39th Meeting of the
Polish Biochemical Society
Gdañsk 16–20 September 2003
SESSION 10
Structure and function of RNA
Organized by Z. Szweykowska-Kuliñska
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Session 10. Structure and function of RNA
2003
407
Lecture
Diseases caused by mitochondrial RNA mutations
Ewa Bartnik
Zak³ad Genetyki, Uniwersytet Warszawski, ul. Pawiñskiego 5A, Warszawa
The human mitochondrial genome is a small circular
DNA molecule encoding the synthesis of 13 proteins,
22 tRNAs and 2 rRNAs. Perturbations in mitochondrial function due to mutations in genes encoded in
both mitochondrial and nuclear DNA can lead to a number of diseases, including blindness, deafness,
neuromuscular disorders and diabetes. Changes in mi-
tochondrial function have been associated with aging,
and somatic mutations in mitochondrial DNA can occur during carcinogenesis. Numerous mutations have
been found in genes encoding tRNA and rRNA. I will
present data on these mutations, including our own results on analyzing mutations involved in mitochondrial
myopaties and mutations found in tumours.
408
Lecture
Ribozymes of the hepatitis delta virus: new findings on catalytic properties of ribonucleic acids
Jerzy Ciesio³ka
Instytut Chemii Bioorganicznej, Polska Akademia Nauk, ul. Noskowskiego 12/14, 61-704 Poznañ
Although the delta ribozymes have been studied for
fifteen years the most important information concerning their structure and mechanism of catalysis were obtained very recently. The determination of the crystal
structure of the genomic ribozyme is, undoubtedly, one
of the most important achievements of ‘ribozymology’
in recent years. Details of the X-ray structure have
greatly stimulated further studies on the folding of
these ribozymes into functionally active molecules as
well as on the mechanism of RNA catalysis. The ability
of the delta ribozymes to carry out general acid-base catalysis by nucleotide side chains has been assumed in
two proposed mechanisms of self-cleavage. Subsequently, a similar strategy has been suggested for the
ribosome and peptide bond formation. General
acid-base catalysis by nucleotide side chains increases
the catalytic repertoire of RNA and suggests that similar strategies could operate in other RNA-catalyzed reactions.
Recently, we have used the in vitro selection approach
to select for antigenomic ribozyme variants that are active in the presence of Mg(II). The following ribozyme regions were randomized: L3, J1/4 and J4/2, and after 10
cycles of selection-amplification, cloning and sequencing
of the enriched ribozyme pool, several functional
ribozymes were identified. Characterization of their catalytic activity, also in the presence of other than Mg(II)
divalent metal ions, revealed that some nucleotide substitutions changed ionic preferences of selected variants. Interestingly, in some variants the cytosine residue
proposed to play a crucial role in the cleavage mechanism of the wild type ribozyme was mutated.
409
Lecture
New mRNA 5’-cap analogues as useful tools in translation studies in eukaryota
1
1
1
1
1
Edward Dar¿ynkiewicz , Jacek Jemielity , Janusz Stêpiñski , Joanna ¯uberek , Anna NiedŸwiecka , Magdalena
1
1
1
2
Lewdorowicz , Dorota Haber , Ryszard Stolarski , Robert Rhoads
1 — Zak³ad Biofizyki IFD, Uniwersytet Warszawski, ul. ¯wirki i Wigury 93, 02-089 Warszawa, 2 — Department of Biochemistry
and Molecular Biology, Louisiana State University Health Sciences Center, Shreveport, USA
Synthetic analogues of 5’-terminal cap of eukaryotic
mRNA and snRNA have played an important role in elucidating such physiological processes as mRNA translation, pre-mRNA splicing, intracellular transport of
mRNA and snRNAs, and mRNA turnover. Particularly
useful are RNAs capped with synthetic analogues, which
are produced by in vitro transcription of a DNA template
using a bacteriophage RNA polymerase in the presence
of ribonucleoside triphosphates and a cap dinucleotide
such as m7Gp3G. Unfortunately, due to the presence of
2003
39th Meeting of the Polish Biochemical Society
a 3’-OH on both the 7-methylguanosine and guanosine
moieties, up to half of the mRNAs contain caps incorporated in the reverse orientation [Pasquinelli et al. (1995)
RNA’ 1: 957–967]. Previously we designed and synthesized two “anti-reverse” cap analogues (ARCAs),
m73’dGp3G and m27,3’OGp3G, that cannot be incorporated in the reverse orientation because of lack of free
3’-OH in 7-methylguanosine moiety [Stepinski et al.
