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Here we present a set of new structural elements formed within the open reading frame of the virus, which are highly probable, evolutionarily conserved and may interact with host proteins. This work focused on the coding regions of the CVB3 genome (particularly the V4-, V1-, 2C-, and 3D-coding regions), which, with the exception of the cis-acting replication element (CRE), have not yet been subjected to experimental analysis of their structures. The SHAPE technique, chemical modification with DMS and RNA cleavage with Pb2+, were performed in order to characterize the RNA structure. The experimental results were used to improve the computer prediction of the structural models, whereas a phylogenetic analysis was performed to check universality of the newly identified structural elements for twenty CVB3 genomes and 11 other enteroviruses. Some of the RNA motifs turned out to be conserved among different enteroviruses. We also observed that the 3′-terminal region of the genome tends to dimerize in a magnesium concentration-dependent manner. RNA affinity chromatography was used to confirm RNA–protein interactions hypothesized by database searches, leading to the discovery of several interactions, which may be important for virus propagation.
Mariola Dutkiewicz; Jakub Kuczynski; Michal Jarzab; Aleksandra Stachowiak; Agata Swiatkowska. New RNA Structural Elements Identified in the Coding Region of the Coxsackie B3 Virus Genome. Viruses 2020, 12, 1232 .
AMA StyleMariola Dutkiewicz, Jakub Kuczynski, Michal Jarzab, Aleksandra Stachowiak, Agata Swiatkowska. New RNA Structural Elements Identified in the Coding Region of the Coxsackie B3 Virus Genome. Viruses. 2020; 12 (11):1232.
Chicago/Turabian StyleMariola Dutkiewicz; Jakub Kuczynski; Michal Jarzab; Aleksandra Stachowiak; Agata Swiatkowska. 2020. "New RNA Structural Elements Identified in the Coding Region of the Coxsackie B3 Virus Genome." Viruses 12, no. 11: 1232.
The p53 protein is one of the transcription factors responsible for cell cycle regulation and prevention of cancer development. Its expression is regulated at the transcriptional, translational and post-translational levels. Recent years of research have shown that the 5ʹ terminus of p53 mRNA plays an important role in this regulation. This region seems to be a docking platform for proteins involved in p53 expression, particularly under stress conditions. Here, we applied RNA-centric affinity chromatography to search for proteins that bind to the 5ʹ terminus of p53 mRNA and thus may be able to regulate the p53 expression profile. We found heterogeneous nuclear ribonucleoprotein K, hnRNP K, to be one of the top candidates. Binding of hnRNP K to the 5ʹ-terminal region of p53 mRNA was confirmed in vitro. We demonstrated that changes in the hnRNP K level in the cell strongly affected the p53 expression profile under various stress conditions. Downregulation or overexpression of hnRNP K caused a decrease or an increase in the p53 mRNA amount, respectively, pointing to the transcriptional mode of expression regulation. However, when hnRNP K was overexpressed under endoplasmic reticulum stress and the p53 amount was elevated no changes in the p53 mRNA level were detected suggesting translational regulation of p53 expression. Our findings have shown that hnRNP K is not only a mutual partner of p53 in the transcriptional activation of target genes under stress conditions but it also acts as a regulator of p53 expression at the transcriptional and potentially translational levels.
Agata Swiatkowska; Mariola Dutkiewicz; Piotr Machtel; Damian M. Janecki; Martyna Kabacinska; Paulina Żydowicz-Machtel; Jerzy Ciesiołka. Regulation of the p53 expression profile by hnRNP K under stress conditions. RNA Biology 2020, 17, 1402 -1415.
AMA StyleAgata Swiatkowska, Mariola Dutkiewicz, Piotr Machtel, Damian M. Janecki, Martyna Kabacinska, Paulina Żydowicz-Machtel, Jerzy Ciesiołka. Regulation of the p53 expression profile by hnRNP K under stress conditions. RNA Biology. 2020; 17 (10):1402-1415.
