ИСТИНА

Retrotransposon L1, a member of the non-LTR retrotransposon (RTP) LINE family, is a mobile element that is extremely widespread in the mammalian genome. The full-length retrotransposition-competent L1 encodes a dicistronic RNA, which is exceptionally rare in eukaryotic cellular mRNAs. The L1 dicistronic RNA serves both as transposition intermediate and an mRNA for the synthesis of two proteins of the RTP. The first cistron codes for an RNA-binding protein of unknown function (p40) and the second one for reverse transcriptase. Both proteins are necessary for L1 retrotransposition and act in cis. This report focuses on the translation initiation of ORF1. The translation of the L1 ORF1 is thought to proceed with a high efficiency (several hundreds copies of p40 per L1 mRNA molecule) unexpected for a 900 nt-long and GC-rich (60%) 5’ UTR. Computer-assisted folding of this 5’ UTR (L1 5’UTR) results in a secondary structure with several highly base paired domains. The presence of such a long leader is accounted for by the fact that the 5’-end of L1 RTP contains an internal promoter for RNA polymerase II. One could suggest that the 5’ UTR of L1 mRNA contained a powerful IRES-element whose activity was comparable to picornavirus IRESs. Using transfection of HEK293 or NT2/D1 cells with the polyadenylated mono- (L1 5’ UTR-Fluc) or dicistronic (Rluc-L1 5’ UTR-Fluc) RNA constructs, capped or uncapped, we have firmly established that the 5’ UTR of L1 does not contain an IRES: the initiation activity of the L1 5’ UTR in the intercistronic position was just 1. 5 – 2 % of that in the 5’ terminal position and critically dependent on the m7G cap. Uncapping reduced the initiation activity of the L1 5’ UTR (150 times in HEK293 and 80 times in NT2/D1 cells, respectively) to background. Furthermore, the initiation activity of the capped L1 5’ UTR was found to be equal to or even slightly exceeding that of the beta-actin 5’ UTR (84 nt-long). Strikingly, the deletion of 80% of the nucleotide sequence of the L1 5’UTR with most of its stem-loops resulted in ~ 2-fold drop rather than the expected enhancement of translation efficiency. The classical model of translation initiation in eukaryotes predicts that the more complex the secondary structure of a 5’ UTR, the less efficient is the cap-dependent initiation on this mRNA. One should recall, however, the scanning model was elaborated and tested for mRNAs with simple and rather short 5’ UTRs. The data presented in this report can significantly modify our ideas on mechanisms used by 40S ribosomal subunits to cope with complex 5’ UTRs and stimulate further studies in this direction.