(2001) RNA, 7: 1486–1495]. In the present study we
have synthesized several new cap analogues modified in
the C2’ and C3’ positions of 7-methylguanosine and in
the number of phosphate residues, m27,2’OGp3G, m72’dGp3G, m72’dGp4G, m27,2’OGp4G, m27,3’OGp4G,
235
m7Gp5G, and m27,3’OGp5G. These were analysed for
conformation in solution, binding affinity to eIF4E, inhibition of in vitro translation, and the ability to stimulate
cap-dependent translation in vitro when incorporated
into mRNA. The results indicate that modifications at
C2’, like those at C3’, prevent reverse incorporation, that
tetra- and pentaphosphate cap analogues bind eIF4E
and inhibit translation more strongly than their
triphosphate counterparts, and that tetraphosphate (but
not pentaphosphate) ARCAs promote cap-dependent
translation more effectively than any cap analogue
tested to date.
Supported by the State Committee for Scientific Research:
PBZ-KBN-059/T09/10.
410
Lecture
Splicing of U12-dependent introns in plants
Artur Jarmo³owski
Department of Gene Expression, Adam Mickiewicz University, ul. Miedzychodzka 5, 60-371 Poznañ
While several animal U12-dependent introns have
been analyzed thoroughly, no significant study of U12
splicing in plants has been made. We scanned the
Arabidopsis genome for U12 introns. Our computational analysis revealed that only 0.06% of all Arabidopsis introns have features characteristic of U12 intervening sequences. The U12 introns were distributed
among 69 genes, all contain one U12-dependent intron
except for two genes that have two U12 introns. There
was no clear preference for the position of U12-type intervening sequences within the genes analyzed.
Three U12-dependent introns were subjected to detailed splicing analysis in planta and in tobacco protoplasts. The introns were derived from three different
Arabidopsis genes, which encode nuclear cap-binding
protein (AtCBP20), glutathione synthetase (Atgsh2)
and LUMINIDEPENDENS (AtLD). The introns studied were spliced efficiently in their normal context
within plant tissues. However, in tobacco protoplasts,
excision from zein pre-mRNA was generally poor.
There were notable differences in efficiency of the U12
introns splicing. While AtCBP20 and Atgsh2 U12 type
introns were spliced only at 10% and 5% respectively,
AtLD intron was spliced with 50% efficiency. Deletion
of natural exonic fragments from the LD construct decreased splicing of the U12 intron to 9%, suggesting the
presence of a putative exonic enhancer within the
exons. We identified by deletion analysis a 40-nucleotide fragment in the upstream exon that stimulates
splicing of the U12 intron more than 5 times. Splicing
of U12 dependent introns in protoplasts was not enhanced by over expression of known plant SR proteins
and U-rich RNA binding protein UBP1. Our experiments suggest that RBP45, a member of the plant
hnRNP-like protein family, can be involved in regulation of U12-dependent splicing in plants.
411
Lecture
Pathways for nuclear mRNA degradation in yeast
1
2
2
2
Joanna Kufel , Laura Milligan , Claire Torchet , Cecile Bousquet-Antonelli , David Tollervey
2
1 — Zak³ad Genetyki, Uniwersytet Warszawski, ul. Pawinskiego 5a, 02-106 Warszawa, 2 — ICMB, University of Edinburgh, EH9
3JR Edinburgh, United Kingdom
Control of mRNA stability is a key step in the regulation of gene expression. During the degradation of cytoplasmic mRNA, deadenylation-dependent removal
of the 5’-cap by the decapping complex Dcp1p/Dcp2p
is activated by the cytoplasmic Lsm1-7p complex.
Pathways that degrade mRNA precursors in the
yeast nucleus have also been identified and different
pathways appear to utilize the same RNA degradation
machinery. Unspliced pre-mRNAs are rapidly degraded predominantly 3’-5’ by the nuclear exosome,
whereas 5’-3’ degradation by the exonuclease Rat1p
plays a minor role. The nuclear exosome also functions in the degradation of pre-mRNAs that have
failed to undergo correct 3’-end formation. However,
236
Session 10. Structure and function of RNA
in this pathway there is no evidence for significant
5’-3’ degradation.