Chicago/Turabian StyleAgata Swiatkowska; Mariola Dutkiewicz; Piotr Machtel; Damian M. Janecki; Martyna Kabacinska; Paulina Żydowicz-Machtel; Jerzy Ciesiołka. 2020. "Regulation of the p53 expression profile by hnRNP K under stress conditions." RNA Biology 17, no. 10: 1402-1415.
In this review, the latest research concerning the structure and function of the 5′-terminal region of p53 mRNA was discussed. Special attention was focused on defined structural motifs which are present in this region, as well as their conservation and plausible functional role in translation. It is known that the length of the 5′-terminal region and the structural environment of initiation codons can strongly modulate translation initiation. The ability of this region of p53 mRNA to bind protein factors was also described with special emphasis on general principles that govern, such RNA-protein interactions. The structural alterations within the 5′-terminal region of p53 mRNA and proteins that bind to this region have a strong impact on the rate of mRNA scanning and on translation efficiency in in vitro assays, in selected cell lines, and under stress conditions. Thus, the structural features of the 5′-terminal region of p53 mRNA seem to be very important for translation and for translation regulation mechanisms. Finally, we suggested topics that, in our opinion, should be further explored for better understanding of the mechanisms of the p53 gene expression regulation at the translational level.
Agata Swiatkowska; Mariola Dutkiewicz; Paulina Zydowicz-Machtel; Joanna Szpotkowska; Damian M. Janecki; Jerzy Ciesiołka. Translational Control in p53 Expression: The Role of 5′-Terminal Region of p53 mRNA. International Journal of Molecular Sciences 2019, 20, 5382 .
AMA StyleAgata Swiatkowska, Mariola Dutkiewicz, Paulina Zydowicz-Machtel, Joanna Szpotkowska, Damian M. Janecki, Jerzy Ciesiołka. Translational Control in p53 Expression: The Role of 5′-Terminal Region of p53 mRNA. International Journal of Molecular Sciences. 2019; 20 (21):5382.
Chicago/Turabian StyleAgata Swiatkowska; Mariola Dutkiewicz; Paulina Zydowicz-Machtel; Joanna Szpotkowska; Damian M. Janecki; Jerzy Ciesiołka. 2019. "Translational Control in p53 Expression: The Role of 5′-Terminal Region of p53 mRNA." International Journal of Molecular Sciences 20, no. 21: 5382.
Transcription initiation sites of Trp53 gene in mice were determined using the 5′RACE method. Based on sequence alignment of the 5′-terminal regions of p53 mRNA in mammals, the site for the most abundant transcript turned out to be essentially identical with that determined for human TP53 gene and slightly differed for the longest transcripts, in mice and humans. Secondary structures of the 5′ -terminal regions of the shorter, most abundant and the longest mouse transcripts were determined in vitro and the shorter transcript was also mapped in transfected mouse cells. For the first time, secondary structure models of the 5′ terminus of two mouse p53 mRNAs were proposed. Comparing these models with the conservativeness of the nucleotide sequence of the 5′-terminal region of mRNA in mouse and other mammals, the possible function of the selected structural domains of this region was discussed. To elucidate the translation mechanisms, the two studied mRNAs were translated in the presence of an increasing concentration of the cap analog. For the longest transcript, the data suggested that IRES element(s) was/were involved in translation initiation. Additionally, changes in p53 synthesis under genotoxic and endoplasmic reticulum stress conditions in mouse cells were analyzed.
Joanna Szpotkowska; Agata Swiatkowska; Jerzy Ciesiołka. Length and secondary structure of the 5′ non-coding regions of mouse p53 mRNA transcripts - mouse as a model organism for p53 gene expression studies. RNA Biology 2018, 16, 25 -41.
AMA StyleJoanna Szpotkowska, Agata Swiatkowska, Jerzy Ciesiołka. Length and secondary structure of the 5′ non-coding regions of mouse p53 mRNA transcripts - mouse as a model organism for p53 gene expression studies. RNA Biology. 2018; 16 (1):25-41.
