ИСТИНА

Telomerase is a key participant of telomere length maintenance machinery; it extends 3'-ends of linear chromosomes in eukaryotic cells. Telomerase is active in stem, germ, embryonic and cancer cells. In human two major components: telomerase RNA (hTR) and reverse transcriptase protein subunit (hTERT) are crucial for telomerase activity. hTR is expressed in a large number of human cell types without detectable telomerase activity, while expression of TERT is a limiting factor for the appearance of active telomerase. hTR contains template for telomere synthesis, several structural elements that are important for telomerase catalytic activity and hTR folding. The 3’-end region of hTR has the H/ACA motif inherent for small nucleolar RNAs (snoRNAs) and small Cajal body RNAs (scaRNAs). In contrast to classical H/ACA containing snoRNAs and scaRNAs hTR is synthesized by RNA polymerase II from its own promoter as a long precursor. Multiple forms of telomerase RNA that differ at 3’-end were identified, but precise position of the transcription stop of pre-hTR remains unknown. The mature form of hTR is produced by 3'-end truncation and 5'-end modification with a trimethylguanosine (TMG) cap. Expression of polyadenylated hTR-transcript from CMV-, CAG- and PGK-promoters led to accumulation of unprocessed form of hTR. The insertion of U1-terminator just after hTR also inhibited hTR processing. hTR containing various sequences at 3’-end of hTR expressed from polymerase II and polymerase III promoters were processed to different extend. These data and recent investigation of the role of H/ACA domain in telomerase RNA maturation indicate that formation of RNP complex at H/ACA domain is crucial for correct hTR processing. However, the question remains whether hTR maturation involves endonuclease cleavage of primary transcript or it is processed by 3’-end exonucleolytic trimming as in classical snoRNAs. To discriminate between these possibilities we have decided to utilize lentiviral vectors to construct stable cell lines in which a region coding for mature hTR (451nt) and its flanking 424 nt from the 3’-end is followed by fluorescent protein coding region with IRES-element. This system allows us to follow the fate of the 3’-end region of primary hTR transcript. The integrity of hTR was analyzed by qRT-PCR method. cDNA was obtained by primer specific to the reporter coding region and RT-PCR was performed with a set of primers specific to the different part of hTR sequence. It was surprised that level of hTR mature form decreased dramatically in comparison with GFP and IRES- region. The analysis of 3’-boarder of hTR by 3’-RACE technique in our cell lines revealed heterogeneity in 3’-end of hTR. Majority of products contain the 3’-end boarder around 851nucleotides. Taken together our data we could hypothesized that the first event in hTR processing is the endonuclease cleavage after that the exonuclease degrades the pre-hTR to the mature form.