5’-3’ degradation of nuclear pre-mRNAs also requires
their decapping prior to exonuclease digestion. Cells
lacking nuclear Lsm2-8 proteins or the decapping factor Dcp1p stabilize pre-mRNA degradation intermediates that would normally be subject to 5’-3’ degradation. These intermediates remain 5’-capped, indicating
that the nuclear Lsm2-8p complex normally targets
RNA substrates for decapping.
Mature HS, GAL and MET mRNAs that are trapped
in the nucleus due to a block in mRNA export are also
stabilized and remain 5’-capped in lsm or dcp1 mutants. These nuclear-restricted mRNAs remain poly-
2003
adenylated until their degradation, indicating that nuclear mRNA degradation does not involve incremental
deadenylation, which is a key feature of cytoplasmic
turnover. Nuclear Lsm8p can be UV cross-linked to nuclear-restricted poly(A)+ RNA. This indicates that the
Lsm2-8p complex interacts directly with mRNA substrates to activate decapping prior to 5’-3’ degradation.
Nuclear-restricted HS and MET mRNAs are also degraded 3’-5’ by Rrp6p, an exonuclease that is specifically associated with the nuclear form of the exosome
complex but not by the core exosome.
These data show that degradation of different nuclear
substrates has distinct features suggesting that they
are targeted by related but distinct turnover pathways.
412
Lecture
New types of RNA within the cell
Maciej Szymañski, Marek ¯ywicki, Miros³awa Barciszewska, Jan Barciszewski
Instytut Chemii Bioorganicznej, Polska Akademia Nauk, ul. Noskowskiego 12/14, 61-704 Poznañ
Recent analyses of the human genome revealed that
only about 1.5% is transcribed and translated into protein. Current data indicate that a considerable fraction
of genomic DNA transcripts is responsible for regulation of gene expression on both transcriptional and
posttranscriptional level. Interestingly both types of
mechanisms involve non-protein coding RNAs.
Noncoding RNAs (ncRNAs) are stable and functional
RNA molecules without coding capacity. They can be
broadly divided into two major group: housekeeping
ncRNAs and regulatory ncRNAs. The RNAs belonging
to the first group are necessary for primary cell functions. The second group can be divided into several subclasses depending on the mechanisms of action. Regulatory ncRNAs are able to affect expression of protein-coding genes on both transcriptional and posttranscriptional levels. These RNAs act by multiple
mechanisms such as RNA-RNA base pairing,
RNA-protein interactions and intrinsic RNA activity
and regulate diverse cellular functions, including chro-
matin structure modification, RNA processing, mRNA
stability and translation. Their involvement in some
key genetic processes, like genomic imprinting or X
chromosome inactivation in higher eukaryotes reveals
their importance in cell metabolism. Noncoding RNAs
have been also implicated in development and their defects can lead to various genetic disorders such as
Prader-Willi syndrome, Beckwith-Wiedemann syndrome or cancerogenesis.
The fact, that over 98% of human genome does not encode proteins stimulates efforts for ncRNAs gene finding. In contrast to protein coding genes, extremely variable structural properties of this type of molecules in addition to lack of open reading frames makes them very
hard targets for computational based genome search.
Till now there is lack of efficient method of ncRNA genes
prediction. The important role of noncoding transcripts
is however evident from the analysis of the mouse
transcriptome, where approximately one third of total
cDNA clones does not show protein-coding potential.
413
Oral Presentation
Analysis of putative human RNA methyltransferase that introduces methyl group on
the first anticodon position in pre-tRNA-Leu(CAA).
1
2
3
Bartosz Brzezicha , Marcin Schmidt , Izabela Maka³owska , Zofia Szweykowska-Kuliñska
1
1 — Zak³ad Ekspresji Genów, Instytut Biologii Molekularnej i Biotechnologii, Uniwersytet im. Adama Mickiewicza,
ul. Miêdzychodzka 5, 60-371 Poznañ, 2 — Katedra Biotechnologii i Mikrobiologii ¯ywnoœci, Akademia Rolnicza im. Augusta
Cieszkowskiego, ul. Wojska Polskiego 48, 60-627 Poznañ, 3 — Computational Genomics Center, The Huck Institute for Life
Sciences, Pennsylvania State University, 506 Wartik Lab, University Park, 16802 Pennsylvania, USA
Yeast m5C34-tRNA-Leu-methyltransferase Trm4 methylates first anticodon position in pre-tRNA-Leu(CAA) in
intron-dependent manner. It is known that the absence of the
enzyme is not lethal for the cell but causes disturbances in
mRNA decoding. Human genes coding for tRNA-Leu(CAA)
contain 22–25 nt long introns. We have shown, that in the nuclear HeLa cell extract human pre-tRNA-Leu(CAA) is, as in
the case of yeast, methylated in the intron-dependent manner.