Chicago/Turabian StyleJoanna Szpotkowska; Agata Swiatkowska; Jerzy Ciesiołka. 2018. "Length and secondary structure of the 5′ non-coding regions of mouse p53 mRNA transcripts - mouse as a model organism for p53 gene expression studies." RNA Biology 16, no. 1: 25-41.
The p53 protein is one of the major cell cycle regulators. The protein is expressed as at least twelve protein isoforms resulting from the use of alternative promoters, alternative splicing or downstream initiation codons. Importantly, there is growing evidence that translation initiation of p53 mRNA may be regulated by the structure and length of the naturally occurring variants of the 5′-terminal region of p53 mRNA transcripts. Here, several mRNA constructs were synthesized with variable length of the p53 5′-terminal regions and encoding luciferase reporter protein, and their translation was monitored continuously in situ in a rabbit reticulocyte lysate system. Moreover, four additional mRNA constructs were prepared. In two constructs, the structural context of AUG1 initiation codon was altered while in the other two constructs, characteristic hairpin motifs present in the p53 5′-terminal region were changed. Translation of the last two constructs was also performed in the presence of the cap analogue to test the function of the 5′-terminal region in cap-independent translation initiation. Superposition of several structural factors connected with the length of the 5′-terminal region, stable elements of the secondary structure, structural environment of the initiation codon and IRES elements greatly influenced the ribosomal scanning and translation efficiency.
Paulina Zydowicz-Machtel; Agata Swiatkowska; Lukasz Popenda; Agnieszka Gorska; Jerzy Ciesiołka. Variants of the 5′-terminal region of p53 mRNA influence the ribosomal scanning and translation efficiency. Scientific Reports 2018, 8, 1 -14.
AMA StylePaulina Zydowicz-Machtel, Agata Swiatkowska, Lukasz Popenda, Agnieszka Gorska, Jerzy Ciesiołka. Variants of the 5′-terminal region of p53 mRNA influence the ribosomal scanning and translation efficiency. Scientific Reports. 2018; 8 (1):1-14.
Chicago/Turabian StylePaulina Zydowicz-Machtel; Agata Swiatkowska; Lukasz Popenda; Agnieszka Gorska; Jerzy Ciesiołka. 2018. "Variants of the 5′-terminal region of p53 mRNA influence the ribosomal scanning and translation efficiency." Scientific Reports 8, no. 1: 1-14.
Enteroviruses are small RNA(+) viruses that encode one open reading frame flanked by two extensive noncoding regions carrying structural RNA regulatory elements that control replication and translation processes. For a long time the central, coding region was thought to remain single-stranded and its only function was supposed to be as the template for polyprotein synthesis. It turned out, however, that the protein coding region also encodes important RNA structures crucial for the viral life cycle and virus persistence in host cells. This review considers the RNA structures in enteroviral genomes identified and characterized to date.
Mariola Dutkiewicz; Aleksandra Stachowiak; Agata Swiatkowska; Jerzy Ciesiołka. Structure and function of RNA elements present in enteroviral genomes. Acta Biochimica Polonica 2017, 63, 623 -630.
AMA StyleMariola Dutkiewicz, Aleksandra Stachowiak, Agata Swiatkowska, Jerzy Ciesiołka. Structure and function of RNA elements present in enteroviral genomes. Acta Biochimica Polonica. 2017; 63 (4):623-630.
Chicago/Turabian StyleMariola Dutkiewicz; Aleksandra Stachowiak; Agata Swiatkowska; Jerzy Ciesiołka. 2017. "Structure and function of RNA elements present in enteroviral genomes." Acta Biochimica Polonica 63, no. 4: 623-630.
The p53 tumour suppressor protein is one of the major factors responsible for cell cycle regulation and protection against cancer development. Which is why it is often referred to as “the guardian of the genome”. On the other hand, mutations in the p53 gene are connected with more than 50% of tumours of various types. The thirty-six years of extensive research into the p53 gene and its protein products have shown how sophisticated the p53-based cell system control is. An additional level of complexity of the p53 research is connected with at least twelve p53 isoforms which have been identified in the cell. Importantly, disturbance of the p53 isoforms expression seems to play a key role in tumorigenesis, cell differentiation and cell response to pathogenic bacteria and RNA and DNA viruses. Expression of various p53 isoforms results from the usage of different transcription promotors, alternative splicing events and translation initiation from alternative AUG codons. The importance of the 5'-terminal regions of different p53 mRNA transcripts in the multi-level regulation of the p53 gene has recently been documented. In this review we focus on the structural features of these regions and their specific role in the p53 translation initiation process.
