) are involved in silencing of
transgenes in the germline in C. elegans.11 Testing these mutants, Tabara et al, found normal
levels of RNAi.9 However, Dudley et al found that mes-3, mes-4 and mes-6 were in fact
needed for RNAi, but that this requirement could be bypassed by using very low levels of
dsRNA.12 Obviously, there are still complexities to be unravelled in the genome's immune
system.13
Overall, this indicates that RNA interference has a role in transposon suppression, at least in
C. elegans. RNAi mutants have also been found in other species such as Drosophila
melanogaster, Neurospora crassa and Arabidopsis thaliana. There are interesting sequence
similarities between certain RNAi genes, especially in certain domains. For example, mut-7
homologues (in the general sense of the word) were found in screens for RNA interference
deficiency in other species, as qde-3 in N. crassa.14 A long series of such conserved RNAi
genes have now been found, as shown in a limited summary table (Table 1).
10
C. elegans (putative
Arabidopsis
Drosophila
N. crassa
H. sapiens
qde-3 14
WRN
function)
mut-7 8 (RNaseD, wrn,
3'-5' exonucleases)
Ego1, Rrf-1, Rrf-2, Rrf-3 18,
Sde1/Sgs2 24
- no homologue-
qde-1 16
- no homologue - 22
Rde-1 9 (initiator...) 52, 53
AGO1,
AGO1, AUB/Sting,
qde-2 15,
HIWI, EIF2C 20
alg1, alg2 20 + 21 homologs
PINHEAD 20
PIWI20 Ago-2 28,
20
20
AGO4 49
Ago-3 29
dcr-1 97 (several RNase-III
CAF, SIN1, SUS1
Dicer + several
proteins 26)
10
others 26
Upf1/Smg-2 22 (transposon
Sde-3 22 (RNA
silencing in C. reinhardthii)17
helicase?)
19
(RdRP in tomato 25 )
(RISC components?!)
hDicer 26, 27
GB110 (mouse) 22
9
rde-4 (initiator ?!, bind
dsRNA,98 bypassed by
injected siRNA 53)
rde-2, rde-3, mut-2,
mut-8-, mut-9 9, 111
mes-2, mes-3, mes-4, mes-6
Enhancer of zeste,
9, 11, 12
extra sex combs 12
(polycomb homologs )
mut-14 (RNA helicase?,
needed for antisense) 52
Table 1 A limited summary of sequence similarity in RNAi mutants
Another interesting sequence similarity group is the one of ego-1, rrf-1, rrf-2 and rrf3 18, 31, as
they are homologues of RNAi genes in other species (Table1), in addition to having sequence
similarity in some domains to a known tomato RNA-dependent RNA polymerase.25 As
double-stranded RNA stands centrally in RNAi, a lot has been made out of this, something
that will be discussed in section 1.4. Furthermore, when rrf-1 is mutated (rrf-1-/-), RNAi
activity was abolished.31, 32 However, rrf-2-/- showed no difference, and rrf-3-/- actually
showed stimulated RNAi interference, a phenomenon that has recently been put to use testing
dsRNA that previously could not induce RNAi phenotypes.32
The most convincing line of evidence, however, for a role for RNAi in virus and transposon
defence, is the discovery of viruses in plants that have evolved proteins to specifically knock
out RNA interference.10 Such parasite-immune system interactions are well known and a
central piece of evidence for RNA interference being a virus defence. Interestingly, Hc-Pro,
one such viral protein that can knock out siRNA production, fails to eliminate the still
mysterious signal that can spread throughout the plant from an infection site, and make the
11
rest of the plant immune.19 Recently it has been shown that viruses also can inactivate RNA
interference in Drosophila, and, impressively, that the inhibitor FHV protein, B2, actually can
inhibit RNA silencing in plants too.33
To conclude this part, the mystery of why Andrew Fire’s double-stranded RNA should be
able to knock out the corresponding mRNA target seems to have been solved: Doublestranded RNA is a symptom of virus entry or transposon activity, and an immune system has
evolved to use this symptom, quite elegantly, to take care of the problem, sometime before the
separation of various single-celled, plant and animal eukaryote lines well over a billion years
ago.21, 30
1.3 Biochemical investigations of dsRNA and siRNA
To solve the problem of mechanism, in vitro reconstitution of RNA interference in
Drosophila embryo lysates was developed by Thomas Tuschl, Phillip Zamore and
coworkers.34 This in vitro assay was used to show a curious dilution phenomena. From the
work of Fire and coworkers it was known that the dsRNA injected into C. elegans would have
been diluted to just a few molecules per cell when the 100 descendant embryos of the F1
generation showed sign of target silencing at their 500-cell stage.5 Some silencing could even
be seen in the F2 generation, something which gave rise to the hypothesis that this resistance
to dilution indicated that an underlying enzymatic system existed, and, possibly, also
propagator proteins replenishing the supply of the silencing agent, especially as rde-2 and
mut-7 were required for F2 generation interference.70
Tuschl et al showed that although 10 nM dsRNA gave good in vitro silencing, a 10-fold
dilution of dsRNA to 1 nM abolished activity. On the other hand, if the dsRNA was diluted by
adding fresh lysate every 30 minutes for 3 hours, for a final dilution of 64-fold, the silencing
activity was as potent as ever. Something seemed to be happening to the dsRNA.
Tuschl and coworkers did not solve the riddle this time around, but they did show that the
hypothetical dsRNA-modifying factors could be titrated out, by adding 200 fold higher
concentration of competitor dsRNA. Furthermore, they showed that the mRNA target was not
mechanically blocked by some association with dsRNA itself, but degraded. Finally, they
showed that the silencing activity declined with the decreasing length of dsRNA, a dsRNA of
only 49 bp having no activity at all.
12
Hamilton and Baulcombe,6 on the other hand, had chosen to search for small RNAs
associated with the silenced state in plants. They reasoned that if these hypothetical antisense
RNA molecules were of a similar size to typical mRNAs, they would have been detected by
routine RNA analysis. They thus started looking for small RNA, finding that four classes of
PTGS in plants, three transgene induced and one virus induced, showed the presence of circa
25 nucleotides long sense and antisense RNA molecules on 15 % PAGE gels blotted to
Hybond filters. This was a vital clue to further progress.
Hamilton and Baulcombe, however, failed to point out that these ~25 nt RNA could be
double-stranded. They proposed that the 25-nt antisense RNA species were not degradation
products of the target RNA because they have antisense polarity, so that a more likely source
of these RNAs is the transcription of an RNA template, thus favouring the RdRP hypothesis
that we will come to later.
Meanwhile Tuschl and Zamore had used their in vitro assay to try to find the dsRNAmodifying specific factors. Presumably spurred on by Hamilton and Baulcombe’s ~25 nt
RNA, they demonstrated breakdown of radioactive dsRNA to radioactive ~21-23 nt sense and
antisense RNA in Drosophila lysates.35 Radioactively labelled, 5’-capped, antisense mRNA
was not degraded, nor was presence of target mRNA necessary. Furthermore, both the target
mRNA and dsRNA was cleaved at 21-23 nucleotide, or basepair, intervals, suggesting that the
21-23 nt RNAs were guiding the cleavage.
In Cold Spring Harbour, another group, led by Gregory Hannon, had started to fractionate
Drosophila S2 cell lysates with dsRNA-induced mRNA degradation activity.36 They found
that a partial purification (using the S100 supernatant fractionated by an anion-exchange
column) contained a clear mRNA degradation peak with a ~25 nt RNA species, that
presumably conferred the target specificity. They termed this enzyme RISC for RNA-induced
silencing complex.
Their lack of observation of stable cleavage endproducts, led them to propose that RISC is an
exonuclease, possibly linked to an endonuclease. Or, alternatively, that an RNAi
endonuclease makes an initial endonucleolytic cut and that non-specific exonucleases
complete the process.36 However, Tuschl and Zamore’s demonstration of obvious cleavage
13
products in Drosophila embryo lysates,35 and, incidentally, our own demonstration of in vivo
cleavage products in human HaCaT cells,2 suggest that RISC is primarily an endonuclease,
although possibly linked to an exonuclease, as discussed in section 2.11.
The first demonstration of RNA interference existing in vertebrates came in zebrafish (Danio
rerio).37 However, there were unspecific effects, as seen by the use of the control lacZ control
dsRNA. Thus the injection procedure was seen as generally impractical and less robust.38, 39
At this point, inspired by the work of Hammond et al,36 we tried to construct a dsRNA hairpin
expressed against a gene of interest, PSKH1,40 as discussed in section 2.1.
Hannon’s group went on to find the dsRNA modifying enzyme and called it Dicer,26 and,
accordingly, also named the not yet characterized RISC nuclease Slicer.41 In this paper,26 they
claim that the RISC activity could be cleared from the extract by a centrifugation of 100 000 g
for 60 minutes, leaving the Dicer activity in the supernatant. Strangely, in the previous
paper,36 such a centrifugation (100 000 g, 60 minutes) removed ribosomes, but left the RISC.
This inconsistency is somewhat confusing. However, in the first paper,36 the 100 000 g
centrifugation that left RISC in the supernatant, was proceeded by a 200 000 g centrifugation
for 3 hours, before RISC was resuspended by use of salt extraction. Presumably the
resuspension process broke up the RISC-ribosome complex and recovering only the so called
soluble RISC.28 There also have been some other indications of ribosome association of
RNAi,42 and Kennerdell et al report that untranslated mRNA remain resistant to RNAi in
Drosophila embryos.43
Hannon has pointed out that their RISC complex varies from other complexes isolated, e.g. it
has not been possible to demonstrate mRNA cleavage fragments, indicating that an
exonuclease exist in this particular complex.128 That RISC complexes consists of several
subunits, that breaks down differently under different conditions, has been amply shown by
the three different estimates of the size of the RISC complex.28,44,45 The main point here,
however, is that Dicer activity and RISC activity could be separated. This point is somewhat
disputed, as some has claimed that Dicer itself is the RISC, in a process termed degradative
PCR,46 as further discussed in section 1.4.