2003
39th Meeting of the Polish Biochemical Society
Full sequence of the methyltransferase gene in yeast
has been established. Searching for orthologous sequences in human GenBank databases revealed the
presence of ESTs with high orthology to yeast enzyme
sequence. We aligned two putative mRNAs that differ
from each other by the presence of longer N-terminus
in the protein after translation. Both have the same locus on fifth human chromosome: 5p15.32.
Both cDNAs have been amplified using Human
HeLa Marathon-Ready cDNA (Clontech) as a template. After sequencing we observed point mutations
that were probably incorporated by Taq Polymerase
during PCR reaction. In longer cDNA some mutations
was responsible for changing encoded amino-acids.
Mutations have been reversed by site-directed mutagenesis. Afterward, both cDNAs have been cloned into
prokaryotic expression vector pET-28a(+) and the
237
shorter one has been also cloned into yeast expression
vector p416 GALS. Overexpression of shorter protein
in E. coli is efficient, contrary to the longer one. Enzymatic tests of shorter protein expressed in E. coli resulted in the lack of the methyltransferase activity.
Expected activity has not been also detected in yeast
extract after transformation of yeast line in which the
endogenous methyltransferase gene has been deleted.
Analogous experiments will be performed for testing
activity of longer protein.
We have established localization of both proteins in
HeLa cells. The proteins have been expressed as a fusion to GFP. We observed localization using confocal
microscopy. Both proteins localize in nucleus with
clearly visible nucleolar localization. This observation
is in accordance to previous data from which we know
that posttranscriptional modifications of tRNA and
rRNA take place mainly in the nucleolus.
414
Oral Presentation
Analysis of the posttranscriptional regulation of DNA polymerase beta expression in
the rat cells
Ewa Grzybowska, Anna Wilczyñska, El¿bieta Sarnowska, Janusz Siedlecki
Zak³ad Biologii Molekularnej, Centrum Onkologii — Instytut, ul. Roentgena 5, 02-781 Warszawa
DNA polymerase beta plays a key role in base excision
repair (BER). Its overexpression leads to a mutator
phenotype, characteristic in certain cancers and its
knockout causes apoptotic death of neurons during embryonic development in mice. Two DNA polymerase
beta transcripts are expressed in rat cells and they differ (due to alternative polyadenylation) only in the
length of their 3’ untranslated regions (3’UTRs). The
transcript containing the shorter 3’UTR is expressed in
most tissues, with significantly higher levels in testis,
while the one with the longer 3’UTR is expressed
mostly in brain and lungs. Both 3’UTRs contain specific motifs — putative protein binding sites — that
could be responsible for DNA polymerase beta posttranscriptional regulation and tissue-specific expression of the two transcripts.
Putative regulatory sequences of the DNA polymerase beta have been isolated and cloned in order to ana-
lyze their ability to bind specific proteins in the yeast
three-hybrid system and EMSA. EMSA performed with
protein fractions from rat cells, confirmed specific protein binding to the chosen mRNA regions. Three-hybrid
analysis of one of these regions has revealed the interaction with HAX-1 protein, a potent anti-apoptotic factor.
The effect of both 3’UTR sequences on transcript stability and translatability has also been analyzed using
both, luciferase and beta-globin reporter systems. We
have established that the presence of short 3’UTR
causes an increase in luciferase expression, in contrast
to the long 3’UTR, which exerts opposite effect. These
results show that 3’UTRs play an important role in regulation of DNA polymerase beta expression and further
identification of specifically binding proteins will help
to elucidate the mechanisms behind it.