Agata Swiatkowska; Paulina Zydowicz; Joanna Sroka; Jerzy Ciesiołka. The role of the 5' terminal region of p53 mRNA in the p53 gene expression. Acta Biochimica Polonica 2017, 63, 1 .
AMA StyleAgata Swiatkowska, Paulina Zydowicz, Joanna Sroka, Jerzy Ciesiołka. The role of the 5' terminal region of p53 mRNA in the p53 gene expression. Acta Biochimica Polonica. 2017; 63 (4):1.
Chicago/Turabian StyleAgata Swiatkowska; Paulina Zydowicz; Joanna Sroka; Jerzy Ciesiołka. 2017. "The role of the 5' terminal region of p53 mRNA in the p53 gene expression." Acta Biochimica Polonica 63, no. 4: 1.
The p53 protein is one of the major factors responsible for cell cycle regulation and stress response. In the 5’-terminal region of p53 mRNA, an IRES element has been found which takes part in the translational regulation of p53 expression. Two characteristic hairpin motifs are present in this mRNA region: G56-C169, with the first AUG codon, and U180-A218, which interacts with the Hdm2 protein (human homolog of mouse double minute 2 protein). 2′-OMe modified antisense oligomers hybridizing to the 5'-terminal region of p53 mRNA were applied to assess the role of these structural elements in translation initiation under conditions of cellular stress. Structural changes in the RNA target occurring upon oligomers’ binding were monitored by the Pb2+-induced cleavage method. The impact of antisense oligomers on the synthesis of two proteins, the full-length p53 and its isoform Δ40p53, was analysed in HT-29, MCF-7 and HepG2 cells, under normal conditions and under stress, as well as in vitro conditions. The results revealed that the hairpin U180-A218 and adjacent single-stranded region A219-A228 were predominantly responsible for high efficacy of IRES-mediated translation in the presence of stress factors. These motifs play a role of cis-acting elements which are able to modulate IRES activity, likely via interactions with protein factors.
Agata Swiatkowska; Paulina Zydowicz; Agnieszka Górska; Julia Suchacka; Mariola Dutkiewicz; Jerzy Ciesiołka. The Role of Structural Elements of the 5'-Terminal Region of p53 mRNA in Translation under Stress Conditions Assayed by the Antisense Oligonucleotide Approach. PLOS ONE 2015, 10, e0141676 .
AMA StyleAgata Swiatkowska, Paulina Zydowicz, Agnieszka Górska, Julia Suchacka, Mariola Dutkiewicz, Jerzy Ciesiołka. The Role of Structural Elements of the 5'-Terminal Region of p53 mRNA in Translation under Stress Conditions Assayed by the Antisense Oligonucleotide Approach. PLOS ONE. 2015; 10 (10):e0141676.
Chicago/Turabian StyleAgata Swiatkowska; Paulina Zydowicz; Agnieszka Górska; Julia Suchacka; Mariola Dutkiewicz; Jerzy Ciesiołka. 2015. "The Role of Structural Elements of the 5'-Terminal Region of p53 mRNA in Translation under Stress Conditions Assayed by the Antisense Oligonucleotide Approach." PLOS ONE 10, no. 10: e0141676.