It should be pointed out that Bernstein et al did not isolate Dicer biochemically.26 Rather they
searched the Drosophila genome databases for candidates with sequence similarity to the
14
known dsRNA nuclease RNase-III family. Three classes of hits were found and
representatives of these three classes were tested. A candidate gene CG4792, having two
RNase-III domains and an amino-terminal helicase domain, showed dsRNA cleavage activity
after having been expressed, in a T7-tagged version, in Drosophila S2 cells. Testing whether
this enzyme, Dicer, really was causing the production of short RNA, an antibody against the
C-terminus of Dicer demonstrated that nuclease activity could be precipitated from cell
extracts. Dicer was shown to require ATP as a cofactor. However, Zhang and coworkers
found that ATP was not required, and suggested that if ATP participates in the Dicer reaction
in mammalian cells, it might be involved in product release needed for the multiple turnover
of the enzyme/substrate complex.109
To establish the link of Dicer with RNAi, dsRNA with Dicer sequence was used to deplete
Dicer mRNA, which resulted in a seven-fold drop in Dicer activity.26 Elegantly, if somewhat
confusing and open to interpretation, RNAi was thus used to demonstrate Dicer requirement
in RNAi. A similar technique was used by Hutvagner et al, utilizing siRNA, to establish the
link of human Dicer to maturation of let-7, an stRNA.27 However, when immunoprecipitating
Dicer, Ago-2, a RISC component, follows, suggesting that Dicer indeed is a component of
RISC, but, in certain cases, fails to process dsRNA.28 Obviously, the exact relationships
between RISC and Dicer is not yet established.
In a very thorough work, using a wide variety of dsRNA, Thomas Tuschl’s new group in
Göttingen finally conclusively proved that the 21-22 nt double-stranded RNA (coined siRNA)
mediated the RNA interference, by demonstrating uncoupling of dsRNA from the siRNA
effect.47 Biochemically isolated siRNA were shown to have a characteristic RNaseIII-like 2-nt
overhang at the 3’ end, and terminal 2’ and 3’ hydroxyl groups. Cloning these small RNA (a
technique later used for microRNA isolation, see section 1.5) and then synthesizing identical
copies by organic chemistry, showed that these synthetic siRNAs had mRNA cleavage
activity in Drosophila embryo lysates in the absence of dsRNA. Furthermore, if these
overhangs were considerably longer or were absent (blunt), the silencing was lost. This in
fact, emphasizes the double-stranded nature of the RNA strands, as it could reasonably be
claimed that it was the single-stranded RNA from spontaneous unwinding in vivo that was the
underlying cause of the RNAi.
A complication arising here, however, is the recent demonstration that the antisense strand of
15
an siRNA, can under certain circumstances work on its own, 2, 3, 45, 48, 52 as will be discussed in
the section 2.15.
Due to responsiveness to alkaline phosphatase treatment, the 5’ end seemed to have a
phosphate group,47 but this was not satisfactorily elucidated before the work of Nykänen et al,
who showed that blocking the 5' end with a methoxy moiety blocks silencing in Drosophila
lysates.44 Tuschl's group also showed that the mRNA cleavage site is situated centrally in the
corresponding siRNA, 47 later determined to be precisely 11 nt from the 5’end of the antisense
strand. 69
It was predicted that it would be possible to cleave a target RNA at almost any position, and
that RNA interference differed from other types of antisense molecules by not having to
search for the optimal target sites.48, 50 This hypothesis we later tested in the first thesis paper,2
as will be discussed in section 2.2. Tuschl’s group went on to use these chemically
synthesized siRNAs in cells,7 a breakthrough that is dealt with in section 1.6.
1.4 The current RNAi model and a challenger
This short review of the central works of the RNA interference field leaves us with the
following model (Fig.1): Aberrant dsRNA is recognized and cleaved by Dicer into short 2123 nt siRNA. These siRNAs are incorporated into RISC, which then can specifically
recognize the target RNA and destroy it. This process can be manipulated to accept foreign
dsRNA or synthetic siRNA, the topic of section 1.6.
A challenge to the current model is the mechanism proposed by Lipardi et al,46 and supported
by some data presented by Sijen et al,31 a mechanism which has been termed degradative
PCR. The idea is that short RNA, complementary to the mRNA, can anneal to mRNA, and
then be extended via the accessible 3’ hydroxyl, by a putative RNAi RNA-dependent RNA
Polymerase (RdRP). This would create a new dsRNA, consisting of the target mRNA and a
newly synthesized strand, which then can again be cleaved by Dicer to produce more siRNA,
and the process is repeated in a chain reaction (Fig.1).
16
17
The idea is attractive. It would serve as a propagating system, giving rise to the lack of
dilution of the silencing in C. elegans descendants of the injected worm, as transcribed
mRNA would continually be converted into siRNA. It would also dispense of the need for the
RISC nuclease, as Dicer would be the active ingredient in RISC.
Lipardi et al demonstrated some expected effects of such a system in Drosophila, such as the
production of secondary siRNA. Hot-labelled siRNA, incubated with S100 fraction of embryo
extracts, led to production of longer transcripts being radioactively labelled, but only when a
RNA template was present that was complementary to the siRNA. Interestingly, these longer
transcripts then are degraded over a period of 180 minutes, something that Lipardi et al
interpret as dsRNA being processed back to siRNA. An objection to these experiments are
that irrelevant viral RdRPs might be present. Other groups have also failed in trying to reestablish the Lipardi-mechanism in Drosophila.
Sijen et al,31 in C. elegans, went a step further, by demonstrating what they called transitive
RNAi, the effect of which is that dsRNA of type X, injected into a worm against a target
transcript YX, not only can knock out YX, but also the independent transcript containing only
sequence Y. In short, silencing seemed to have spread from X sequences, towards the 5’ end,
into Y sequences, and from there to the separate transcript Y. Also, the inverse was shown not
to be true, as a target transcript with the sequence XY will not give rise to transitive silencing
of Y. More specifically, X here stands for unc22 and Y is GFP.31 Spreading of silencing, even
through promoter sequences, has also been seen in Drosophila,90 as discussed in section 2.10.
Unfortunately this mechanism has been perceived as inadequate for several reasons: Firstly,
nobody has yet biochemically demonstrated any RNAi RdRP-activity, any tortuous argument
of sequence similarity to tomato RdRP notwithstanding. Also, humans, and more importantly,
Drosophila, have in any case no such RdRP homologue, even if C. elegans does.22 Secondly,
Dicer needs ATP and cleavage in vitro can proceed without it,44, 66 even if this argument is
weakened by a recent study on human Dicer showing no need for ATP.109 Thirdly, Bernstein
et al claimed to be able to separate Dicer and RISC activity by centrifugation,26 although what
kind of centrifugal steps performed was not stated clearly.26 An allegedly inactive Dicer was
later co-immunoprecipitated with Ago-2, as noted above.
Fourthly, propagative effects are not seen with siRNA in mammalian cells as RNAi silencing
18
fades out over time, in our case at 3-4 days post-transfection.2 This has been reproduced in
other works.54 Finally, an siRNA with a blocked 3’ end, by a FITC-group, that therefore can
not be elongated, had as potent silencing activity as the wild-type siRNA.2 This has been done
by Zamore's group, who also showed that 3' blocked siRNA also worked in Drosophila. To
control for the possible regeneration of 3'-hydroxyl by loss of the terminal nucleotide, a series
of progressively shorter antisense siRNA was tested. The activity was then lower, thus
showing that loss of the 3' nucleotide could not possibly give as high activity as the wild-type
21 nt siRNA.48 Recemtly, we have also repeated this experiment, obtaining the same strong
silencing, with a siRNA blocked at the 3' end by using a 3' deoxy nucleotide, as discussed in
section 2.9.
Obviously, the alternative mechanism proposed by Sijen et al,31 whereby the dsRNA is
produced by self-priming by the cleaved 5' end of the mRNA, is not invalidated by blocked 3'
ends on siRNA antisense strand. However, the absence of transitive silencing when knocking
down one isoform, and then reintroducing an almost identical isoform,55 in addition to the loss
of siRNA silencing over time,2 would indicate that in human cells this propagative
mechanism is not active either. Thus, whatever mechanisms that underlie the data of Lipardi
et al and Sijen et al, it can not be universal, as silencing in mammalian cells does not seem to
need it.
1.5 Non-complimentary short RNA, stRNA, inhibit translation
Yet, there is obviously much that is not known. The investigations into microRNA started
with Victor Ambros’ description of heterochronic C. elegans embryo development mutants in
1989. He showed that lin-4 was a regulator of the larva-to-adult molting stage switch. 56
Strangely, the loci this gene was mapped to, turned out to be empty of functional open reading
frames. Lee et al thus proposed that lin-4 was expressed as a short RNA that interacted with
complementary sequences in the 3’ UTR of the downstream gene of lin-14.57 Later, another
such microRNA (also called stRNA (short temporal RNA), after the molting-time effects in
C. elegans embryos), let-7, was found.58 This gene has also a homologue that is expressed in
humans,59 although the function here is unknown.
Using Tuschl’s technique for cloning of small RNA,47 in addition to bioinformatic methods,
this class of microgenes was shown to contain at least hundreds of different genes in different
organisms.60-62 Their structure seem to be a ~70 nt hairpin RNA that is expressed, and then
19
processed by Dicer into 21-22 nt RNA similar to siRNA, but with only the antisense strand
being retained.27 However, recent results investigating temperature dependence of RNA
silencing, indicate that temperature-labile siRNA and non-affected stRNA are generated by
different nuclease complexes.63 The plant system might be different in this respect, although,
since it also has two populations of siRNA with different sizes,64 having recently been shown
to originate from dsRNA by plant Dicer.65 Furthermore, in a very recent report, an
Arabidopsis Dicer-like mutant, carpel factory (CAF), did not compromise PTGS.68
The stRNA, paired with the mRNA target, forms an interesting non-basepairing bulge in the
central region,58 as will be discussed in section 2.8. The functions of these microgenes are
unknown. The mechanism of action is only known in the case of lin-4 and let-7 in C. elegans,
where they seem to be inhibiting the translation of the target mRNA. By adding pre-let-7 to
Drosophila lysates and also in HeLa lysates RNA cleavage activity can be generated if stRNA
can basepair perfectly to a target in an mRNA target strand. The function of this bizarre spillover from stRNA to siRNA was proposed by the authors to serve no function in human
cells.66 Further research is needed.
