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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.
The 5ʹ-UTR of the actin-related protein 2/3 complex subunit 2 (ARPC2) mRNA exists in two variants. Using a bicistronic reporter construct, the present study demonstrates that the longer variant of the 5ʹ-UTR harbours an internal ribosome entry site (IRES) which is lacking in the shorter one. Multiple control assays confirmed that only this variant promotes cap-independent translation. Furthermore, it includes a guanine-rich region that is capable of forming a guanine-quadruplex (G-quadruplex) structure which was found to contribute to the IRES activity. To investigate the cellular function of the IRES element, we determined the expression level of ARPC2 at various cell densities. At high cell density, the relative ARPC2 protein level increases, supporting the presumed function of IRES elements in driving the expression of certain genes under stressful conditions that compromise cap-dependent translation. Based on chemical probing experiments and computer-based predictions, we propose a structural model of the IRES element, which includes the G-quadruplex motif exposed from the central stem-loop element. Taken together, our study describes the functional relevance of two alternative 5ʹ-UTR splice variants of the ARPC2 mRNA, one of which contains an IRES element with a G-quadruplex as a central motif, promoting translation under stressful cellular conditions
Munir A. Al-Zeer; Mariola Dutkiewicz; Annekathrin Von Hacht; Denise Kreuzmann; Viola Röhrs; Jens Kurreck. Alternatively spliced variants of the 5’-UTR of the ARPC2 mRNA regulate translation by an internal ribosome entry site (IRES) harboring a guanine-quadruplex motif. RNA Biology 2019, 16, 1622 -1632.
AMA StyleMunir A. Al-Zeer, Mariola Dutkiewicz, Annekathrin Von Hacht, Denise Kreuzmann, Viola Röhrs, Jens Kurreck. Alternatively spliced variants of the 5’-UTR of the ARPC2 mRNA regulate translation by an internal ribosome entry site (IRES) harboring a guanine-quadruplex motif. RNA Biology. 2019; 16 (11):1622-1632.
Chicago/Turabian StyleMunir A. Al-Zeer; Mariola Dutkiewicz; Annekathrin Von Hacht; Denise Kreuzmann; Viola Röhrs; Jens Kurreck. 2019. "Alternatively spliced variants of the 5’-UTR of the ARPC2 mRNA regulate translation by an internal ribosome entry site (IRES) harboring a guanine-quadruplex motif." RNA Biology 16, no. 11: 1622-1632.
At the 3’ end of genomic hepatitis C virus (HCV) RNA there is a highly conserved untranslated region, the 3’X-tail, which forms part of the 3’UTR. This region plays key functions in regulation of critical processes of the viral life cycle. The 3’X region is essential for viral replication and infectivity. It is also responsible for regulation of switching between translation and transcription of the viral RNA. There is some evidence indicating the contribution of the 3’X region to the translation efficiency of the viral polyprotein and to the encapsidation process. Several different secondary structure models of the 3’X region, based on computer predictions and experimental structure probing, have been proposed. It is likely that the 3’X region adopts more than one structural form in infected cells and that a specific equilibrium between the various forms regulates several aspects of the viral life cycle. The most intriguing explanations of the structural heterogeneity problem of the 3’X region came with the discovery of its involvement in long-range RNA-RNA interactions and the potential for homodimer formation. This article summarizes current knowledge on the structure and function of the 3’X region of hepatitis C genomic RNA, reviews previous opinions, presents new hypotheses and summarizes the questions that still remain unanswered.
Mariola Dutkiewicz; Jerzy Ciesiołka. Form confers function: Case of the 3’X region of the hepatitis C virus genome. World Journal of Gastroenterology 2018, 24, 3374 -3383.
AMA StyleMariola Dutkiewicz, Jerzy Ciesiołka. Form confers function: Case of the 3’X region of the hepatitis C virus genome. World Journal of Gastroenterology. 2018; 24 (30):3374-3383.
Chicago/Turabian StyleMariola Dutkiewicz; Jerzy Ciesiołka. 2018. "Form confers function: Case of the 3’X region of the hepatitis C virus genome." World Journal of Gastroenterology 24, no. 30: 3374-3383.
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 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.