415
Oral Presentation
Efficiency of RNA polyadenylation by poly(A) polymerase in Escherichia coli depends
on bacterial growth rate
Jacek Jasiecki, Grzegorz Wêgrzyn
Katedra Biologii Molekularnej, Uniwersytet Gdañski, ul. K³adki 24, 80-822 Gdañsk
RNA polyadenylation occurs not only in eukaryotes
but also in bacteria. In prokaryotes, polyadenylated
RNA molecules are usually degraded more efficiently
than non-modified transcripts. Here, we demonstrate
that two transcripts, which were shown previously to be
substrates for poly(A) polymerase I (PAP I), Escherichia
coli lpp mRNA and bacteriophage lambda oop RNA, are
polyadenylated more efficiently in slowly growing cells
than in rapidly growing bacteria. Intracellular levels of
PAP I varied in inverse proportion to bacterial growth
rate. Moreover, transcription from a promoter for the
pcnB gene (encoding for PAP I) was shown to be more efficient under conditions of low bacterial growth rates.
We conclude that efficiency of RNA polyadenylation in
E. coli is higher in slowly growing bacteria due to more
efficient expression of the pcnB gene. This may allow
regulation of stability of certain transcripts (those subjected to PAP I-dependent polyadenylation) in response
to various growth conditions.
416
Poster
Synthesis of nucleosides tri- and tetraphosphates resistant to hydrolysis
Jacek Jemielity, Marcin Ka³ek, Janusz Stêpiñski, Edward Dar¿ynkiewicz
Zak³ad Biofizyki IFD, Uniwersytet Warszawski, ul. ¯wirki i Wigury 93, 02-089 Warszawa
The series of nucleosides tri- and tetraphosphates
containing methylene group instead of terminal
pyrophosphoric bond has been synthesized. Such modified compounds are resistant to either chemical and enzymatic hydrolysis [1, 2]. These derivatives may be applied in studies of decapping process and other biochemical researches. The method used to insert the
methylene bridge to the phosphate chain consists in
coupling an imidazole derivative NpnIm with
bisphosphonate[3]. The elaborated synthetic method
enables connecting the substrates with high yields and
does not require a usage of any protective groups, what
makes it very useful in synthesis of unstable compounds like for instance modified derivatives of
7-methylguanosisne. Preparation of analogue contain-
ing two CH2 groups is far more complicated. Currently,
we are carrying out a synthesis of GpCH2pCH2pG.
Crucial step in route to this compound is a condensation of protected guanosine with bis(O-benzyl
phosphonomethyl)phosphonic acid benzyl ester, via
Mitsunobu reaction[4].
References:
1. Thevenod F, Anderie I, Schulz I (1994) J Biol Chem, 269:
24410.
2. Tobin T, Akera T, Lee CY, Brody TM (1974) Biochem
Biophys Acta, 345: 102.
3. Jemielity J et al, RNA — in press.
4. Klein E, Mons S, Valleix A, Mioskowski C, Lebeau L (2002)
J Org Chem, 67146.
Supported by the State Committee for Scientific Research:
PBZ-KBN-059/T09/10.
417
Poster
HIV RT-mediated site-specific nonhomologous RNA recombination
Anna Kurzynska-Kokorniak, Paulina Jackowiak, Marek Figlerowicz
Instytut Chemii Bioorganicznej, Polska Akademia Nauk, ul. Noskowskiego 12/14, 61-704 Poznañ
Our preliminary studies conducted in vivo on brome
mosaic virus (BMV) and in vitro on human immunodeficiency virus (HIV) showed that local hybridization between RNA molecules (local RNA-RNA duplex formation) can induce site-specific nonhomologous recombination if a double-stranded region is accompanied by
short homologous sequences. A specific localization of
the latter causes the local RNA-RNA duplex to exist in
two different conformations. We hypothesized that the
observed conformational dynamism of recombining
molecules is responsible for the precise transfer of the
viral polymerase from one template (called RNA donor)
to the other (called RNA acceptor).
To verify the above hypothesis we have created an in
vitro system enabling effective studies on the mechanism of nonhomologous site-specific RNA recombina-
tion. Using this system we have tested the recombination activity of HIV-1 reverse transcriptase (HIV-1 RT ?
p51/p66 heterodimer) and of the p66/p66 homodimer.
To establish the role of RNA structure in the studied
process, we have constructed several RNA donor and
RNA acceptor molecules sharing short homologous sequences of various length (3, 5 and 10 nucleotides). We
have also tried to determine the enzymes’ ability to
switch from the 5’ end of the donor template to the 3’
end of the acceptor template. Moreover, we have tested
HIV-1 RT and p66/p66 homodimer recombination activities in the system that offers optimal conditions for
site-specific template switching by reverse transcriptase.