1.6 Synthetic siRNA versions
A limiting obstacle to use of RNA-induced silencing in mammalian cells was the induction of
PKR/interferon/panic response to double-stranded RNA.72 This virus defence shuts down
non-specifically gene activity in a cell, even if some mammalian cell types seem to lack this
response and thus permit the use of long dsRNA.73, 74 Tuschl and coworkers' breakthrough
article demonstrated that synthetic siRNA, as developed in their earlier work in Drosophila,47
could bypass this virus defence and be able to knock down targets in mammalian cells.7
Elbashir et al prepared three siRNA against three different luciferase reporters, pGL2, pGL3
and pRL, and cotransfected these along with two reporter plasmids into various Drosophila,
mouse, monkey and human cell lines. In all cases there were clear knock-down of the targeted
reporter, as seen by standardization with the control reporter, although the effect was
somewhat variable for the human cell lines, something that inspired us to start looking at
position effects with siRNA, as discussed in section 2.2.
The double-stranded siRNA had 21 nt RNA strands, ending in 2 nt non-basepairing termini.
These overhanging 3' ends Elbashir et al replaced with DNA thymidines instead of uridine,
20
with the rationale that it was cheaper and could perhaps give better protection against
degradation. A control, using UU instead of TT, showed no significant difference, thus siRNA
was shown for the first time to tolerate chemical modifications, as further discussed in section
2.9. It should also be noted that the last T, or U, in the antisense strand of GL2, uGL2 and
GL3, did not basepair with the mRNA target, as this is CA in both X65324 and U47296, for
pGL2-Control and pGL3-Control, respectively, and thus constituted a kind of mutation, as
further discussed in section 2.8.
In contrast, longer dsRNA, ~50 bp or ~500 bp, did induce a 10-20 fold and 200 fold
reduction, respectively, of all reporters in a non-specific fashion, assumedly by the PKR
system. Even so, a specific depletion of the target reporter could be seen superimposed onto
the non-specific results. Why this cohabitation of the RNAi system and the PKR system
should exist, is still unclear. Elbashir et al then used siRNA to knock out endogenous proteins
lamin A/C, lamin B1, NuMA and vimentin.7
Caplen et al presented much the same siRNA results a few months later,75 with the additional
claim that 22-23 nt siRNAs were somewhat more efficient, of the 21-27 nt strand range tested,
in contrast with Elbashir et al, who found that 21 nt duplexes were the most efficient, of the
20-25 nt strands tested.69 Caplen et al also demonstrated directly that the PKR response was
not activated by siRNA, something that have been supported by later studies.76-78
Furthermore, these oligos had no DNA 3' end overhangs, but were no more efficient.
Interestingly, siRNA antisense oligos were found to have no activity, as also many others
have reported later, something that will be discussed in section 2.15 on antisense siRNA.
Boutla et al were the first to publish a report showing siRNA to work in vivo in Drosophila
embryos, and that a central mutation was partly tolerated.79 However, Elbashir et al also
published an article on the functional anatomy of siRNA late in 2001, reporting that a central
mutation abolished silencing activity in their Drosophila embryo lysate,69 in apparent contrast
with both Boutla et al 79 and our own results,2 as discussed in section 2.8. Furthermore,
Elbashir et al found that DNA nucleotide substitution was tolerated in the 3’ end of the RNA
strands (up to 4 DNA nucleotides at 3' end), but that full substitution of DNA or 2’-O-methyl
nucleotides abolished activity, as will be discussed in section 2.9. They also found support for
their earlier finding that 2 nt 3’ overhang siRNA were the most effective,47, 69 while finding
that some overhang sequences in the antisense strand were more effective than others (UG,
21
UU, TdG and TT), possibly due to the penultimate nucleotide contributing to target
recognition.69
Nykänen et al showed by fractionation of cell lysate that it is not the siRNA incorporated into
the RISC complex that is the active complex, but a RISC complex containing the singlestranded antisense strand of the siRNA, a complex designated RISC*.44 This active complex
size was found to be less than 232 kDa (inactive RISC ca. 360 kDa) something that clashes
with Hammond et al’s size of 500 kDa,28 and Tuschl’s recent report of 90-160 kDa.45
Furthermore, it was reported that two of the four sequential steps found (Fig.1), needed ATP,
both the siRNA unwinding and mRNA cleavage, in addition to there seeming to be a pair of
putative gateway kinases and phosphatases that cycled phosphates on the 5’ end of free
siRNA. As noted above, when this 5'-hydroxyl is blocked by a methoxy moiety the activity of
the siRNA is lost.44
1.7 Endogenously expressed short hairpin RNA (shRNA)
A third class of RNA molecules, in addition to siRNA and stRNA, must briefly be mentioned.
Although it has been shown that dsRNA as short as 36 basepairs are inactive,47 and that
dsRNA longer than 30 bp will induce the PKR/interferon/response,72, 75 many groups
commenced to try expressing hairpin RNA shorter than 30 bp. Transcription in vivo, would be
an advantage, as delivery of siRNA can be a problem, although use of viral vectors developed
for gene therapy have its own problems.80, 131
Use of shRNA gave good results.76, 81-89 The basis of this technology is that RNA polymerase
III will terminate transcription of a transcript after 5 successive thymidines in the template
and the transcript is cleaved after two uridines.81 Thus a short transcript, in the form of a
hairpin, or with two complementary and separate strands, is possible.82, 86 The shRNA seems
to be processed by Dicer to siRNA, whether they were longer mimics of let-7 hairpins, or
simple, 29 bp hairpins.84
Although Brummelkamp et al found that the 9 nt loop is clearly better than a 5 nt and 7 nt
loop, 81 something that also is corroborated by Castanotto et al,89 a wide range of loops
connecting the two strands have been used with good results: Paul et al and Paddison et al
used 4-loops (UUCG and UUAA, respectively),83, 84 while Yu et al and Yang et al used 3loops (AUG and UCU, respectively).87, 88 Sui et al reports that a 6-loop works well,85 while
22
Jacque et al find no difference between 3-, 5- and 7-loops.76 More research seems to be
needed also here.
Ohkawa and Taira, significantly, have constructed a RNA polymerase III promoter that is
regulatable, by fusing this promoter with a Tetracycline Response Element (TRE).71 This
system will obviously find many applications for tissue and time-point specific induction of
RNAi silencing. In fact, Taira is in the process of commercialising this plasmid, as requests
for it recently have become too exceptionally intense and time consuming to respond to.
1.8 Using siRNA and shRNA against viruses
A long series of impressive results against virus targets have been announced the last 12
months. The first report was from John Rossi's group, where targeting HIV-1 rev transcripts,
in an cotransfection assay, gave an impressive 10 000-fold reduction of p24, using siRNA
expressed from two complementary U6 snRNA promoter constructs, a construct that is now
in the process of being reconstructed for delivery by lentivirus.86 Jacque et al, using synthetic
siRNA against several regions of the HIV-1 genome such as LTR, vif and nef, found that in a
cotransfection assay, virus production was reduced 30-50 fold. More importantly, even if the
HIV-1 infection process itself was not interupted, production of viral cDNA intermediates
were dramatically inhibited if the cells had been transfected by siRNA against the HIV-1
genome, even if the virus was added up to 4 days after the siRNA transfection.76 A third
group, led by Phillip A. Sharp, demonstrated siRNA inhibition against HIV-1 cellular receptor
CD4, Gag and GFP-for-Nef targets,91 and recently Martinez et al demonstrated inhibited
HIV-1 replication by using siRNA against CXCR4 and CCR5.92
In a very interesting piece of work, Gitlin et al, targeted polio-virus by transfecting synthetic
siRNA into HeLa cells before infection with the cytolytic and apoptosis-inducing Mahoney
strain.77 Remarkably, siRNA inhibited plaque formation completely. In co-electroporation
assays, where target mRNA was introduced along with siRNA, maximum silencing was seen
already after 3 hours. However, the silencing was lost over time, and when the emerging
strains were sequenced, it was found that they contained mutations in the siRNA-targeted site,
as will be discussed in section 2.8. The first use of siRNA against viruses in a full animal, was
done by Hu et al, where co-electroporation of RSV proviral DNA and siRNA into the
developing chick neural tube, inhibited viral replication.93 Recently, Kapadia et al has
reported inhibition of hepatitis C replication in Huh-7 cells.78
23
2 Discussion of results in thesis papers I, II & III
2.0 Results introduction - a short summary of ribozyme experiments
This thesis work intended to use ribozymes for functional genomic studies on PSKH1.94 A
year of artefact-prone work ensued, where the luciferase-based assay system repeatedly
demonstrated good results (ca 60-80 % depletion of target signal), a silencing signal which
was partially lost when the assay was tried out under different conditions, e.g. like the shift
from COS-1 cells to HeLa cells or the shift from using total protein content to using Renilla
Luciferase as an internal control (data not shown, unpublished). Such assay dependence will
be discussed in section 2.5.
It was finally decided to do inactivation mutations to the ribozyme core. It is known that
hammerhead ribozymes have the conserved core (3-9) sequence CUGANGA, where G-5, G-8
and A-9 are vital for cleavage activity.95,96 Interestingly, our mutated ribozymes turned out to
be just as active, or even more active, than the wildtype ribozymes against the Tissue Factor
reporter construct TF-Luc (Fig.2B, unpublished). Also note that different hTF ribozymes
seem to have different depletion capability (Fig.2A, unpublished), a position effect similar to
the one seen previously with ribozymes against PSKH1-Luc.103 We went on to investigate
position effects with siRNA, as discussed in section 2.2. That mutated ribozymes were just as
effective as the non-mutated ribozymes, probably means that the Tissue Factor ribozymes had
no significant catalytic effect upon their target mRNA.
Some irregularities discovered in ribozyme synthesis and storage, threw some doubt upon
these conclusions, as ribozymes had been exposed to unbuffered, acidic conditions. This
could possibly account for both the lack of catalysis, the position effect seen, and some of the
non-specific effects. Routine testing of the ribozymes for degradation with PAGE gels had
disclosed no significant degradation, but there seemed to be a possibility of acidic conditions
having led to depurination in certain positions. MALDI-TOF mass spectrometry, however,
although no optimalization was performed, and only rough peaks were obtained, showed no
sign of peak shift due to loss of purines, and thus seemed intact (data not shown,
unpublished). In addition, theoretical judgement expected that such depurination would have
led to breakdown and cleavage of the RNA strand, supporting the hypothesis of the ribozymes
indeed having no significant enzymatic activity.