In the replication process of RNA(+) viruses both the positive-strand template and the newly synthesized negative strand appear in a double-stranded form, RF. It has been shown for poliovirus that prior to the initiation of positive-strand synthesis, the 5'-terminus of the positive strand must adopt a cloverleaf structure. When that happens, the 3'-terminal region of the negative strand is released from the RF form and is able to form into its own defined structure. In order to determine the secondary structure of this region, a comprehensive approach consisting of experimental mapping methods, phylogenetic analysis and computer predictions was applied. Here we propose the first structural model of the 3'-terminal region of the coxsackievirus B3 (CV-B3) negative strand, approximately 450 nucleotides in length. The region folds into three highly defined structural domains, I'-III'. The most 3'-terminal part of this region is domain I', which folds into a cloverleaf structure similar to that found in the viral RNA strand of positive-polarity. Remarkably, this motif is conserved among all analyzed viral isolates of CV-B3 despite the observed sequence diversity. Several other conserved structural motifs within the 3'-terminal region of the viral negative strand were also identified. The structure of this region may be crucial for the replication complex assembly.
Mariola Dutkiewicz; Agata Ojdowska; Agnieszka Gorska; Agata Swiatkowska; Jerzy Ciesiołka. The structural and phylogenetic profile of the 3′ terminus of coxsackievirus B3 negative strand. Virus Research 2014, 188, 81 -89.
AMA StyleMariola Dutkiewicz, Agata Ojdowska, Agnieszka Gorska, Agata Swiatkowska, Jerzy Ciesiołka. The structural and phylogenetic profile of the 3′ terminus of coxsackievirus B3 negative strand. Virus Research. 2014; 188 ():81-89.
Chicago/Turabian StyleMariola Dutkiewicz; Agata Ojdowska; Agnieszka Gorska; Agata Swiatkowska; Jerzy Ciesiołka. 2014. "The structural and phylogenetic profile of the 3′ terminus of coxsackievirus B3 negative strand." Virus Research 188, no. : 81-89.
The p53 protein is a key player in cell response to stress events and cancer prevention. However, up-regulation of p53 that occurs during radiotherapy of some tumours results in radio-resistance of targeted cells. Recently, antisense oligonucleotides have been used to reduce the p53 level in tumour cells which facilitates their radiation-induced apoptosis. Here we describe the rational design of antisense oligomers directed against the 5′-terminal region of p53 mRNA aimed to inhibit the synthesis of p53 protein and its ΔNp53 isoform. A comprehensive analysis of the sites accessible to oligomer hybridization in this mRNA region was performed. Subsequently, translation efficiency from the initiation codons for both proteins in the presence of selected oligomers was determined in rabbit reticulocyte lysate and in MCF-7 cells. The antisense oligomers with 2′-OMe and LNA modifications were used to study the mechanism of their impact on translation. It turned out that the remaining RNase H activity of the lysate contributed to modulation of protein synthesis efficiency which was observed in the presence of antisense oligomers. A possibility of changing the ratio of the newly synthetized p53 and ΔNp53 in a controlled manner was revealed which is potentially very attractive considering the relationship between the functioning of these two proteins. Selected antisense oligonucleotides which were designed based on accessibility mapping of the 5′-terminal region of p53 mRNA were able to significantly reduce the level of p53 protein in MCF-7 cells. One of these oligomers might be used in the future as a support treatment in anticancer therapy.
Agnieszka Gorska; Agata Swiatkowska; Mariola Dutkiewicz; Jerzy Ciesiolka. Modulation of p53 Expression Using Antisense Oligonucleotides Complementary to the 5′-Terminal Region of p53 mRNA In Vitro and in the Living Cells. PLOS ONE 2013, 8, e78863 .
AMA StyleAgnieszka Gorska, Agata Swiatkowska, Mariola Dutkiewicz, Jerzy Ciesiolka. Modulation of p53 Expression Using Antisense Oligonucleotides Complementary to the 5′-Terminal Region of p53 mRNA In Vitro and in the Living Cells. PLOS ONE. 2013; 8 (11):e78863.
Chicago/Turabian StyleAgnieszka Gorska; Agata Swiatkowska; Mariola Dutkiewicz; Jerzy Ciesiolka. 2013. "Modulation of p53 Expression Using Antisense Oligonucleotides Complementary to the 5′-Terminal Region of p53 mRNA In Vitro and in the Living Cells." PLOS ONE 8, no. 11: e78863.