Our results indicate that neither local hybridization
between template molecules nor the presence of short
2003
39th Meeting of the Polish Biochemical Society
homologous sequences, can alone induce site-specific
template switching by HIV-1 RT or p66/p66 homodimer. Only the simultaneous occurrence and the
239
proper localization of both motifs lead to recombinant
formation.
418
Poster
Appearance of novel PSTVd variants upon coinfection with two non-infectious
PSTVd clones
Wojciech Podstolski, Anna Góra-Sochacka, W³odzimierz Zagórski-Ostoja
Instytut Biochemii i Biofizyki, Polska Akademia Nauk, ul. Pawiñskiego 5A, 02-106 Warszawa
In the course of studies on the genetic stability of the
infectious PSTVd variants I2 and I4, among a diverse
population of progeny molecules, two non-infectious
clones, I2-50 and
I4-37, were found. The I2-50 and I4-37 variants are
considered non-infectious on the basis of lack of visible
symptoms on Lycopersicon esculentum ‘Rutgers’ test
plants and negative dot blot hybridization results.
The presence of such mutants among the population
of viable variants remains a puzzling observation. To
investigate the biological properties and possible interactions between these molecules, two types of experiments were carried out.
Double inoculation experiments were performed in
which monomeric full-length cDNA copies of the I2-50
and I4-37 variants were used. None of the twenty plants
inoculated with either variant (I2-50 or I4-37) used alone
as controls, or a mixture of the variants showed visible
symptoms. However, dot blot hybridization analyses revealed that 7 out of the 30 doubly (I2-50 and I4-37) inoculated plants were PSTVd positive. The PSTVd progeny
isolated from three selected plants was analyzed revealing the presence of 10 different PSTVd variants. Eight of
them have not been previously described.
The same experiment was conducted, but using
monomeric (+) RNA transcripts. Contrary to the results obtained with the cDNA copies, all inoculations
yielded positive signals (in 3/15 plants for I2-50, 2/15
for I4-37 and 10/30 for the mixture of transcripts). This
result shows that transcripts of apparently non-infectious cDNA can induce infection. Analysis of PSTVd
clones isolated from three selected plants revealed that
all the progeny of I2-50 and the mixture of both transcripts reversed to the infectious I2 parental type. In
contrast in the progeny of I4-37, 8 sequences were detected, 6 of them were not previously described.
It must be stressed that in neither of these two types
of experiments, were I2-50 nor I4-37 sequences detected. Analysis and comparison of the sequences identified in both experiments with sequences retrieved
from the NCBI database made it possible to identify
mutations responsible for the apparent lack of
infectivity and draw general conclusions on nucleotide
preferences at certain positions in the PSTVd genome.
Our experiments show that mixed infection with two
apparently non-infectious PSTVd variants is followed
by the appearance of progeny recombinants with restored regions allowing systemic infection.
419
Poster
AtCBP20 — the cap-binding protein from Arabidopsis thaliana has an nuclear localization signal
Artur Rogowski, Maciej Kmieciak, Artur Jarmo³owski
Department of Gene Expression, Adam Mickiewicz University, ul. Miedzychodzka 5, 60-371 Poznañ
All transcripts made by eukaryotic RNA polymerase
II are co-transcriptionally modified at their 5’ ends by
the addition of a cap structure. It has been shown that
the cap structure is involved in several different aspects of RNA metabolism, such as pre-mRNA splicing,
RNA export from the nucleus to the cytoplasm and
polyadenylation. These functions are mediated by a nuclear complex of cap-binding proteins called CBC. It
consists of two cap-binding proteins: CBP20 and
CBP80. We have recently identified and characterized
a similar cap-binding complex from A. thaliana.
AtCBP20 has a predicted molecular mass of 30 kDa.
This protein contains a highly conserved canonical
RNA-binding domain and a plant specific long
C-terminal tail. The C-terminal part of AtCBP20 is rich
in glycine and arginine residues. It has been shown that
CBC from HeLa cells is transported into the nucleus
due to nuclear localization signal (NLS) located in the
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Session 10. Structure and function of RNA
2003
ments. Mutations in this motifs have revealed that they
function in nuclear transport independently. Our results indicate that this NLS is located in the plant specific C-terminal tail of AtCBP20. This data suggest that
in plants CBP20 is responsible for transport of the
cap-binding complex from the cytoplasm to the nucleus.