24
A
120
100
80
60
40
20
hT
F1
78
hT
F2
61
hT
F2
70
hT
F3
34
hT
F3
83
hT
F4
52
hT
F4
89
hT
F5
07
hT
F5
24
hT
F5
73
hT
F7
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co
nt
ro
l
0
B
120
100
80
60
40
20
0
control
rbz
hTF383
383mut1 383mut2 hTF489
489mut1 489mut2
Fig.2 Tissue Factor ribozymes exhibit position effects (A) and strong activity after having the
conserved core mutated (B). Cotransfection of ribozymes and TF-Luc reporter was done
essentially as described for siRNA in paper I. Ribozymes 383mut1 and 489mut1 have
methylated G-5. Ribozymes 383mut2 and 489mut2 have a G5A mutation in addition to
methylation of this A.
To explain the sporadic depletion effects seen with ribozymes in certain assays, one should
look to the long list of improvements of ribozymes tha have been tried out, one of them using
hybridizing DNA arms on a RNA ribozyme core.95 This, in in vitro reactions on short RNA
25
targets, will improve both enzymatic turnover (DNA:RNA duplexes bind less tight, allowing
higher turnover) and protect against degradation. In addition, RNaseH, is known to recognize
and cleave RNA annealed to DNA. Furthermore, these DNA arms can also be synthesized
with phosphorothioate nucleotides, with the intention of improving the degradation resistance
even more. Finally, RNaseH is known to be very promiscuous in its cleavage of such thiophosphodiester DNA:RNA hybrids, in some cases only needing 5-7 hybridizing basepairs.39,
99, 100
Thus, our ribozymes were probably in fact depending on RNaseH action.
Although there seemed to be some weak activity against endogenous Tissue Factor mRNA
(data not shown, unpublished), probably due to RNase H action, control ribozymes actually
stimulated Tissue Factor expression 2-3 fold (data not shown, unpublished). Tissue Factor, in
its role as the principal initiator of blood coagulation, is known to respond to a wide range of
agents causing perturbation of vascular endothelium, among them bacterial
lipopolysaccharide, lectins, phorbol ester, endotoxins and inflammatory cytokines,101 thus the
response to extensively modified nucleic acids is not surprising, as this might be perceived as
foreign material. As will be discussed in section 2.5, siRNA, interestingly did not induce
endogenous Tissue Factor.
2.1 An attempt at RNA interference in mammalian cells using long dsRNA
Our first ribozyme target was PSKH1,40 and a lot of effort was spent to get PSKH1 ribozymes
to work reliably both in our cotransfection assay 103 (and data not published) and against
endogenous PSKH1 mRNA in HeLa cells, with only limited success (data not shown,
unpublished). Thus, in the wake of Hammond et al’s paper,36 a plasmid expressing a dsRNA
of the protein coding region, was attempted constructed.
A second copy 1275 bp coding region of PSKH1 was attempted ligated bidirectionally into a
BamHI site after the first copy of the PSKH1 coding region, in what should have produced 50
% clones of each variant. However, it turned out that our standard E. coli strain Dh5alpha
only produced direct repeats after selection for clones. In all 12 clones with direct repeats
were isolated, but no inverted repeats. Likewise, an attempt to construct a similar TF dsRNA
construct was aborted. The problems was solved by trying out the RecABC-deficient E.coli
strain SURE-R, and a PSKH1 dsRNA hairpin construct was finally obtained, indicating that
our standard strain Dh5alpha indeed rejected inverted repeats by recombination (data not
shown, unpublished).
26
Tested in our cotransfection assay against our PSKH1-LUC reporter plasmid, the dsRNAexpressing construct showed no activity (data not shown, unpublished). Why this didn’t work
was unclear, at least to us, until Tuschl’s group published their paper on short interfering
siRNA avoiding the PKR-response.7 The methodology, however, was later proved to be
sound, as two plasmids expressing two versions of shRNA (hTFsh and hTFsh+6) against
Tissue Factor, based on John Rossi’s plasmid construct,86 proved to deplete TF-LUC in our
assay by ca. 85 % and 70 %, respectively, in our cotransfection assays (data not shown,
unpublished).
Sites on mRNA targets can be differentially accessible to ribozymes and
oligodeoxynucleotides.103 Therefore, Thomas Tuschl’s breakthrough, showing that RNA
interference could be obtained with short dsRNA (siRNA), made us consider a comparison
between ribozyme and siRNA position effects, as it seemed to us that certain siRNA species
had limited efficiency.7,47
2.2 Positional effects in a series of siRNA
A series of siRNA was synthesized to test whether our ribozyme position effects also would
occur with siRNA (Fig.1A - paper I). Four siRNA were made against each of our two human
genes of interest, PSKH1 and Tissue Factor (hTF), with two of these four targeting the best
ribozyme sites and two targeting the least active sites, on each mRNA. Surprisingly, using
Tissue Factor siRNA and reporter construct (Fig.1B - paper I), a strong position effect was
found (Fig.1C - paper I), but it did not correlate with the ribozyme data. This has some
implications for certain ideas of mRNA target accessibility, as discussed in section 2.3.
To further investigate the positional effect, we synthesized several series of siRNAs against
new target sites that could be thought to have different degrees of secondary mRNA structure
or general accessibility. The translation initiation site (hTF77i) and the 3'-end of the coding
region (hTF929i) were both essentially inaccessible targets. Two other siRNA targeted
heavily basepaired regions as predicted by MFold.104 One (hTF256i) demonstrated
intermediate activity while the other (hTF459i), had low activity (Fig. 1C - paper I).
From this position effect, supported by further position effect data from other measurement
assays (discussed in section 2.4, 2.5 and 2.15), springs the main hypothesis of this thesis, that
27
siRNA exhibits natural variation in activity, as explicitly posed in section 2.6. Further
discussion of possible targeting rules are deferred to section 2.4.
Support for the existence of siRNA position effects is emerging from the literature, as several
examples of inactive or less active siRNAs in mammalian cells have been described,78, 82, 84, 86,
105-107, 119, 124, 134
while a weak positional effect in Drosophila lysates can be inferred from
published data.47, 69
In C. elegans, Simmer et al managed to activate dozens of previously inactive dsRNA
stretches, using the RNAi-enhancing rrf-3 negative strain, and in some cases created RNAi
knockout phenotypes from genes that had hitherto not been responsive to dsRNA.32
Yang et al, working with a wide range of different techniques, reported some inactive
chemically synthesized and in vitro transcribed siRNA, as well as inactive shRNA.
Interestingly, the authors also established the new technique of esiRNA (endoribonucleaseprepared siRNA), in which an overlapping set of siRNAs are produced in vitro by partial
digestion with E. coli RNase III, a technique that can be superior even to long dsRNA.88
Processing of dsRNA by Dicer starts from a fixed end and proceeds in a sequential
manner,47,109 and is thus expected to produce a largely non-overlapping set of siRNAs.
Cleavage of a dsRNA by E.coli RNase III will thus create a larger and more complete set of
siRNAs than the non-overlapping set produced by Dicer from dsRNA. The higher activity of
the esiRNA supports the argument that different siRNAs have different activities. Otherwise
any set of siRNA from a dsRNA would have the same activity. That esiRNA might not be a
wholly superior technique to siRNA, as esiRNA will average out the siRNA performence, can
possibly be inferred from our competition studies, as discussed in section 2.10.
Recently Taira's group also has used recombinant Dicer to produce siRNA from dsRNA in
vitro (calling it diced siRNA), finding that both puromycin and H-ras mRNA could be more
depleted using diced siRNA than any other of 10 specific positions using siRNA. Some of
these 20 siRNA had also low activity, giving further support of the existence of position
effects.110
28
2.3 On the possibility of finding local targeting rules
One way to deal with RNA target selection, has been to postulate open areas more vulnerable
for attack and then try to find these theoretical local targets by computational methods based
on mRNA sequence and measured energies for such structures as hairpins. An example is
such secondary structure prediction software as Mfold.104 We found no correlation between
MFold predictions and our siRNA data, nor, as noted above, with our ribozyme data. This
supports other reports in the literature finding MFold inadequate as a target prediction tool.110
In any case, mRNA is not naked in vivo, but packed with RNA binding proteins. These RNA
binding proteins, in addition to, and in cooperation with, secondary RNA structure, is possibly
responsible for our observed position effects, as also pointed out previously for certain less
than fully active siRNA.7 Furthermore, probably few antisense oligo can work in an
exquisitely specific manner without protein effectors, making mRNA target cleavage a
complex interaction between mRNA structure, mRNA binding proteins, antisense basepairing
and effector protein(s).
Such a complicated process, involving many factors, seems less likely to have so simple local
rules that they can be inferred from experiments typically featuring a few mRNA targets and a
few dozen antisense agents. More to the point, this multifactor process might easily be
imagined to be the physical basis for the main hypothesis of this thesis, namely a continual
range of siRNA with differing silencing activity, as posed in section 2.6.
Even the pharmaceutical company ISIS, which routinely screens hundreds of targets with
massive tiling of oligos, has found no rules increasing the success rate to better than one good
hit in 50, although later generations of oligos, like 2’MOE, have a somewhat higher success
rate. A review of the literature found that at best, in large studies, one in eight antisense
oligonucleotides are active. Despite this, 1655 citations reported success with only a single
oligo.99 In this perspective, effective targeting rules might still be some way off.
2.4 Investigating local area around hTF167i
Going on to investigate the target positions close to our best agent hTF167i, we found a
phenomenon illustrating this local targeting rule problem very nicely. A series of siRNA, each
sharing 18 out of 21 nucleotides with each neighbour (Fig.2A - paper I) had very different
activities (Fig.2B - paper I). E.g. hTF158i and hTF161 differed by only 9 and 6 nucleotides,
29
respectively, from hTF167i, thus sharing 12 and 15 nucleotides, respectively, yet they were
practically inactive.
