Chapter 16 Other RNA
Processing Events
Trans-splicing, Editing, RNAi, miRNAs
Trans-splicing
section 16.3
First seen in a parasitic protozoa
Trypanosomes, protozoan that causes
African sleeping sickness
trans-splicing used to generate changing
surface coat proteins that help outwit the
immune system
trans-splicing
Figure 16.12
200 copies of a 35 n leader encodes in a different
place in the genome.
Editing
Focus on this one
protozoa = U-insertion
protozoa = U-deletion
mammals, insects & plants = nucleotide deaminiation
16.4
RNA editing by deamination
ADAR = Adenosine deaminase acting on
RNA
adenosine -> inosine
inosine bp with cytidine
So codons change
ACG codon (threonine) changes to an
ICG codon which is read as GCG
(alanine)
pg 493 4th ed.
Results in major changes in properties of the
protein
Example
Glutamate receptor ion channel
GluR-B changes glutamine->arginine
Reduces Ca2+-permeability.
How?
Usually codons to be changed are near
introns. A guide RNA molecule base
pairs to an intron and then points ADAR
at the correct codon.
So what?
Not a trivial change.
It is extremely important for the normal
development and function of the nervous
system.
In mammals, it appears to be part of the
way that the nervous system generates
diversity and complexity (ADAR 3
unique to brain).
Cytidine deaminaton
CDAR cytidine deaminase acting on
RNA
C-->U
Discovery of post-transcriptinal
gene silencing (PTGS) or posttranscriptional control of gene
expression
• Involved attempts to manipulate pigment synthesis genes in petunia
• Genes were enzymes of the flavonoid/anthocyanin pathway:
CHS: chalcone synthase
DFR: dihydroflavonol reductase
When these genes were introduced into petunia using a strong
viral promoter, mRNA levels dropped and so did pigment levels in
many transgenics.
Discovery of PTGS
First observed in plants
(R. Jorgensen, 1990)
Introduction of a transgene homologous
to an endogenous gene resulted in
both genes being suppressed!
Also called Co-suppression
involved enhanced degradation of the
endogenous and transgene mRNAs
DFR construct introduced into petunia
CaMV - 35S promoter from Cauliflower
Mosaic Virus
DFR cDNA – cDNA copy of the DFR
mRNA (intronless DFR gene)
T Nos - 3’ processing signal from the
Nopaline synthase gene
Flowers from 3 different transgenic petunia plants carrying copies of the
chimeric DFR gene above. The flowers had low DFR mRNA levels in the
non-pigmented areas, but gene was still being transcribed.
RNAi
RNA interferance
Discovered in a control experiment
pg 501 Weaver 4th edition
RNAi
RNAi discovered in C. elegans
(first animal) while attempting to
use antisense RNA in vivo
Control “sense” RNAs also
produced suppression of target
gene!
sense (and antisense) RNAs were
contaminated with dsRNA.
dsRNA was the suppressing
agent.
Craig Mello
Andrew Fire
2006 Nobel Prize in Physiology & Medicine
2. The experiment.
unc22 gene
nonessential
myofilament
protein.
Mutations in unc22 cause a
twitching
phenotype.
dbstded unc-22
RNA phenocopies.
Double-stranded RNA (dsRNA) induced interference
of the Mex-3 mRNA in the nematode C. elegans.
Inject antisense RNA (c)
or dsRNA (d) for the
mex-3 (mRNA) into C.
elegans ovaries.
mex-3 mRNA was
detected in embryos by
in situ hybridization with
a mex-3 probe.
Fig. 16.29
Weaver 4th Ed.
negative control
positive control
no probe
mex-3 antisense
mex-3 dsRNA
Conclusions: (1) dsRNA reduced mex-3 mRNA better than antisense
mRNA. (2) the suppressing signal moved from cell to cell.
Hammond et al. 2000. Nature 404:293-296.
An RNA-directed nuclease is purified from Drosophila cells
that seems to specifically degrade mRNAs.
S2 cells
extract destroys
cognate RNAs
T7
dsRNA
T7
As others have seen,
notice the
accumulation of a 25
nt RNA which can bp
to the target mRNA.
Destruction of 25 nt RNA with micrococcal nuclease blocks reaction.
Hammond et al. 2000. An RNA-directed nuclease mediates post-trancriptional gene silencing in Drosophila cells. Nature 404:293-296
Figure is not in Weaver 4th but is mentioned on pg 501-502.
Short interfering RNAs -siRNAs
Drosophila embryo lysate system simplifies step by step analysis.
Processes the trigger to the 21-23nt fragments.
Both strands of the trigger are cut. - show by radiolabelling one strand and then the other strand (sense,
antisense).
Processing of trigger is not dependent on mRNA.
dsRNA
Zamore et al. 2000. Cell 101:25-33
p
p
p
Fig 16.30 4th ed
p
The dsRNA that is added dictates
where the destabilized mRNA is
cleaved.
The dsRNAs A, B, or C were added to
the Drosophila extract together with
a Rr-luc mRNA that is 32P-labeled at the
5’ end. The RNA was then analyzed on a
polyacrylamide gel and
autoradiographed.
Results: the products of Rr-luc mRNA
degradation triggered by dsRNA B are
~100nt longer than those triggered by
dsRNA C (and ~100 nt longer again for
dsRNA A-induced degradation).
Fig 16.31
High resolution gel analysis of the
products of Rr-luc mRNA
degradation from the previous
slide.
Results: the cleavages occur mainly
at 21-23 nt intervals; 14 of 16
cleavage sites were at a U.There is
an exceptional cleavage only 9 nt
away from the adjacent site (induced
by dsRNA C); this site had a stretch
of 7 Us.
