| Abstract: | The standard method of translation initiation, where the ribosome binding onto mRNA is mediated by initiation factors that congregate at the 5' "cap-nucleotide" of the RNA, is at times, partially disabled. For example, during viral infection, mitosis, and cellular stress, the efficiency of this form of initiation is reduced relative to an alternate mechanism of initiation that utilizes Internal Ribosome Entry Sites (IRESes) contained in the 5' UTR sequence of viral and cellular RNA transcripts. These IRESes often are bound by non-canonical initiation factors that help to recruit the ribosomes to the mRNA upstream of the start codon without the need of a 5' cap structure. In viral IRESes, Eke HCV and EMCV, RNA structures have been shown to be critical for IRES function. These structures are also similar in other viruses that do not share sequence similarity. HCV for example, is similar to CSFV and BVDV, while the EMCV IRES structure is shared with other cardio-picornaviruses. In contrast, although several cellular IRESes' RNA secondary structures have been determined, no similarities have yet been identified. In order to establish if structure similarities could be found in cellular IRESes, the secondary structure of the X-linked inhibitor of apoptosis (XIAP) IRES was determined and used for an in silico search of all human 5' UTRs using the RSEARCH program. Five of the top computational matches were cloned and tested for IRES activity in beta-Gal/CAT bicistronic constructs. Of these, the 5' UTRs from Aquaporin 4 and the uncharacterized ELG1 transcripts exhibited IRES activity although at a much lower level than that of the MAP IRES. The actual RNA secondary structure of the ELG1 IRES did not match the predicted match from RSEARCH but the AQP4 IRES had a similar structure surrounding a polypyrimidine tract. Importantly, deletion of this motif removed all IRES activity similar to the analogous polypyrimidine tract deletion of the XIAP IRES. As only a slight similarity was found, it seems that RNA structures of IRESes in cellular messages are not shared and do not play the same role as those in viral mRNAs. |