RNA target accessibility is one of the most important factors limiting the efficiency of RNA interference-mediated RNA degradation. However, targeting RNA viruses in their poorly accessible, highly structured regions can be advantageous because these regions are often conserved in sequence and thus less prone to viral escape. We developed an experimental strategy to attack highly structured RNA by means of pairs of specifically designed small interfering RNAs and helper antisense oligonucleotides using the 5’ untranslated region (5’UTR) of coxsackievirus B3 as a model target. In the first step, sites accessible to hybridization of complementary oligonucleotides were identified using two mapping methods with random libraries of short DNA oligomers. Subsequently, the accessibility of the mapped regions for hybridization of longer DNA 16-mers was confirmed by an RNase H assay. Using criteria for the design of efficient small interfering RNAs (siRNA) and a secondary structure model of the viral 5’UTR, several DNA 19-mers were designed against partly double-stranded RNA regions. Target sites for DNA 19-mers were located opposite the sites which had been confirmed as accessible for hybridization. Three pairs of DNA 19-mers and the helper 2’-O-methyl-16-mers were able to effectively induce RNase H cleavage in vitro. For cellular assays, the DNA 19-mers were replaced by siRNAs, and the corresponding three pairs of siRNA-helper oligomer tools were found to target 5’UTR efficiently in a reporter construct in HeLa cells. Addition of the helper oligomer improved silencing capacity of the respective siRNA. We assume that the described procedure will generally be useful for designing of nucleic acid-based tools to silence highly structured RNA targets.
Mariola Dutkiewicz; Agata Ojdowska; Jakub Kuczyński; Vanessa Lindig; Heinz Zeichhardt; Jens Kurreck; Jerzy Ciesiołka. Targeting Highly Structured RNA by Cooperative Action of siRNAs and Helper Antisense Oligomers in Living Cells. PLOS ONE 2015, 10, e0136395 .
AMA StyleMariola Dutkiewicz, Agata Ojdowska, Jakub Kuczyński, Vanessa Lindig, Heinz Zeichhardt, Jens Kurreck, Jerzy Ciesiołka. Targeting Highly Structured RNA by Cooperative Action of siRNAs and Helper Antisense Oligomers in Living Cells. PLOS ONE. 2015; 10 (8):e0136395.
Chicago/Turabian StyleMariola Dutkiewicz; Agata Ojdowska; Jakub Kuczyński; Vanessa Lindig; Heinz Zeichhardt; Jens Kurreck; Jerzy Ciesiołka. 2015. "Targeting Highly Structured RNA by Cooperative Action of siRNAs and Helper Antisense Oligomers in Living Cells." PLOS ONE 10, no. 8: e0136395.
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.
Agnieszka Górska; Leszek Błaszczyk; Mariola Dutkiewicz; Jerzy Ciesiołka. Length variants of the 5′ untranslated region of p53 mRNA and their impact on the efficiency of translation initiation of p53 and its N-truncated isoform ΔNp53. RNA Biology 2013, 10, 1726 -1740.
AMA StyleAgnieszka Górska, Leszek Błaszczyk, Mariola Dutkiewicz, Jerzy Ciesiołka. Length variants of the 5′ untranslated region of p53 mRNA and their impact on the efficiency of translation initiation of p53 and its N-truncated isoform ΔNp53. RNA Biology. 2013; 10 (11):1726-1740.
Chicago/Turabian StyleAgnieszka Górska; Leszek Błaszczyk; Mariola Dutkiewicz; Jerzy Ciesiołka. 2013. "Length variants of the 5′ untranslated region of p53 mRNA and their impact on the efficiency of translation initiation of p53 and its N-truncated isoform ΔNp53." RNA Biology 10, no. 11: 1726-1740.
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.
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.
Picornaviruses are a class of RNA viruses with a single-stranded genome in positive orientation. Since the prospects of treatment are limited, we employ RNA interference (RNAi) as an antiviral tool to inhibit different picornaviruses. We identified small interfering RNAs (siRNAs) against the 3D RNA dependent RNA polymerase of coxsackievirus B3 that were capable of efficiently inhibiting the virus. Targeting of the conserved 5′ UTR of the virus turned out to be a challenging task since stable structures of this region are detrimental to silencing. We developed a rational strategy to solve this problem and found an siRNA containing locked nucleic acids (LNAs) to possess high antiviral potency. To analyse the mechanism of virus inhibition in more detail, LNAs were incorporated into the siRNA to inactivate either of the siRNA strands. These experiments clearly revealed that only the genomic plus-strand but not the intermediary synthesised minus-strand can be targeted by siRNAs. Furthermore, siRNAs were employed to silence the virus receptor on the host cell and thus prevent viral spread.