Pre-ribosomal particles undergo numerous structural changes during maturation, but their high complexity and short lifetimes make these changes very difficult to follow in vivo. In consequence, pre-ribosome structure and composition have largely been inferred from purified particles and analyzed in vitro. Here we describe techniques for kinetic analyses of the changes in pre-ribosome structure in living cells of Saccharomyces cerevisiae. To allow this, in vivo structure probing by DMS modification was combined with affinity purification of newly synthesized 20S pre-rRNA over a time course of metabolic labeling with 4-thiouracil. To demonstrate that this approach is generally applicable, we initially analyzed the accessibility of the region surrounding cleavage site D site at the 3′ end of the mature 18S rRNA region of the pre-rRNA. This revealed a remarkably flexible structure throughout 40S subunit biogenesis, with little stable RNA–protein interaction apparent. Analysis of folding in the region of the 18S central pseudoknot was consistent with previous data showing U3 snoRNA–18S rRNA interactions. Dynamic changes in the structure of the hinge between helix 28 (H28) and H44 of pre-18S rRNA were consistent with recently reported interactions with the 3′ guide region of U3 snoRNA. Finally, analysis of the H18 region indicates that the RNA structure matures early, but additional protection appears subsequently, presumably reflecting protein binding. The structural analyses described here were performed on total, affinity-purified, newly synthesized RNA, so many classes of RNA and RNA–protein complex are potentially amenable to this approach.
Agata Swiatkowska; Wiebke Wlotzka; Alex Tuck; J. David Barrass; Jean D. Beggs; David Tollervey. Kinetic analysis of pre-ribosome structure in vivo. RNA 2012, 18, 2187 -2200.
AMA StyleAgata Swiatkowska, Wiebke Wlotzka, Alex Tuck, J. David Barrass, Jean D. Beggs, David Tollervey. Kinetic analysis of pre-ribosome structure in vivo. RNA. 2012; 18 (12):2187-2200.
Chicago/Turabian StyleAgata Swiatkowska; Wiebke Wlotzka; Alex Tuck; J. David Barrass; Jean D. Beggs; David Tollervey. 2012. "Kinetic analysis of pre-ribosome structure in vivo." RNA 18, no. 12: 2187-2200.
During the final stages of yeast ribosome synthesis, immature translation-incompetent pre-40S particles that contain 20S pre-rRNA are converted to the mature translation-competent subunits containing 18S rRNA. In vitro and in vivo data now demonstrate that processing of 20S pre-rRNA is stimulated by translation initiation factor Fun12, and that its interaction with 60S ribosomal subunits is required for efficient 20S pre-rRNA processing. In the final steps of yeast ribosome synthesis, immature translation-incompetent pre-40S particles that contain 20S pre-rRNA are converted to the mature translation-competent subunits containing the 18S rRNA. An assay for 20S pre-rRNA cleavage in purified pre-40S particles showed that cleavage by the PIN domain endonuclease Nob1 was strongly stimulated by the GTPase activity of Fun12, the yeast homolog of cytoplasmic translation initiation factor eIF5b. Cleavage of the 20S pre-rRNA was also inhibited in vivo and in vitro by blocking binding of Fun12 to the 25S rRNA through specific methylation of its binding site. Cleavage competent pre-40S particles stably associated with Fun12 and formed 80S complexes with 60S ribosomal subunits. We propose that recruitment of 60S subunits promotes GTP hydrolysis by Fun12, leading to structural rearrangements within the pre-40S particle that bring Nob1 and the pre-rRNA cleavage site together.
Simon Lebaron; Claudia Schneider; Rob W.. van Nues; Agata Swiatkowska; Dietrich Walsh; Bettina Böttcher; Sander Granneman; Nicholas Watkins; David Tollervey. Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits. Nature Structural & Molecular Biology 2012, 19, 744 -753.
AMA StyleSimon Lebaron, Claudia Schneider, Rob W.. van Nues, Agata Swiatkowska, Dietrich Walsh, Bettina Böttcher, Sander Granneman, Nicholas Watkins, David Tollervey. Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits. Nature Structural & Molecular Biology. 2012; 19 (8):744-753.
