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

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.

Modulation of p53 Expression Using Antisense Oligonucleotides Complementary to the 5′-Terminal Region of p53 mRNA In Vitro and in the Living Cells

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.

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.

Recently, we have determined the secondary structure of the 5′-terminal region of p53 mRNA that starts from the P1 transcription initiation site and includes two major translation initiation codons responsible for the synthesis of p53 and ΔNp53 isoform. Here, we showed that when this region was extended into 5′ direction to the P0 transcription start site, the two characteristic hairpin motifs found in this region were preserved. Moreover, the presence of alternatively spliced intron 2 did not interfere with the formation of the larger hairpin in which the initiation codon for p53 was embedded. The impact of the different variants of p53 5′-terminal region, which start at P0 or P1 site and end with the initiation codon for p53 or ΔNp53, on the translation of luciferase reporter protein was compared. Strikingly, the efficiency of translation performed in rabbit reticulocyte lysate differed by two orders of magnitude. The toe-printing analysis was also applied to investigate the formation of the ribosomal complex on the model mRNA constructs. The relative translation efficiencies in HeLa and MCF-7 cells were similar to those observed in the cell lysate, although some differences were noted in comparison with cell-free conditions. The results were discussed in terms of the role of secondary structure folding of the 5′-terminal region of p53 mRNA in translation and possible modes of p53 and ΔNp53 translation initiation.

Structure and function of RNA elements present in enteroviral genomes

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 the host cells. This review considers the RNA structures in enteroviral genomes identified and characterized to date.

Targeting Highly Structured RNA by Cooperative Action of siRNAs and Helper Antisense Oligomers in Living Cells

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.

Form confers function: Case of the 3’X region of the hepatitis C virus genome

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.