N-terminal domain of CBP80. Human CBP20 does not
contain any NLS. Using transient expression in
transfected tobacco protoplasts and fluorescent microscopy, we have shown that CBP20 from A. thaliana has
its own nuclear localization signal. We have recently
shown that this signal consist of two separated ele-
420
Poster
Studies of SIAH1, potential mRNA target for NANOS1-PUMILIO2 protein complex
in the human germ cells
1
2
3
1
3
Anna Spik , S³awomir Oczkowski , Jacek B³a¿ewicz , Maciej Kotecki , Piotr Formanowicz , Jadwiga
1
Jaruzelska
1 — Polska Akademia Nauk, Instytut Genetyki Cz³owieka, ul. Strzeszyñska 32, 60-479 Poznañ, 2 — Instytut Informatyki,
Politechnika Poznañska, ul. Piotrowo 3A, Poznañ, 3 — Polska Akademia Nauk, Instytut Chemii Bioorganicznej,
ul. Noskowskiego 12, Poznañ
Nanos and Pumilio in Drosophila. We found that one of
these candidates is SIAH1. This gene is homologous to
Sina, the fertility gene of Drosophila. Indeed, our experiments demonstrated that SIAH1 is expressed in human testes. The bend-shift approach has shown that
NANOS1 and PUMILIO2 bind 3’UTR of the SIAH1
mRNA. In the next step, the conserved 3’UTR motifs of
SIAH1 were mutated by introducing nucleotide substitutions using site directed mutagenesis. The binding efficiency of NANOS1-PUMILIO2 complex to 3’UTR mutated variants was substantially diminished. Although
further tests are needed we believe that SIAH1 is
strong candidate for the NANOS1-PUMILIO2 complex
mRNA target in the human germ cells.
The Nanos-Pumilio complex is Drosophila translational repressor in morphogenesis, and in development of the germ cells. We have recently identified homologous protein complex (NANOS1-PUMILIO2) in
the male human germ-line. The high conservation of
the RNA-binding domains of these proteins suggests
that they may function as translational regulators also
in humans. Therefore, identification of target mRNAs
seems crucial to understand the role of NANOS1 and
PUMILIO2 in development of the human germ-line.
Electronic searches of the GeneBank using software
we have generated enabled us to retrieve candidate human mRNA targets of the NANOS1-PUMILIO2 complex. These candidate mRNAs contain 3’UTR motifs
identical to motifs of the known mRNA targets of
421
Poster
Study of cleavage of the phosphodiester bond of mRNA 5’-cap analogs promoted by
copper ion and copper complexes
1
2
1
2
1
Alicja Stachelska , Satu Mikkola , Zbigniew Wieczorek , Sanna Anderson , Monika Pietrzak , Satu Valakoski
2
1 — Department of Physics and Biophysics, University of Warmia and Mazury in Olsztyn, ul. Oczapowskiego 4, 10-719 Olsztyn,
2 — Department of Chemistry, University of Turku, FIN-20014 Turku, Finland
The aim of this studies was to investigate the hydrolysis of mRNA compound ApppA and 5’-cap analogs:
2+
m7GpppG and m7GppppG in the presence of Cu ions
2+
2+
and of Cu TerPy and Cu BiPy complexes to find efficient catalysts for cleavage its phosphodiester bound.
Comparison of rate constants of hydrolysis of ApppA,
2+
m7GpppG in the presence and absence of Cu ion
shows its significant role in the cleavage of the
phospodiester bond of studied compounds. The results
show that hydroxoligand of metal ion catalyst is involved in this reaction. Comparison between the hydro-
lysis of ApppA, m7GpppG and m7GppppG in the pres2+
ence of Cu complexes show that the complex of
2+
2+
Cu TerPy is more effective catalyst than Cu BiPy for
hydrolysis of the cap-analogs at pH 8.0. The hydrolysis
2+
of ApppA in presence Cu TerPy proceeds about 2.7
2+
times faster than in the presence of Cu BiPy. However, m7GpppG hydrolysis in the presence of
2+
Cu TerPy is about 1.33 times faster than that in the
2+
presence of Cu BiPy. The hydrolysis of m7GppppG is
about one order of magnitude faster in the presence of
2+
2+
Cu TerPy than in the presence of Cu BiPy.