Chicago/Turabian StyleSimon Lebaron; Claudia Schneider; Rob W.. van Nues; Agata Swiatkowska; Dietrich Walsh; Bettina Böttcher; Sander Granneman; Nicholas Watkins; David Tollervey. 2012. "Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits." Nature Structural & Molecular Biology 19, no. 8: 744-753.
Coxsackievirus B3 (CVB-3) belongs to the Picornaviridae family of enterovirus genus of pathogens that cause a great number of human diseases. A viral infection is associated with many pathological states such as: myocarditis , dilated cardiomyopathy, pericarditis, pleurodynia , systemic...
Mariola Dutkiewicz; Agata Swiatkowska; Agata Ojdowska; Barbara Smólska; Teresa Dymarek-Babś; Anna Jasińska; Jerzy Ciesiołka. Molecular mechanisms of genome expression of coxsackievirus B3 that belongs to enteroviruses. BioTechnologia 2012, 4, 414 -423.
AMA StyleMariola Dutkiewicz, Agata Swiatkowska, Agata Ojdowska, Barbara Smólska, Teresa Dymarek-Babś, Anna Jasińska, Jerzy Ciesiołka. Molecular mechanisms of genome expression of coxsackievirus B3 that belongs to enteroviruses. BioTechnologia. 2012; 4 ():414-423.
Chicago/Turabian StyleMariola Dutkiewicz; Agata Swiatkowska; Agata Ojdowska; Barbara Smólska; Teresa Dymarek-Babś; Anna Jasińska; Jerzy Ciesiołka. 2012. "Molecular mechanisms of genome expression of coxsackievirus B3 that belongs to enteroviruses." BioTechnologia 4, no. : 414-423.
Understanding of eukaryotic ribosome synthesis has been slowed by a lack of structural data for the pre‐ribosomal particles. We report rRNA‐binding sites for six late‐acting 40S ribosome synthesis factors, three of which cluster around the 3′ end of the 18S rRNA in model 3D structures. Enp1 and Ltv1 were previously implicated in ‘beak’ structure formation during 40S maturation—and their binding sites indicate direct functions. The kinase Rio2, putative GTPase Tsr1 and dimethylase Dim1 bind sequences involved in tRNA interactions and mRNA decoding, indicating that their presence is incompatible with translation. The Dim1‐ and Tsr1‐binding sites overlap with those of homologous Escherichia coli proteins, revealing conservation in assembly pathways. The primary binding sites for the 18S 3′‐endonuclease Nob1 are distinct from its cleavage site and were unaltered by mutation of the catalytic PIN domain. Structure probing indicated that at steady state the cleavage site is likely unbound by Nob1 and flexible in the pre‐rRNA. Nob1 binds before pre‐rRNA cleavage, and we conclude that structural reorganization is needed to bring together the catalytic PIN domain and its target.
Sander Granneman; Elisabeth Petfalski; Agata Swiatkowska; David Tollervey. Cracking pre-40S ribosomal subunit structure by systematic analyses of RNA–protein cross-linking. The EMBO Journal 2010, 29, 2026 -2036.
AMA StyleSander Granneman, Elisabeth Petfalski, Agata Swiatkowska, David Tollervey. Cracking pre-40S ribosomal subunit structure by systematic analyses of RNA–protein cross-linking. The EMBO Journal. 2010; 29 (12):2026-2036.
Chicago/Turabian StyleSander Granneman; Elisabeth Petfalski; Agata Swiatkowska; David Tollervey. 2010. "Cracking pre-40S ribosomal subunit structure by systematic analyses of RNA–protein cross-linking." The EMBO Journal 29, no. 12: 2026-2036.