Diana Rothe; Denise Werk; Mariola Dutkiewicz; Steffen Schubert; Hans-Peter Grunert; Heinz Zeichhardt; Volker A. Erdmann; Henry Fechner; Jens Kurreck. Inhibition of picornaviruses by means of RNA interference. Nucleic Acids Symposium Series 2008, 52, 63 -64.
AMA StyleDiana Rothe, Denise Werk, Mariola Dutkiewicz, Steffen Schubert, Hans-Peter Grunert, Heinz Zeichhardt, Volker A. Erdmann, Henry Fechner, Jens Kurreck. Inhibition of picornaviruses by means of RNA interference. Nucleic Acids Symposium Series. 2008; 52 (1):63-64.
Chicago/Turabian StyleDiana Rothe; Denise Werk; Mariola Dutkiewicz; Steffen Schubert; Hans-Peter Grunert; Heinz Zeichhardt; Volker A. Erdmann; Henry Fechner; Jens Kurreck. 2008. "Inhibition of picornaviruses by means of RNA interference." Nucleic Acids Symposium Series 52, no. 1: 63-64.
This study describes a strategy to develop LNA-modified small interfering RNA (siRNAs) against the highly structured 5′ UTR of coxsackievirus B3 (CVB-3), which is an attractive target site due to its high degree of conservation. Accessible sites were identified based on structural models and RNase H assays with DNA oligonucleotides. Subsequently, LNA gapmers, siRNAs, siLNAs and small internally segmented interfering RNA (sisiLNAs) were designed against sites, which were found to be accessible in the in vitro assays, and tested in reporter assays and experiments with the infectious virus. The best siLNA improved viability of infected cells by 92% and exerted good antiviral activity in plaque reduction assays
Mariola Dutkiewicz; Hans-Peter Grunert; Heinz Zeichhardt; Suzy W. Lena; Jesper Wengel; Jens Kurreck. Design of LNA-modified siRNAs against the highly structured 5′ UTR of coxsackievirus B3. FEBS Letters 2008, 582, 3061 -3066.
AMA StyleMariola Dutkiewicz, Hans-Peter Grunert, Heinz Zeichhardt, Suzy W. Lena, Jesper Wengel, Jens Kurreck. Design of LNA-modified siRNAs against the highly structured 5′ UTR of coxsackievirus B3. FEBS Letters. 2008; 582 (20):3061-3066.
Chicago/Turabian StyleMariola Dutkiewicz; Hans-Peter Grunert; Heinz Zeichhardt; Suzy W. Lena; Jesper Wengel; Jens Kurreck. 2008. "Design of LNA-modified siRNAs against the highly structured 5′ UTR of coxsackievirus B3." FEBS Letters 582, no. 20: 3061-3066.
Guanine-rich sequences can adopt intramolecular four-stranded structures, called G-quadruplexes. These motifs have been intensively investigated on the DNA level, but their overall biological relevance remains elusive. Only recently has research concerning the function of G-quadruplexes in RNAs commenced. Here, we demonstrate for the first time, that an RNA G-quadruplex structure inhibits translation in vivo in eukaryotic cells. We investigated the function of a highly conserved, thermodynamically stable RNA G-quadruplex in the 5′-UTR of the mRNA of the human Zic-1 zinc-finger protein. Using dual luciferase reporter assay, we demonstrate that the Zic-1 RNA G-quadruplex represses protein synthesis inside eukaryotic cells. Quantitative RT-PCR assays confirmed that the reduction of protein synthesis is due to regulation of the translation process and not a consequence of reduced transcription. Western blot analysis revealed that expression of Zic-1 is strongly reduced by a 73 nucleotides-long fragment of the UTR containing the G-quadruplex motif. These structures might add to the more recently discovered elements in untranslated regions of mRNAs that regulate their translation.