Alluringly, hTF167i lies in the middle of a window of very low mRNA GC content,
suggesting that target accessibility might be due to this easily determined factor. Furthermore,
Tuschl et al, in addition to licensed manufacturers of siRNA, have previously recommended
approximately 50 % GC content.124 However, a review of the siRNA in the published
literature and their targets, found only a weak bias of uncertain biological significance in
favour of low GC content. Recently Taira's group also found no GC bias in their sample of 20
siRNA against a puromycin-resistance gene and H-ras. Interestingly, one of their best siRNA
against H-ras had a GC-percentage of 84 %, while one of the worst had 53 % GC.110
2.5. Endogenous hTF mRNA targets differ markedly in siRNA accessibility
An effort had been going on for a while to try out our best ribozymes against endogenous
mRNA targets, with very little success, as the best Tissue Factor ribozyme, capable of ca. 75
% target depletion in our cotransfection assay (Fig.2A, unpublished), had only limited activity
against the endogenous target (data not shown, unpublished). For PSKH1 the results were
similar.
Such lack of robustness in activity, can also be inferred from the literature, where good initial
results seldom seem to be followed up by application in more stringent and complicated assay
systems. An interesting example from the Tissue Factor field is Stephens & Rivers obtaining
already in 1997 an impressive 80.4 % (+/- 2.2 %) suppression of Tissue Factor activity in
primary monocytes with an antisense oligodeoxy nucleotide (ODN).113 This remarkable
success has not been followed up by the authors, or anybody else, even if almost 1600 articles
with keyword Tissue Factor has been published in the six years hence.
Such an assay system dependence, or contextual dependency, is perceived to be chronic in the
antisense field.99,100 With this system dependence in mind we turned from reporter constructs
to endogenous targets for siRNA. There we demonstrated that the pattern of depletion in our
cotransfection assays were mirrored in results on endogenous Tissue Factor mRNA targets,
both on a global (Fig.3A - paper I) and on a local (Fig.3B - paper I) mRNA target scale.
Reassuringly, endogenous Tissue Factor expression, known to respond to many
30
environmental stimuli,101 was not stimulated by the introduction of siRNA, as seen by the
transfection of the irrelevant control siRNAs PSK314i, PSK546i, PSK566i, PSK739i (Fig.3Apaper I). The position effects were in fact more clear-cut with our Northern assay than with
the cotransfection assay, as the siRNA with some activity in cotransfections had practically no
activity in Northerns.
That there might be false positives in the cotransfection assay, something that we have seen
indications of, emphasizes a certain weakness of a reporter system dependent on forced
expression of fusion constructs from plasmids transiently transfected. The overall similar
pattern of depletion shows that the cotransfection assay is of value as a fast and convenient
tool for initial investigation. However, the cotransfections results must later be backed up with
data on endogenous targets. An illustrating example, among several others in the RNAi field,
of trusting cotransfection data perhaps a bit too far, can be seen in a recent paper by Chiu and
Rana, where they claim that crosslinking of siRNA does not completely abolish silencing, as
30 % silencing was still retained, in a cotransfection assay.114
The similar pattern in cotransfection assays and with endogenous targets, have implications
also for the search for local targeting rules, as the fusion mRNA, hTF-LUC, was targeted
much the same way as the hTF mRNA itself. Furthermore, preliminary results from mouse
cells indicate that position effects are strong also in mTF, and, interestingly, targets
corresponding to our best hTF siRNAs are also the best in mouse cells, even if the conserved
sequence similarity between hTF and mTF is only ca. 66% (data not shown, unpublished).
This probably means that there are local rules, shaped by the local nucleotide pattern,
although they might not be easily elucidated or generalized.
2.6 The main hypothesis: Natural variation in siRNA activity
Thus far we have demonstrated inactive siRNA and active siRNA. Furthermore, there
probably exists a balance between RNAi mRNA depletion and mRNA production during the
long decline of silencing from 24 h till the effect fade completely 2-3 days later, as discussed
in section 2.13. But what is the situation for our best candidate hTF167i at the point of
maximum silencing at 24 h? A major hypothesis of this thesis is that this siRNA at this point
has excess capacity, as in able to cleave more mRNA if it was present.
Two good siRNA candidates would generally obtain the same high level of silencing, as
31
transfection efficiency limits in the silencing assay obscure any differences. Thus, the
existence of the excess capacity class of siRNA must be demonstrated indirectly by the high
tolerance for mutational and chemical modifications, as will be discussed in section 2.8 and
2.9, respectively. Dosage-response experiments discussed in section 2.12 and 2.15, can show
more directly that at certain siRNA concentrations silencing capacity is saturated, and could
be used to differentiate excess capacity siRNA. Finally, time-course experiments, as discussed
in section 2.13, can show that certain agents silence faster and more persistently, making it
possible to differentiate candidates that all give complete silencing under standard conditions.
More theoretically and abstractly, a continuum of siRNA with differing activities from very
high activity to inactive there can be imagined. Practically it might be easier to think of
siRNA as falling into three broad categories, and more importantly, this is easier to
demonstrate:
1) Inactive siRNA.
2) Intermediate activity siRNA.
3) Excess capacity siRNA.
Certain observations in our papers, and in the literature, become explainable in the light of
this hypothesis.
2.7 Tissue Factor protein and procoagulant activity also show position effects
Going on to measure the downstream protein level, and, more importantly, the physiological
process of blood clotting, we transfected HaCaT cells 48 h before harvest, to allow for protein
half-life of 8 hours.115 Again there was a pronounced position effect, with Tissue Factor
protein being depleted ten-fold by the best candidate hTF167i and five-fold by hTF372i
(Fig.3C - paper I), while hTF478i had no activity (data not shown).
Finally, using a procoagulant assay,116 we showed that hTF167i reduced activity five-fold,
with hTF372i also here having less effect (Fig.3C - paper I), thus demonstrating siRNA
position effect in four different assays. This robust effect shows that RNA interference holds
great promise for downregulation, not only of gene transcripts, but of gene function.
32
2.8 Tolerance of siRNA for mutations
In line with the ribozyme inactivation experiments (Fig.2B, unpublished), we wanted to try
mutational inactivation of siRNA. Two versions of hTF167i, with one or two central
mutations, were synthesized and named M1 and M2 (later renamed s10 and ds10/11 in paper
II: Fig.6a - paper I, Fig.1 - paper II). Later, this central position was shown to be close to the
putative active site of the RISC, as the cleavage site was found to be exactly 11 nt from the 3'
end of antisense strand.69 In addition, GC pairs were chosen for mutation to disrupt more
hydrogen bonds possibly involved in hybridisation.
Satisfyingly, these two mutations showed a gradual decline of activity (Fig.6C - paper I).
Interestingly, M2 had higher activity in the Northern assay than in the TF-LUC assay, where
M2 was almost inactive. In the perspective of a recent prediction by Hutvagner & Zamore,
where it was proposed that bulges in the stRNA is causing the translational inhibition rather
than mRNA degradation,66 our experiment indicated that the nature of the bulge is also
important.
If a central bulge was all that is needed, as indeed seems to be the case for the AU-bulge for
let-7 targeting two targets in lin-41,58 there would not be much degradation of mRNA,
(although there would be some, as there seems to be spill-over into RISC by stRNA),66 while
the protein would be silenced. This is actually the opposite of what we see in our experiment
with M1 and M2, where endogenous TF mRNA is being degraded, while TF-LUC is not
significantly silenced (Fig.6 B,C - paper I). Further support of this is found in a very recent
report, where Zeng & Cullen performed a systematic series of mutations on the human
microRNA miR-30, showing that the most sensitive area was not the bulge, but the stemstructure at the 5' and 3' end, just outside the processed stRNA.117
Other combinations of modified and wild-type strands, thus with a bulge in the siRNA itself,
were also tolerated well, with the combination M1/wt (sense/antisense) and M2/wt siRNA not
having much less activity than wild-type, while the mutation in the antisense strand was more
deleterious (Fig.6B,C - paper I). This has previously been shown for dsRNA,67 and for 3' end
overhangs,69 and has recently been reproduced.112 DNA:RNA, or DNA:DNA oligos, on the
other hand, had little activity, although the antisense RNA had some interesting weak activity
possibly due to antisense siRNA effects (Fig.6B,C - paper I), as will be discussed in section
2.15.
33
Wanting to explore the tolerance for mutations further, we did study introducing a series of
G:C mutations from one end of hTF167i to the other. We found that hTF167i had a general
tolerance to mutations, with less tolerance for mutations at 3’ end of the siRNA (Fig.2 - paper
II), possibly facilitating design of single-nucleotide polymorphism (SNP) specific siRNA.
However, preliminary results from recent targeting of A:T pairs in the 3' end, show that s14
and s17 seem to be well tolerated (data not shown, unpublished). Tuschl and coworkers found
the diametrically opposite effect, namely that double mutations in the 3' end of the siRNA
were less deleterious to silencing than mutations in the 5' end of the siRNA, in a Drosophila
lysate assay.69 Overall, our best agent hTF167i seem to exhibit great tolerance for mutation,
supporting the existence of siRNA with excess capacity. Further support for this view can be
found in preliminary data showing the intermediate activity siRNA hTF372i and hTF173i
being more affected by s7-mutations than hTF167i (data not shown, unpublished).