Here we present the results of a structural analysis of the 3′-terminal region of the replicative strand of hepatitis C virus (HCV), IRES(−), by the Pb2+-induced cleavage approach and partial digestion with T1 ribonuclease. Oligoribonucleotides that represent selected domains of the earlier proposed in the literature secondary structure models of this region were also synthesized, their structures were analyzed in solution, and the results were compared to those obtained with the full-length molecule. Such “structural fingerprinting” gave better insight into the structure of the IRES(−) region. We showed that in the case of the IRES(−) fragment, which consists of 374 nucleotides, its three domains, D3 (nucleotides 1−104), DM (nucleotides 105−222), and D5 (nucleotides 223−374), independently fold on one another. However, when the IRES(−) molecule is extended by 25 nucleotides of the upstream viral sequence, domains D3 and DM fold autonomously, but a part of domain D5 interacts with that additional RNA stretch. Analysis in silico suggests that similar interactions involving the IRES(−) region and upstream sequences are also possible in other fragments of viral RNA, several hundreds of nucleotides in length. The results of experimental probing are supported by secondary structure predictions in silico and phylogenetic analysis.
Mariola Dutkiewicz; Agata Świa̧tkowska; Marek Figlerowicz; Jerzy Ciesiołka. Structural Domains of the 3′-Terminal Sequence of the Hepatitis C Virus Replicative Strand. Biochemistry 2008, 47, 12197 -12207.
AMA StyleMariola Dutkiewicz, Agata Świa̧tkowska, Marek Figlerowicz, Jerzy Ciesiołka. Structural Domains of the 3′-Terminal Sequence of the Hepatitis C Virus Replicative Strand. Biochemistry. 2008; 47 (46):12197-12207.
Chicago/Turabian StyleMariola Dutkiewicz; Agata Świa̧tkowska; Marek Figlerowicz; Jerzy Ciesiołka. 2008. "Structural Domains of the 3′-Terminal Sequence of the Hepatitis C Virus Replicative Strand." Biochemistry 47, no. 46: 12197-12207.
The aim of this work was to shed some more light on factors influencing the effectiveness of delta ribozyme cleavage of structured RNA molecules. An oligoribonucleotide that corresponds to the 3‘-terminal region X of HCV RNA and yeast tRNAPhe were used as representative RNA targets. Only a few sites susceptible to ribozyme cleavage were identified in these targets using a combinatorial library of ribozyme variants, in which the region responsible for ribozyme−target interaction was randomized. On the other hand, the targets were fairly accessible for binding of complementary oligonucleotides, as was shown by 6-mer DNA libraries and RNase H approach. Moreover, the specifically acting ribozymes cleaved the targets precisely but with unexpectedly modest efficacy. To explain these observations, six model RNA molecules were designed, in which the same seven nucleotide long sequence recognized by the delta ribozyme was always single stranded but was embedded into different RNA structural context. These molecules were cleaved with differentiated rates, and the corresponding k2 values were in the range of 0.91−0.021 min-1; thus they differed almost 50-fold. This clearly shows that cleavage of structured RNAs might be much slower than cleavage of a short unstructured oligoribonucleotide, despite full accessibility of the targeted regions for hybridization. Restricted possibilities of conformational transitions, which are necessary to occur on the cleavage reaction trajectory, seem to be responsible for these differences. Their magnitude, which was evaluated in this work, should be taken into account while considering the use of delta ribozymes for practical applications.
Agata Swiatkowska; Mariola Dutkiewicz; Jerzy Ciesiołka. Structural Features of Target RNA Molecules Greatly Modulate the Cleavage Efficiency of trans-Acting Delta Ribozymes. Biochemistry 2007, 46, 5523 -5533.
AMA StyleAgata Swiatkowska, Mariola Dutkiewicz, Jerzy Ciesiołka. Structural Features of Target RNA Molecules Greatly Modulate the Cleavage Efficiency of trans-Acting Delta Ribozymes. Biochemistry. 2007; 46 (18):5523-5533.
Chicago/Turabian StyleAgata Swiatkowska; Mariola Dutkiewicz; Jerzy Ciesiołka. 2007. "Structural Features of Target RNA Molecules Greatly Modulate the Cleavage Efficiency of trans-Acting Delta Ribozymes." Biochemistry 46, no. 18: 5523-5533.