2003
39th Meeting of the Polish Biochemical Society
The results support suggestion that a hydroxoligand
of a phosphate-bound metal ion acts as a nucleophile in
2+
the hydrolysis reaction [2]. The role of the Cu complex catalysts is significant, but is more complicated
2+
than for Cu ion promoted hydrolysis [1]. The acidic
aquoligand may be needed for the phospodiester bond
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cleavage, more than one process. In some cases sufficient catalysis can be induced be one metal ion, but
sometimes two separate catalysts are required.
References:
1. Anderson S, LDhde M, Mikkola S, Morris G, Stachelska A,
Valakoski S, Williams NH (2002) Collect Symp, Ser 5.
2. Valakoski S, Heiskanen S, Anderson S, LDhde M, Mikkola
S (2002) J Chem Soc, Perkin Trans, 2604–610.
422
Poster
Homologous RNA recombination as a factor stabilizing and destabilizing a viral genome
1
1
1
Anna Urbanowicz , Magdalena Alejska , Marek Figlerowicz , Jozef Bujarski
2
1 — Instytut Chemii Bioorganicznej, Polska Akademia Nauk, ul. Noskowskiego 12/14, 61-704 Poznañ, 2 — Plant Molecular
Biology Center and Biology Department, Northern Illinois University, DeKalb, USA
Enormous genetic variability is one of the most unusual features of RNA viruses. There are two main
sources of the genetic polymorphism of RNA genomes:
error prone replication and RNA recombination.
Error prone replication introduces into a viral genome a wide spectrum of point mutations. They can
arise so frequently because RNA polymerases lack
proofreading activity. The estimated average mutation
–4
rate amounts to 10 per nucleotide per replication cycle. RNA recombination is responsible for more profound changes within the viral genome like sequence
deletion, sequence insertion or the exchange of the entire coding domain. In the course of our studies we are
trying to establish what the real role of homologous recombination in the virus life cycle is and this way to
learn more about the mechanisms generating new viral
strains or species. The object of our study is brome mosaic virus (BMV) — a model (+)RNA virus of plants. Its
genome consists of three RNA segments, RNA 1, 2 and
3. To determine the frequency of homologous cross-
overs between the same genomic BMV molecules, we
induced mixed infections in plants, using pairs of BMV
genomic RNAs bearing marker mutations in coding
and noncoding regions.
We observed that marker mutations in 3’ and 5’
noncoding regions of BMV RNAs were very efficiently
removed by imprecise replication. An analysis of the
distribution of the stable marker mutations in the nascent virus revealed that homologous recombination
events occur very often during virus genome replication. The frequency of recombination in the coding regions ranged from 10% to 25%. We also identified a recombination hot spot in the intergenic region of RNA3,
where the frequency of crossovers reached 71%. That
region might serve as a place for the exchanging of
whole genes. In general our results suggest that the majority of nascent viruses are recombinants. This indicates that homologous RNA recombination is one of the
main factors responsible for both the stabilization and
destabilization of the viral genome.
423
Poster
A procedure to generate in vitro RNA transcripts with homogenous 3’ ends
Agnieszka Wich³acz, Jerzy Ciesio³ka
Instytut Chemii Bioorganicznej, Polska Akademia Nauk, ul. Noskowskiego 12/14, 61-704 Poznañ
It is known that during in vitro run–off transcription
RNA polymerases often generate heterogeneous 3’
ends. In particular, T7 RNA polymerase attaches additional nucleotides to the nascent RNA transcript and at
the same time may show premature termination close
to its 3’ end. This results in decreased number of the
full-length, correctly transcribed products. However, in
many applications such as NMR, crystallization studies
and ligation reactions homogeneous transcripts are indispensable.
One approach for the preparation of RNA transcripts
with homogeneous 3’ terminus is the use of ribozymes
which are attached to their 3’ ends. These constructs
can cleave-off themselves acting in cis. On the contrary
to hammerhead or hairpin ribozymes, delta ribozymes
do not have particular requirements on the sequence
242
Session 10. Structure and function of RNA
upstream of the cleavage site and then can be applied to
any RNA. In this communication we have shown that
the antigenomic delta ribozyme could also be applied in
trans to correctly process the 3’ ends of RNA transcripts. Two different transcripts were extended with
the sequence of only 7 nucleotides complementary to
2003
the ribozyme recognition site. Following the cleavage
reaction with the ribozyme acting as ‘molecular scissors’ homogeneity of transcripts was proven by TLC
analysis of their 3’ terminal nucleotides. The proposed
procedure is useful for virtually any RNA molecule that
we desire to be homogeneous at its 3’ end.