The most recent review of RNAi still claims that one of the unique features of RNAi is its
exquisite sequence specificity, where a single mismatch can abolish activity,118 while Tuschl’s
latest review conclude that the silencing effect is extraordinarily specific, because one
nucleotide mismatch between the target RNA and the central region of the siRNA is
frequently sufficient to prevent silencing.54
However, more reports on tolerance of single mutations are now available. In the first
published paper on siRNA mutations, other than the 3' end mismatches of Tuschl's siRNA,7, 47
Boutla et al reported that a mutated siRNA with a single centrally located mismatch relative
to the mRNA target sequence, retained substantial activity when injected into Drosophila
embryos.79 Jacque et al found that a single mismatch in siRNA targeting the HIV LTR
resulted in only partial loss of activity, while another siRNA targeting the HIV VIF exhibited
almost full activity.76 In a GFP-fluorescence cotransfection assay, Yu et al 's single mutation
in the antisense strand was tolerated, while, paradoxically, the corresponding mutation in the
sense strand led to reduced silencing, although against the antisense target this mutated
siRNA had good activity.87
In contrast, Elbashir et al found that a single mismatch, in the tenth or eleventh basepair
(position), counting from the 5' end of the siRNA, was deleterious to activity in a Drosophila
embryo lysate assay.69 That there is no mRNA production in such in vitro assays complicates
34
matters, as an siRNA that in vivo had not sufficient capacity to influence the mRNA level,
might still be effective against limited amounts of RNA target. However, abolishment of in
vivo activity by a single mutation has been reported. Brummelkamp et al,81 using a short
hairpin RNA (shRNA), processed to siRNA by Dicer,84 achieved inactivation by single
mutations in either the second or ninth position from the putative 5’ end of the shRNA. Gitlin
et al, argued the case for single mutation inactivation more strongly by isolating siRNAresistant polio virus strains containing single mutations in the target site on the genomic RNA,
corresponding to the sixth or ninth nucleotide of the siRNA, counted from the 5’ end of the
sense strand.7 The ninth position was also mutated by Martinez et al, resulting in strong
inhibition of a mutant form of p53, while leaving wild-type p53 untouched.108
In the context of the hypothesis of natural variation in siRNA activity, it is possible that these
results are explainable by these particular siRNA falling into the intermediate capacity group,
and thus having no excess capacity to resist mutations. To support this view, it can be
observed that Gitlin et al also tested antisense siRNA, showing this to have no activity. Since
antisense siRNA do have activity, although at a lower efficiency, as discussed in section 2.15,
it might be argued that if Gitlin et al's siRNA had excess capacity, its antisense siRNA partner
should also have had some remaining activity.
In stark contrast to single mutations, four mutations abolished activity completely for Jaques
et al.76 Complete abolishment of activity has also been reported by Gitlin et al, Klahre et al
and Garrus et al, for siRNAs with 5, 6 and 7 mutations, respectively.77, 120, 121 Paddison et al
reported that a triple mutation in an shRNA abolished activity in a cotransfection assay,84 as
did Elbashir et al for the pair of GL2 and GL3 siRNA.7,124
Thus, a double mutation is perhaps the threshold for toleration for most siRNA. Kisielow et al
reported that an siRNA resulting in essentially complete knockdown of the expression of its
target gene, was unable to inhibit the expression of an isoform from a transiently transfected
plasmid, an isoform having two non-contiguous mutations in the recognition sequence of the
siRNA.55 The positions of these mutations correspond to our ds10/13 double mutant, which,
along with two other double mutations (Fig.1 - paper II), also exhibited low activity in our
assay (Fig.2 - paper II). Yu et al, Lassus et al and Wilda et al also reported abolishment of
activity with a double mutation.87,122,123
35
Therefore, our results, and the literature, so far, is consistent with the hypothesis of siRNA
falling into three general groups of siRNA ranked on their activity, as posed in section 2.6:
Inactive siRNA, intermediate activity siRNA and excess capacity siRNA. This latest group of
excess capacity siRNA, seen in conjunction with our results showing a gradual decline of
activity when introducing modifications in our best agent hTF167i (section 2.9), might be
interesting for siRNA-based drug development.
2.9 Tolerance of siRNA for chemical modifications
As noted in the introduction, the siRNAs first used by Elbashir et al in mammalian cells,
were not wildtype siRNA, as they were synthesized with two 3’ end overhangs of deoxyribose
thymidine nucleotides (TT), one of which did not basepair with the mRNA target.7 Our
siRNA, having to target ribozyme sites, and following Tuschl's example, also did not basepair
with mRNA in these overhangs in the penultimate nucletide.
At the time we worried that this might be the reason for some of our inactive siRNAs (Fig.1 paper I), and the loss of silencing seen in time-course experiments, as discussed in section
2.13. Since a major objective initially was to try to detect such long-term silencing (or
propagative effects) that had been seen in C. elegans,5 we decided to test both base-pairing,
non-TT deoxyribose overhangs for hTF167i (hTF167i-BO), in addition to resynthesizing pure
RNA strand versions of hTF167i, hTF173i, hTF256i, hTF372i, hTF478i and hTF562i with
base-pairing overhangs. In all cases, TT-nonbasepairing overhangs, DNA-basepairing
overhangs and all-RNA siRNA, we found no significant change of activity between versions
measured at 24 h, or in time-course experiments, using either Northerns (Fig.6D, and data not
shown - paper I) or reporter plasmids in serial transfection assays (data not shown).
Thus our work supported Tuschl's conclusions of some tolerance for modifications in the 3'
end of siRNA.7, 47, 69 More extensive tolerance for DNA modifications in the 3' end was also
found by Tuschl's group, as even 4 DNA nucleotides, at each 3' end, did not affect activity
much, although full DNA or 2-O-methyl substitution abolished activity.69 This tolerance has
recently been exploited very elegantly by Martinez et al to isolate RISC complexes by way of
photo-cleavable linkers from the 3' end of the RNA strand to a biotin marker.45 More 5' and 3'
modifications will be discussed in section 2.16.
Wanting to use fluorescent siRNA, we synthesized an antisense oligo version, with a FITC
36
group tagged to the 3' end, targeting position hTF167. In a siRNA duplex, hTF167-FITC, this
oligo had no significant loss of activity (Fig.6B,C - paper I), something which has attracted
some interest in the RNA interference field, because this is not consistent with the mechanism
proposed by Lipardi et al,46 as mentioned in the introduction.
Also, since we had been bothered somewhat by the thought of synthesis failures being
responsible for the lack of activity in some of our siRNA, even if they were tested for
annealing on non-denaturing PAGE gels, these repeated rounds of synthesis gave additional
support for the observed position dependence. During the mutational screening (section 2.8),
we indeed ran into two cases of documented difficulties with synthesis failure. We were able
to detect which synthesized RNA strand was faulty, when they failed to anneal in our routine
PAGE controls, by annealing each to different sense and antisense versions of the two strands.
In two cases, one of the two RNA strands failed this cross-annealing test, and were then
resynthesized (data not shown, unpublished).
Our time-course experiments (section 2.13) and the observed tolerance for 3' end
modifications, in the context of our earlier experiences with ribozymes, led us to the thought
of trying out a series of experiments modifying the ends of the RNA oligos, with the objective
of reducing the vulnerability to degradation. However, since RNA oligos in the RNA
interference system must be incorporated into a protein complex, we expected chemical
modifications to affect the silencing activity in a negative way. This in contrast with the
antisense field generally, where the appropriate modifications can improve oligo activity.95, 132
We wanted first to test three different chemical modifications that earlier have been used in
the ribozyme field for improvement of nuclease resistance, namely phosphothioates, 2-Omethylation and 2-O-allylation.95, 103 With the possible loss of activity in mind, oligos were
chosen with double substitution in the 3' end only. We expected the 5' end to be vulnerable,
since this is where putative RNAi kinases and RNAi phosphatases cycle a phosphate group.44
A series with a single modification in the 5' end and the 3' end each was also synthesized. In
all, 6 siRNA were synthesized, P0+2, P1+1, M0+2, M1+1, A0+2, A1+1. In this pilot
experiment the primary objective was to estimate the activity loss, as such minor
modifications can not be expected to stabilize oligos against degradation.
Interestingly, five of these siRNAs showed no significant loss of activity (Fig.3 - paper II),
37
while the siRNA with an allyl group on the 5' nucleotide had markedly lower activity than
wildtype siRNA and the other modified siRNA. We proposed that this might be due to
disturbance of 5' phosphate regulating enzymes,44 although it should be noted that A1+1 still
had good silencing capability. This general tolerance to these and other modifications, in our
best agent hTF167i, can be interpreted as supporting the existence of the excess capacity
siRNA.
Abandoning allylation, another series of siRNA were synthesized with more extensive 2-Omethylation or phosphorothioate backbones: M2+2, M2+4, P2+2 and P2+4. At this point
oligo degradation should start to be inhibited,95 but the question was whether the silencing
activity loss was acceptable. Satisfyingly, these siRNAs still showed strong activity (Fig.3 paper II), although somewhat lower than wildtype, and more persistent silencing in our timecourse assay (Fig.4 - paper II). Unfortunately, the phosphorothioate-modified siRNAs now
exhibited cytotoxicity, in particular P2+4, which was removed from further analysis.
Interestingly, oligos with more extensive phosphorothioation have been used with good
effect.95,99,132 Indeed, the pharmaceutical company ISIS have drugs in clinical trials with such
modifications.132 Why our oligos had strong toxicity when others use them without major side
effects even in preclinical and initial clinical tests, is unclear, although it is possible that this is
an example of the assay system dependence discussed in section 2.5, or, perhaps, our
modified oligos interfered with the RNAi components themselves.
A final series of modification was tried out, as we had in mind that Tuschl's fully 2-Omethylated siRNA had no activity at al.69 A series of siRNA were prepared: M4+4, M4+6,
M6+6, M6+8. A gradual decline of activity would support the hypothesis of the existence of a
continual range of siRNA activities rather than abrupt thresholds. Indeed, this last series of
modifications finally seemed to exhaust the activity of our best candidate hTF167i, as M6+8
(having only 7 pure RNA basepairs left in the centre of the siRNA) had almost no silencing
effect (figure 3A - paper III). In one representative experiment, the activities of these siRNA
exhibited 28%, 42%, 68% and 75% residual expression, respectively (data not shown - paper
II).
The last chemically modified oligo produced so far, a 3' deoxy version of the antisense strand
of hTF167i, also had activity indistinguishable from the wildtype, or, indeed, hTF167-FITC,
when incorporated in an siRNA duplex hTF167-3d (Fig.3C - paper III). Working on its own,
38
as an antisense siRNA oligo, it had severely limited activity compared with wildtype
antisense siRNA, as will be discussed in section 2.16.
2.10 Competition from inactive siRNA can block active siRNA
That the observed limit (at ca. 10 % residual mRNA) to depletion is probably caused by
untransfected cells is now well established, but at the time we wished for a stronger effect. It
was reasoned that the use of single siRNA is not a biologically relevant situation, as Dicer
would produce a range of different siRNA. It was possible that there existed cooperative
effects between pairs of siRNA, as also suggested by others.136 One scenario was that multiple
RISC complexes could be stimulating each other to give faster mRNA cleavage when several
of them were situated near each other on the same mRNA.
