Projects / Programmes source: ARIS

Strukturne študije RNK oligonukleotidov udeleženih v regulaciji genov (Slovene)

Research activity

Code Science Field Subfield
1.05.00  Natural sciences and mathematics  Biochemistry and molecular biology   
Evaluation (rules)
source: COBISS
Researchers (1)
no. Code Name and surname Research area Role Period No. of publicationsNo. of publications
1.  24448  PhD Mirko Cevec  Biochemistry and molecular biology  Head  2009 - 2011  65 
Organisations (1)
no. Code Research organisation City Registration number No. of publicationsNo. of publications
1.  0104  National Institute of Chemistry  Ljubljana  5051592000  21,357 
Significance for science
siRNA and miRNA are two classes of non-coding RNAs, which are involved at the post-transcriptional gene regulation by binding to the target mRNA. The structural features of their complexes with mRNA play the main role in the expression and evolution of protein-coding genes. miRNA binds to the target mRNA with the imperfect complementarity and mostly represses translation, while siRNA binds to the target mRNA with perfect complementarity and cleaves the bond in the middle of the mRNA. Numerous studies on the functional characterization of the miRNAs are done, but only few information is available on the structural features of the miRNA:mRNA complex. To our knowledge, only our two NMR structures of the miRNA:mRNA complexes (LCS1co and LCS2co) has been reported so far. The possible reasons are probably in the partial complementarity and high conformational flexibility of the complexes, which hurdle the experimental determination of miRNA:mRNA structures with the other research methods. Beside the determination of the 3D structure of RNA complexes the main aim of our project was also the comparison of the structural features of siRNA:mRNA and miRNA:mRNA complexes. For siRNA:mRNA complexes was know from before that they have a standard A-RNA form. Mismatched base pairs and bulges, which are present in miRNA:mRNA complexes, induce a kinked structure. It seems that in the siRNA:mRNA structure, the scissile phosphate of the mRNA strand is more accessible to the catalytic site in the Argonaute protein as compared to the miRNA:mRNA structures. We studied let-7 miRNA target site within 3'-UTR lin-41 mRNA. It belongs to 3'-compensatory miRNA target sites, which represent 1% of all conserved target sites in mammals. Despite the very low frequency this target site is interesting due to the structural features, such as asymmetric internal loop and bulge. let-7 was one of the first discovered miRNAs and at the beginning it was understood that all miRNAs have this type of target site within mRNAs. Results of this study are important for the better understanding of the role of miRNA structure in gene silencing and for the discovery of new target sites on mRNA. Thus the NMR structure of LCS1co indirectly allows better understanding of the structures of miRNA and mRNA inside the RNA-induced silencing complex (RISC). The structure of LCS1co shows that the nucleobase of adenine bulge A30 does not stack into the helix, but points into the minor groove and could represent a protein binding site. Also asymmetric internal loop is interesting from structural point of view, because this kind of loop was not studied before. All this structural details are useful at the development of the software, which is used for the prediction of miRNA structures and target sites in mRNAs.
Significance for the country
The project was carried out at Slovenian NMR centre at National Institute of Chemistry Ljubljana. The project allowed us to disseminate and expand the knowledge of the Slovenian scientific community in the field of multidimensional NMR spectroscopy. The project leader collaborated with a B.Sc. student, who prepared a thesis about the mutants of double stranded let-7 miRNA:lin-41 mRNA complexes. Modern methods for RNA structural determination and dynamics were used, such as nuclear magnetic resonance, ultraviolet and visible absorption spectroscopy, and gel electrophoresis. miRNA:mRNA complexes are conformational flexible due to internal loops and bulges, and are thus interesting to study by solution NMR. Currently only two NMR structures of miRNA:mRNA complexes are known (LCS1co and LCS2co) and both are determined by our research group. These results place us to the top European scientists studying RNA structure with NMR spectroscopy in solution. Likewise, the results show, what can be studied in Slovenian NMR centre and encourage Slovenian researchers and companies that they start collaborations with the centre, in particular on the research fields, which cover investigation of relationships between the structure and activity of larger RNAs. miRNA:mRNA complexes could be interesting for Slovenian pharmaceutical companies for the development, preparation and improvement of new drugs, which will base on the function of miRNA. There is an increase in the number of biotech firms in the world, which were founded to treat diseases caused by miRNAs and this trend could also come to Slovenia.
Most important scientific results Annual report 2010, final report, complete report on dLib.si
Most important socioeconomically and culturally relevant results Annual report 2010, final report, complete report on dLib.si
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