Enzyme cleaves at ~23-nt intervals &
after U.
In 2001 Hammond et al purify the
enzyme and name it DICER.
Fig. 16.32
dsRNA
Weaver 4th edition pg 501-507
DICER - RNase III family member
ATP
ADP+Pi
p
p
p
Dicer leaves 2nt 3’
overhangs &
phosphorylated 5’ ends
p
Argonaute has a PAZ and a PIWI domain.
p
21-23 nt siRNP
-R2D2
p
-Armitrage
The 2 domains
of Argonaute
RISC loading
complex
p
ATP
ADP+Pi
p
Argonaute
PIWI PAZ
p
-Dicer
RISC=RNAinduced
silencing
complex.
RISC - one of the proteins is SLICER. In
Drosophila SLICER is the product of the
Argonaute gene.
Dicer
mRNA
In mice there are 4 Ago genes but only Ago2
appears to be SLICER.
RISC
Target recognition
p
p
Target
cleavage
mRNA
p
PIWI domain forms a shape like an RNase H.
p
Dicer participates in selecting the guide RNA
that is passed on to Argonaute.
Roles of R2D2 and Armitrage are not clear.
Argo2 is Sliceris shown by building highly specfic siRNA complexes in vitro using bacterially expressed Argo2.
Bizarre
figure see
next one for
explanation.
Argo2 is Sliceris shown by building highly specfic siRNA complexes in vitro using bacterially expressed Argo2.
RNA transcript
made
siRNA1 could bp about 140n from 5’ end of transcript
siRNA2 could bp about 180n 3’ end of transcript
Argo2 that has been produced in bacteria
lane1
lane 1 transcript + siRNA2 + Argonaute + MgCl2
lane2
lane 2 transcript + siRNA1 + Argonaute + MgCl2
Argo2 is Sliceris shown by building highly specfic siRNA complexes in vitro using bacterially expressed Argo2.
Ago2 knock out in mice
embryonic lethal with severe defects
important for RNAi & miRNA
Function of RNAi
Antiviral - Double stranded RNA is an
intermediate in the replication of some
RNAi viruses.
Suppress transposon activity
Great research tool because it provides a
way to experimentally eliminate a gene
product
Might be a useful therapy for cancer, etc.
How to evoke RNAi
• Inject double stranded RNA
• Express or inject antisense RNA inside a cell
• Express a gene which has an inverted repeat.
• Two promoters which point at one other.
• Expression of 2 different genes whose
mRNAs can base-pair over a short region.
But wait there’s (too much )more
Amplification of siRNA
Role of RNAi machinery in the
formation of heterochromatin
miRNAs - inhibition of translation
miRNAs - stimulation of translation
But wait there’s (too much )more
Amplification of siRNA
Tiny amounts of a trigger can have
a very large and long lasting effect.
Occurs in Plants, Drosophila and
C. elegans.
Role of RNAi machinery in the
formation of heterochromatin
miRNAs - inhibition of translation
miRNAs - stimulation of translation
dsRNA
Amplification (pg508 4th ed)
mRNA
ATP
Dicer
ADP+Pi
p
p
p
Dicer leaves 2nt 3’
overhangs &
phosphorylated 5’ ends
p
p
p
-Armitrage
ATP
ADP+Pi
mRNA
RISC
Target recognition
p
p
Target
cleavage
mRNA
p
Dicer
p
RISC loading
complex
p
p
Argonaute
ATP
ADP+Pi
21-23 nt siRNP
-R2D2
RdRp
p
(RNA directed RNA polymerase)
p
p
-Dicer
RISC=RNAinduced
silencing
complex.
NTPs
PPi
p
p
p
p
p
p
Potential for exon spreading
Reference: Nishikura 2001 Cell 107:415-418.
But wait there’s (too much )more
Amplification of siRNA
Role of RNAi machinery in the
formation of heterochromatin
miRNAs - degradation of mRNA or
inhibition of translation
miRNAs - stimulation of translation
Role of RNAi machinery in the
formation of heterochromatin
Heterochromatin - condensed chromatin, silenced chromatin
Centromeres - include much heterochromatin
Centromeres - One does not observe transcription from material
adjacent to the centromeres.
In yeast, mutations in Dicer, Argonaute and RdRp cause such
transcripts to appear.
meH3lys4 - associated with active genes
meH3lys9 - associated with inactive genes.
Normally centromeres would have low meH3lys4 and high meH3lys9.
Mutants have the opposite.
RdRP found associated with centromere (but called RDRC there).
Swi6 is required to form heterochromatin. It is attracted to meH3lys9
outer edge of a centromere
Histone methyl
transferase bound
by RITS.
Supposed to
indicate that the
RDRC copies
(amplifies) the
siRNA
RITS - RNA-induced initiator of
transcriptional gene silencing
contains Ago1 + siRNA
RDRC - RNA-directed RNA polymerase
complex contains RdRp
But wait there’s (too much )more
Amplification of siRNA
Role of RNAi machinery in the
formation of heterochromatin
miRNAs - degradation of mRNA or
inhibition of translation
miRNAs - stimulation of translation
Comparison of Mechanisms of MiRNA Biogenesis and Action
Better complementarity of MiRNAs and targets in plants.
40
Fig. 16.45
Source of miRNA’s
Why RNA silencing?
• Original view is that RNAi evolved to protect the
genome from viruses, and perhaps transposons or
mobile DNAs.
• Some viruses have proteins that suppress
silencing:
45
References
• Baulcombe, D. (2004) RNA silencing in
plants. Nature 431: 356-363.
• Millar, A.A. and P.M. Waterhouse (2005)
Plant and animal microRNAs: similarities
and differences. Functional & Integrative
Genomics 5: 129-135.