In animals, microRNAs (miRNAs) bind to the 3 UTRs of their target mRNAs and interfere with translation, although the exact mechanism of inhibition of protein synthesis remains unclear. Functional miRNA-binding sites in the coding regions or 5 UTRs of endogenous mRNAs have not been identified. We studied the effect of introducing miRNA target sites into the 5 UTR of luciferase reporter mRNAs containing internal ribosome entry sites (IRESs), so that potential steric hindrance by a microribonucleoprotein complex would not interfere with the initiation of translation. In human HeLa cells, which express endogenous let-7a miRNA, the translational efficiency of these IRES-containing reporters with 5 let-7 complementary sites from the Caenorhabditis elegans lin-41 3 UTR was repressed. Similarly, the IRES-containing reporters were translationally repressed when human Ago2 was tethered to either the 5 or 3 UTR. Interestingly, the method of DNA transfection affected our ability to observe miRNA-mediated repression. Our results suggest that association with any position on a target mRNA is mechanistically sufficient for a microribonucleoprotein to exert repression of translation at some step downstream of initiation.internal ribosome entry sites ͉ repression ͉ method of transfection ͉ let-7 miRNP ͉ translation efficiency

We have found that RNase P from HeLa cells specifically and efficiently cleaves hepatitis C virus (HCV) transcripts in vitro. The evidence includes identification of the 5-phosphate polarity of the newly generated termini at position A 2860 as well as immunological and biochemical assays. Active cleavage has been shown in five dominant sequences of HCV "quasispecies" differing at or near the position of cleavage, demonstrating that this is a general property of HCV RNA. During the analysis, a second cleavage event was found in the 3 domain of the internal ribosome entry site. We have found that HCV RNA competitively inhibits pre-tRNA cleavage by RNase P, suggesting that HCV RNA has structural similarities to tRNA. This finding sets HCV apart from other pathogens causing serious human diseases and represents the first description of human RNase P-viral RNA cleavage. Here we discuss the possible meaning of these RNase P-accessible structures built into the viral genome and their possible role in vivo. Moreover, such structures within the viral genome might be vulnerable to attack by therapeutic strategies.

The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) RNA is known to interact with the 40S ribosomal subunit alone, in the absence of any additional initiation factors or Met-tRNAi. Previous work from this laboratory on the 80S and 48S ribosomal initiation complexes involving the HCV IRES showed that stem-loop III, the pseudoknot domain, and some coding sequence were protected from pancreatic RNase digestion. Stem-loop II is never protected by these complexes. Furthermore, there is no prior evidence reported showing extensive direct binding of stem-loop II to ribosomes or subunits. Using direct analysis of RNase-protected HCV IRES domains bound to 40S ribosomal subunits, we have determined that stem-loops II and III and the pseudoknot of the HCV IRES are involved in this initial binding step. The start AUG codon is only minimally protected. The HCV-40S subunit binary complex thus involves recognition and binding of stem-loop II, revealing its role in the first step of a multistep initiation process that may also involve rearrangement of the bound IRES RNA as it progresses.

The internal ribosome entry site (IRES) of hepatitis C virus (HCV) RNA contains >300 bases of highly conserved 5'-terminal sequence, most of it in the uncapped 5'-untranslated region (5'-UTR) upstream from the single AUG initiator triplet at which translation of the HCV polyprotein begins. Although progress has been made in defining singularities like the RNA pseudoknot near this AUG, the sequence and structural features of the HCV IRES which stimulate accurate and efficient initiation of protein synthesis are only partially defined. Here we report that a region further upstream from the AUG, stem-loop II of the HCV IRES, also contains an element of local tertiary structure which we have detected using RNase H cleavage and have mapped using the singular ability of two bases therein to undergo covalent intra-chain crosslinking stimulated by UV light. This pre-existing element maps to two non-contiguous stretches of the HCV IRES sequence, residues 53-68 and 103-117. Several earlier studies have shown that the correct sequence between bases 45 and 70 of the HCV IRES stem-loop II domain is required for initiation of protein synthesis. Because features of local tertiary structure like the one we report here are often associated with protein binding, we propose that the HCV stem-loop II element is directly involved in IRES action.