Amit Arora; Mariola Dutkiewicz; Vinod Scaria; Manoj Hariharan; Souvik Maiti; Jens Kurreck. Inhibition of translation in living eukaryotic cells by an RNA G-quadruplex motif. Structure of the 30S ribosomal decoding complex at ambient temperature 2008, 14, 1290 -1296.
AMA StyleAmit Arora, Mariola Dutkiewicz, Vinod Scaria, Manoj Hariharan, Souvik Maiti, Jens Kurreck. Inhibition of translation in living eukaryotic cells by an RNA G-quadruplex motif. Structure of the 30S ribosomal decoding complex at ambient temperature. 2008; 14 (7):1290-1296.
Chicago/Turabian StyleAmit Arora; Mariola Dutkiewicz; Vinod Scaria; Manoj Hariharan; Souvik Maiti; Jens Kurreck. 2008. "Inhibition of translation in living eukaryotic cells by an RNA G-quadruplex motif." Structure of the 30S ribosomal decoding complex at ambient temperature 14, no. 7: 1290-1296.
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.
Leszek Błaszczyk; Mariola Dutkiewicz; Jerzy Ciesiołka. [Translation of eukaryotic mRNA in a cap-independent mode]. Postępy Biochemii 2007, 53, 1 .
AMA StyleLeszek Błaszczyk, Mariola Dutkiewicz, Jerzy Ciesiołka. [Translation of eukaryotic mRNA in a cap-independent mode]. Postępy Biochemii. 2007; 53 (4):1.
Chicago/Turabian StyleLeszek Błaszczyk; Mariola Dutkiewicz; Jerzy Ciesiołka. 2007. "[Translation of eukaryotic mRNA in a cap-independent mode]." Postępy Biochemii 53, no. 4: 1.
Mariola Dutkiewicz; Agata Swiqtkowska; Jerzy Ciesiołka. [Structure and function of the non-coding regions of hepatitis C viral RNA]. Postępy Biochemii 2006, 52, 1 .
AMA StyleMariola Dutkiewicz, Agata Swiqtkowska, Jerzy Ciesiołka. [Structure and function of the non-coding regions of hepatitis C viral RNA]. Postępy Biochemii. 2006; 52 (1):1.
Chicago/Turabian StyleMariola Dutkiewicz; Agata Swiqtkowska; Jerzy Ciesiołka. 2006. "[Structure and function of the non-coding regions of hepatitis C viral RNA]." Postępy Biochemii 52, no. 1: 1.
Oligoribonucleotides that corresponded to the X regions of the (+) and (-) polarity strands of HCV RNA, as well as several shorter oligomers comprising defined stem-loop motifs of their predicted secondary structure models, were analyzed by Pb2+-induced cleavage, partial digestion with specific nucleases and chemical modification. Patterns characteristic of the motifs were compared with those obtained for the full-length molecules and on the basis of such 'structural fingerprinting' conclusions concerning folding of regions X were formulated. It turned out that the secondary structure model of X(+) RNA proposed earlier, the three-stem-loop model composed of hairpins SL1, SL2 and SL3, was only partially consistent with our experimental data. We confirmed the presence of SL1 and SL3 motifs and showed that the single-stranded stretch adjacent to the earlier proposed hairpin SL2 contributed to the folding of that region. It seemed to be arranged into two hairpins, which might form a hypothetical pseudoknot by changing their base-pairing systems. These data were discussed in terms of their possible biological significance. On the other hand, analysis of the X(-) RNA and its sub-fragments supported a three-stem-loop secondary structure model for this RNA.
Mariola Dutkiewicz. Structural characterization of the highly conserved 98-base sequence at the 3' end of HCV RNA genome and the complementary sequence located at the 5' end of the replicative viral strand. Nucleic Acids Research 2005, 33, 693 -703.
AMA StyleMariola Dutkiewicz. Structural characterization of the highly conserved 98-base sequence at the 3' end of HCV RNA genome and the complementary sequence located at the 5' end of the replicative viral strand. Nucleic Acids Research. 2005; 33 (2):693-703.
Chicago/Turabian StyleMariola Dutkiewicz. 2005. "Structural characterization of the highly conserved 98-base sequence at the 3' end of HCV RNA genome and the complementary sequence located at the 5' end of the replicative viral strand." Nucleic Acids Research 33, no. 2: 693-703.