Testing this idea by performing a range of experiments using several siRNA simultaneously,
we obtained some initial success that did not hold up to scrutiny. It seemed possible, however,
that the excess capacity of hTF167i in the transfected cells masked the cooperative effect. One
way to differentiate siRNA that have excess capacity, would be to measure the persistence of
the silencing, therefore the idea of RISC cooperation was considered falsified when timecourse experiments showed that a triplet of hTF167i, hTF372i and hTF562i did not give a
significant increased persistence (data not shown, unpublished data).
During these experiments it was noticed, however, that inactive siRNA could affect active
siRNA in a negative fashion. Exploring this effect, in a cotransfection assay, we found that
with a ratio of 2:1 (20 nM inactive: 10 nM active siRNA) a weak inhibition could be seen,
even if 10 nM active siRNA was well into the saturation area. Furthermore, when the ratio
was increased to 9:1 (27 nM inactive : 3 nM active siRNA), the silencing was practically
abolished even if 3 nM hTF167i normally should have full silencing capacity (Fig.4A - paper
I). The competition effect could also be demonstrated with different active siRNA and
blocked by different inactive siRNAs (Fig.4B, C - paper I). In addition siRNA competition
was demonstrated in the three other assays, endogenous mRNA, protein and procoagulation
(Fig.4D - paper I). Where along the RNAi pathway the competition occurred (Fig.1), we were
not able to determine
but the order of siRNA transfection did not affect hTF167i activity in competition
experiments. This suggests that siRNA incorporation into RISC is not irreversible (data not
shown). Two other studies using siRNA competition have now been published. McManus et
39
al report that inactive siRNA can compete with active siRNA when electroporated into E10
cells, finding an inability to silence both CD4 and CD8alpha targets simultaneously,134 while
Martinez et al found that siRNA incorporation into RISC was irreversible, in a HeLa S100
extract, and thus impregnable to competition.45
Biologcially, this competition effect poses some interesting questions. How can RNA
interference cope with more than 9 targets if a 9:1 ratio (inactive:active siRNA) can block
siRNA silencing? Virus and transposons also can produce large amounts of dsRNA, so how
will a prodigious production of newly Dicer-cleaved siRNA affect suppression of other
targets? How can RNAi silence a target that has very few open target positions, such as Tissue
Factor, and PSKH1 (section 2.14), seemingly have? Would not Dicer then produce many
inactive siRNA competing with the active siRNA produced? Furthermore, as siRNA has been
proposed a role in clinical applications, will not a flood of siRNA against a drug target led to
transposon (and/or virus) release and the mutational effects seen in C. elegans mutator
strains? Finally, will not the above considerations, if valid, make necessary a link from RNA
interference system to a more permanent silencing, such as chromosomal silencing, with RNA
interference being an analogue to the innate immune system compared with immune
memory?
Data for such a link between RNAi and chromosomal silencing are scarce and scattered.
Phillip A. Sharp has proposed a link to the Polycomb-system,135 based on the data, among
others, from James Birchler and coworkers' series of experiments in Drosophila,90,129,130
where silencing (cosuppression) seems to flow from homologous sequences in the expressed
mRNA region, and through promoter sequences to silence a gene with only sequence
similarity in non-expressed regions. More specifically, the transgene construct w-Adh, with
promotor sequence from w and transcribed region from Adh, silenced not only endogenous
Adh, but also affected Adh-w. The latter, consisting of promotor sequence from Adh and
transcribed region from w, had no sequence similarity with w-Adh in the transcribed region,
and only promoter sequences in common with Adh. Furthermore, Adh-w is only silenced
when endogenous Adh is present.90
Birchler’s group also demonstrated a partial loss of silencing when using Polycombgroup
(PcG) mutants, a group of genes implicated in chromosomal silencing in Drosophila. Also,
certain RNAi mutants in C. elegans have sequence similarity with Polycombgroup genes,12
40
something that is especially interesting as RNAi homologues have now been implicated in
centromere silencing in S. pombe.141 Furthermore, methylation of DNA induced by dsRNA in
plants is well established.142 Still, the mechanism of RNA induced DNA silencing is yet very
unclear.
A last note on competition must be made, as the esiRNA technique have in two distinct cases
proven to be superior to siRNA.88, 110 However, our competition data indicate that esiRNA
will average out the siRNA performance, as the postulated few fully superior siRNA would be
outnumbered by siRNA of a more average activity. On the other hand, there might be
mechanisms for selection of the best siRNA, as an analogue to the classical immune system's
antibody selection and affinity maturation. Verification or falsification of this idea must await
a study testing a full tiling of an mRNA target, to see whether the best siRNA still are better
than esiRNA, as yet a very expensive exercise.
2.11 Cleavage fragment production from siRNA suggests the existence of an RNAi
exonuclease
There seems to be a relation between the phenomena of siRNA excess capacity and siRNA
cleavage fragment production. The appearances of the latter (Fig.3A - paper I) are puzzling.
The 3’ end cleavage fragments that we would be isolating with our poly-dT beads, thus an
mRNA not protected by a 5’ CAP, should not be stable more than a few minutes in vivo.138, 139
Likewise, single-stranded siRNA oligos are rapidly degraded in lysates.27,48 So why do we see
a signal that appears to be as strong as the main band at maximum depletion (10 % +/- 1.2 %
residual signal) there at all? We have been forced to consider that there must be some kind of
stabilization after endonucleolytic cleavage, perhaps due to a rate-limiting factor at a later
stage in the RNAi pathway.
More specifically, the appearance of the cleavage fragment only occurs when the silencing
effect is the strongest. It disappeared when the concentration of siRNA is decreased (Fig.6E paper I), when the siRNA is made less efficient by mutations (Fig.6C - paper I), or when the
siRNAs are targeting less accessible positions like hTF173 (data not shown).
Interestingly, this cleavage fragment appearance thus seems to correlate with the speed with
which the silencing is established (Fig.5A - paper I). If one can assume that our best agents
hTF167i, hTF167-M1 and hTF372i have a higher depletion speed, that is, higher mRNA
41
cleavage rate than hTF173i and hTF167-M2, it would seem reasonable that the cleavage
product is a classical rate-limiting result due to a saturated, and mRNA-stabilizing, downstream factor.
We proposed that this limiting factor might be the RNAi exonuclease postulated, as the RISC,
by Hammond et al, in Drosophila.36 One candidate would be the human mut-7 homologue,
Werner syndrome protein (WRN), as this has an 3'-5' exonuclease domain.8 Some effort, over
a dozen experiments, have been spent to try develop an in vitro assay in HaCaT cells to
investigate this cleavage fragment phenomena, so far with little success. However, when
mRNA transcription was turned off using Actinomycin D, it was found that natural turnover
of Tissue Factor was faster than RNA interference, as could have been expected by the halflife of Tissue Factor and the depletion speed of RNAi (Fig.5A - paper I). More interestingly,
the two effects are additive (Fig.5A - paper III). This can be seen to support the existence of
an independent RNAi exonuclease, which possibly can be saturated. Whereas if RNAi tagged
mRNA for degradation by non-specific exonuclease, as proposed by Hammond et at,8 such
saturation would not seem possible. A system, like the degradosome, which handles the
continual mRNA degradation of thousands of different genes, would not be saturated this
easily. Incidentally, the requirement for DEAD-box RNA helicase in the degradosome for 3'5' degradation,140 also suggests other roles for the RNAi RNA helicases than making initial
mRNA cleavage possible or unwinding siRNA in RISC.
2.12 Dose-response experiments
The excess capacity siRNA hypothesis has been supported by dose-response experiments,
demonstrating a saturation level of circa 5 nM in the cotransfection assay (Fig.4A, lower
curve - paper I), and circa 30 nM in the Northern assay, above which more siRNA will not
result in more silencing, and thus can be said to have excess silencing capacity. Saturation of
silencing above a certain threshold has been seen also by others.7, 48, 124, 134
However, our dose-response experiments have been criticized by an anonymous referee to
paper I. The referee's main objection was that at lower concentration, the complexation
between nucleic acids and the lipid transfection agents might be less efficient, but this
argument founders on the fact that all siRNA doses were complexed together in one volume,
at standard complexation concentration. A range of transfection concentrations was obtained
when this complexation solution was diluted into different volumes laid upon the cells.
42
Complexation creates small lipid micelles, containing siRNA, as can be visualized using
fluorescent tagged siRNA. These, presumably, will fuse with cells, when hitting the cell
membrane, creating a speckled appearance, with many speckles per cell (Fig.5B - paper I). It
is still possible that there might be threshold effects, but the main point here is that the doseresponse curves actually seem continuous, from full activity to no activity (Fig.4A - paper I).
Lowering the transfection concentration will probably only led to fewer hits per cell, and not
any abrupt threshold effects, although this experiment has not yet been done.
2.13 Propagation of silencing not detected in human cells
Silencing induced by siRNA is slow to reach a maximum (Fig.5A - paper I) and fades out 3-4
days post-transfection (Fig.5C - paper I). Similar results have recently been obtained by
Phillip Sharp's group.134 Other groups have measured later recovery of expression. 54, 88 The
relatively rapid recovery of expression is perhaps due to the high transcription rate and rapid
turnover of Tissue Factor mRNA, which has a half-life of only 0.5-1.5 hours.101 Support of
this notion can be found in the fact that challenge with greater amounts of TF-Luc reporter
plasmids, using the serial transfection assay, showed less silencing when using more plasmid
(data not shown - paper I), as already noted in section 2.5. In addition, the HaCaT cells
typically expanded 10-fold during the 6 day period of these experiments, leading to lowering
of siRNA concentration in the cells. The significance of this loss of silencing is that it
constitutes an argument against a system of propagation (as in the Lipardi-mechanism or
transitive RNAi) in human cells, as noted in section 1.4.
