RNase III and RNase P are structurally dependent endoribonucleases which recognize dsRNA and precursor tRNA substrates, respectively. Using both enzymes as molecular tools, we have been able to detect the presence of mimetic motifs inside mRNA of viral and cellular origin. These structural RNA motifs may represent signals within the cellular context in which they are found.
It has been proposed that the hepatitis C virus internal ribosome entry site resides in a locked conformation due to annealing of its immediate flanking sequences (Beguiristain, 2005-NAR). In the first part of the results section, structure probing using Escherichia coli dsRNA-specific RNase III and other classical tools shows that this region switches to an open conformation triggered by a liver-specific microRNA (miR-122). This structural transition, which is observed in vitro, may be important for essential functions involving stem loop VI and may also suggest a role for liver-specific miR-122 in HCV infection. In addition, induced RNA switching at the 5¿ untranslated region could ultimately represent a new mechanism of action of micro-RNAs.
In the second part of the results section we explore whether liver mRNA species have structural features which, in common with HCV mRNA, can specify the RNase P cleavage reaction in vitro. We observed that human liver mRNA competes with pretRNATyr cleavage in a human RNase P competition essay, and subsequently identified a set of mRNA species that could, at least in part, be responsible for human RNase P competition. The presence of tRNA mimics in three mRNA transcripts (coding for the ribosomal protein RPS9, the histone variant H2AFJ.2 and the interferon IFNA5) was confirmed individually by RNase P cleavage and competition essays, and one of these, namely IFNA5 mRNA, was further evaluated to obtain rigorous proof of its specificity. One of the cleavage sites in this mRNA, and its minimal substrate length, lie within the reported IFNA1 cytoplasmic accumulation region signal (Lei, 2011-PNAS).
Finally, in silico predictions have allowed us to find a target for miR-122 within the 3¿ UTR of IFNA5. As a result, in the third part of the results section we analyse the RNA structure in this region using a number of RNases. miR-122 induces changes in the digestion pattern of various of these RNases close to their hybridisation region. These modifications affect the structure of the codon that denotes the end of the translation and the possible appearance of a pseudoknot. The conformational change resulting upon hybridisation of miR-122 generates a tRNA-mimic structure that can be recognised by human RNase P in vitro.
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