There had been a report that RNAi silencing could be mediated by the growth medium.133
Furthermore, RNAi silencing does cross cellular boundaries in C. elegans.5 Therefore we tried
incubating cells in the presence of siRNA. No unaided uptake was found, however, as the
cells showed no silencing when transfected with reporter plasmid the following day. Recently,
functional, unaided uptake has been observed with rat cholangiocytes.119 Furthermore, when
co-culturing cells transfected with siRNA alongside cells transfected with reporter plasmids,
no flow of silencing from the siRNA transfected cells were detected (data not shown).
Another apparent ambiguity in siRNA uptake is the already noted inability of siRNA to
silence the last 10 % of the mRNA in mammalian cells. The most plausible reason is that
Northerns include the cells that resist transfection. This is supported by the work of Tuschl’s
43
group when it was observed that cells are being silenced in a binary manner, single cells
appearing totally unsilenced in a field of silenced cells.7 Experiments with a fluorescent
siRNA, hTF167-FITC, showed a speckled distribution on all cells examined. The fluorescent
signal was detectable from 2 h post-transfection until and beyond 22 h post-transfection
(Fig.5B - paper I). This probably reflects small lipid micelles fused to the cells, as noted in
section 2.12.
That all cells showed speckles, could mean that the cells immune to silencing have micelles
fused to them, but that the siRNA are not released from the lipid micelles for some reason,
perhaps due to lack of lipid rearrangment dependent upon cell cycle. Also, slow or absent
release could partly explain the slow build-up of silencing (Fig.5A - paper I). Some support
for this view can be found in experiments done by Gitlin et al where electroporation of the siL
siRNA into HeLa cells, led to almost full silencing of both luciferase target already after 2-3
hours.77 The mRNA target was co-electroporated into the cells, although, and might lack the
protein coverage, or at least the same kind of protein coverage resulting from RNA
polymerase II transcription and processing by the splicing machinery. However, McManus et
al also used electroporation in E10 cells and observed maximum silencing at 36 h.134
A series of experiments testing a non-liposome transfection agents, calcium phosphate
precipitation, to see whether this would affect speed of silencing, showed in prelimenary
experiments strong silencing in cotransfection experiments (data not shown, unpublished).
However, calcium levels needed for efficient transfection were somewhat toxic to HeLa cells.
Furthermore, HaCaT cells, which express our endogenous target TF mRNA, is not compatible
with high calcium levels, as this will disturb the TF expression.101
We have also performed a series of pilot experiments on primary monocytes, which can be
stimulated to express hTF,101 but these have proved much harder to transfect than cell lines
(unpublished, data not shown). However, there have been many reports of natural uptake of
nucleic acid oligos in vivo,132 in addition to reported results with siRNA in whole mice,51,102
thus we remain optimistic that using siRNA against hTF, and the corresponding mTF in
whole mice, should still be possible.
2.14 PSKH1 siRNA had not much activity against endogenous mRNA
One siRNA in the first series of four siRNA against PSKH1 had good activity (60-80 %)
44
against PSKH1-Luc in our cotransfection assay (data not shown, unpublished). Several series
of new siRNA against PSKH1, in total 12, all ended up showing no substantial activity
against endogenously expressed PSKH1 in HeLa cells. Interestingly, PSKH1 mRNA is
expressed at a low level, even in HeLa cells, possibly indicating that RNAi might have a
lower threshold for mRNA. Alternatively, PSKH1 mRNA might be particularly packed with
RNA binding proteins. A third possibility is that the high GC percentage of PSKH1 is
influencing the silencing. However, testing this hypothesis of low GC content being more
accessible, by synthesizing a series of six siRNA against low GC sites in PSKH1 had no more
success (data not shown, unpublished).
2.15 Antisense siRNA compared with double-stranded siRNA
Our best siRNA candidate, hTF167i, has a corresponding antisense siRNA (as-167) that has
strong silencing activity at a standard transfection concentration of 200 nM (RNA
concentration) (Fig.5 - paper II). This is interesting in the context of a series of papers
reporting no activity for antisense siRNA, including Caplen et al, Gitlin et al, Lee et al,
Castanotto et al, Novina et al, Hu et al, Chiu et al and Elbashir et al.75, 77, 86, 89, 91, 93, 114, 124
We found that as-167 has a 5-6 fold higher IC50 than its partner hTF167i, and is already
approaching a saturation of silencing at 100 nM (Fig.1A - paper III), in a manner similar to
saturation effects seen previously (Fig.4A - paper I). The 5' phosphorylation status, crucial for
activity in an earlier report,45 did not seem to matter (Fig.5 - paper II). Furthermore, we find
that the pattern of position effects are very similar for antisense siRNA targeting same sites as
double-stranded siRNA (Fig.1B,C - paper III).
Thus, siRNA with intermediate activity, being used as antisense siRNA, could possibly lose
all its activity, yielding the results seen in the above cited publications. 75, 77, 86, 89, 91, 93, 114, 124
Excess capacity siRNA, on the other hand, would have spare activity to tolerate the 5-6 fold
lower efficiency of antisense siRNA, in a manner seen by our best candidate pair hTF167i /
as-167, thus being consistent with our main thesis of natural siRNA variation, as posed in
section 2.6.
However, the prevailing results regarding the relative efficiencies of siRNA and RNAi
antisense are quite contradictory in the literature. Martinez et al states that RISC activity
could not be induced by RNAi antisense in Drosophila lysates,45 while Schwarz et al found
45
that it could.48 Furthermore, Martinez et al claim as good antisense effects against the protein
A/C lamin in vivo in HeLa cells, as with double-stranded siRNA.45 On the other hand,
Schwarz et al report that they need eight-fold more antisense to approach the potency of
siRNA.48
A long series of RISC-complexes and RISC-like complexes have been described. 28, 44, 45, 66,
125-127
The hypothesis of a shared RNAi pathway, despite all the cited possible effector
complexes, we reasoned, would be falsified if major differences in antisense and doublestranded siRNA were found. We therefore compared a series of antisense siRNA and doublestranded siRNA, and found a close similarity with regards to position effects (Fig.1B,C paper III), appearance of mRNA cleavage fragments (Fig.1C - paper III) and tolerance for
mutational and chemical backbone modifications (Fig.2, Fig.3A - paper III). These
experiments therefore did not falsify the hypothesis of a shared RNAi pathway.
Furthermore, we found that in competition experiments, double-stranded siRNA could block
antisense siRNA (Fig.4 - paper III), something which support the notion of a shared RNAi
pathway. Finally, when comparing the speed with which silencing is obtained, we found that
antisense siRNA worked significantly faster than double-stranded siRNA (Fig.5C - paper III),
suggesting that antisense siRNA enters the RNAi pathway at a later stage, as schematically
illustrated in Figure 1.
2.16 Blocking 3' OH blocks activity in antisense siRNA
Constructing a fluorescent version of hTF167i for use in uptake studies (section 2.17),
hTF167-FITC, we found that even an siRNA tagged at the 3’ end of the antisense strand with
a bulky FITC group, somewhat surprisingly, had only marginally lower activity than the
wildtype (Fig. 6B, 6C). These data were challenged by Zamore's group, on the grounds that
we had not proved that all siRNA had been FITC-tagged.48 However, the synthesis of our
FITC-tagged oligo was done using nucleotides which already had the FITC group on the 3'
hydroxyl. Furthermore, our dose-response experiments show clearly that even if a small
fraction did escape FITC-tagging, the concentration of this untagged fraction would be too
small to account for the robust silencing both at 24 h and in the time-course studies, as seen in
the dose-response experiment (Fig.6E - paper I).
However, if the terminal nucleotide was completely removed, in vivo, and thus regenerated
46
the hydroxyl group, the silencing observed could still be due to a free 3' hydroxyl. Zamore
and coworkers controlled for this by showing that if the hydroxyl was regenerated by loss of
the 3' terminal nucleotide, the siRNA would have lost activity.48 Additional support of the 3'
modification tolerance, as noted earlier, comes from Tuschl's group, which used biotin-linkers
at the 3' end to isolate RISC complexes, without loss of activity, something that even worked
on the 5' end on the sense strand.45
Some closing notes on antisense siRNA must be added, as several experiments indicate that
the mechanisms of antisense siRNA and double-stranded siRNA are not entirely identical. We
found, as noted in the introduction, that double-stranded siRNA could very well have the 3'
end (of the antisense strand) blocked by a FITC-group (Fig.6B,C - paper I). Furthermore, in
time-course studies, hTF167-FITC had the same silencing decay found with hTF167i in serial
transfection assays at least up to 72 h (data not shown, unpublished data). However, when
testing the corresponding antisense siRNA (as-FITC), we found that it had severely limited
activity (Fig.3C - paper III). Reasoning that this could be due to inefficient incorporation into
RISC*, because of the bulky fluorescent group, we synthesized a version having a hydrogen
group (3'deoxy, termed as-3d) replacing the 3' hydroxyl on the 3' terminal nucleotide. This too
had practically full activity as a double-stranded siRNA (hTF167-3d), but low activity as an
antisense siRNA (Fig.3C - paper III).
In apparent contrast with this, Schwarz et al found that in HeLa lysates there was no
difference between the cleavage activity of 3' hydroxyl and 3' blocked antisense siRNA.48 The
cleavage rate was much weaker than with duplex siRNA, however, but this was accredited to
degradation, and they concluded that the 3' hydroxyl had no role in RNAi in flies or humans.
Interestingly, they tested 5' blockage of antisense siRNA, but not their 3' blocked antisense
siRNA, in vivo in HeLa cells, thus there is no direct inconsistency between our results.
Degradation should not be a direct factor, unless a 3' deoxy and 3' FITC are, for some strange
reason, particularly vulnerable compared to wild-type as-167.
Other results in the literature also indicate a role for the 3' hydroxyl, as Tijsterman et al report
that a 3' hydroxyl on antisense siRNA are needed in C. elegans.52 Taira's group report even of
inactivated double-stranded siRNA due to 3' modifications.112 So there still might be a role for
the 3' hydroxyl. Furthermore, the published results on the 5' end modifications were not
completely satisfactory. Schwarz et al's 5' amino-modifier was less destructive of silencing
47
than the 5' methoxy, although these results were obtained in cotransfection assays that might
be vulnerable to non-specific effects.48 These paradoxes will possibly yield to further
research. But further enquiry is necessary.
48
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