Human telomerase catalytic subunit hTERT is subjected to alternative splicing results in loss of its function and leads to decrease of telomerase activity. However, very little is known about the mechanism of hTERT pre-mRNA alternative splicing. Apoptotic endonuclease EndoG is known to participate this process. The aim of this study was to determine the role of EndoG in regulation of hTERT alternative splicing. Increased expression of b-deletion splice variant was determined during EndoG over-expression in CaCo-2 cell line, after EndoG treatment of cell cytoplasm and nuclei and after nuclei incubation with EndoG digested cell RNA. hTERT alternative splicing was induced by 47-mer RNA oligonucleotide in naked nuclei and in cells after transfection. Identified long non-coding RNA, that is the precursor of 47-mer RNA oligonucleotide. Its size is 1754 nucleotides. Based on the results the following mechanism was proposed. hTERT pre-mRNA is transcribed from coding DNA strand while long non-coding RNA is transcribed from template strand of hTERT gene. EndoG digests long non-coding RNA and produces 47-mer RNA oligonucleotide complementary to hTERT pre-mRNA exon 8 and intron 8 junction place. Interaction of 47-mer RNA oligonucleotide and hTERT pre-mRNA causes alternative splicing.
Kataliticheskaia sub"edinitsa telomerazy cheloveka hTERT podvergaetsia al'ternativnomu splaĭsingu, chto privodit k potere ee funktsiĭ i snizheniiu telomeraznoĭ aktivnosti. Izvestno, chto v al'ternativnom splaĭsinge mRNK hTERT prinimaet uchastie apoptoticheskaia éndonukleaza EndoG. Nami izuchena rol' EndoG v reguliatsii al'ternativnogo splaĭsinga mRNK hTERT. Pokazano, chto sverkhékspressiia EndoG v kletkakh kartsinomy kishechnika CaCo-2, a takzhe obrabotka kletochnykh iader i tsitoplazmy rekombinantnoĭ EndoG i inkubatsiia iader s RNK, rasshcheplennoĭ rekombinantnoĭ EndoG, privodit k povysheniiu urovnia varianta s b-deletsieĭ. V izolirovannykh kletochnykh iadrakh i transfitsirovannykh kletkakh al'ternativnyĭ splaĭsing mRNK hTERT indutsirovalsia 47-chlennym RNK-oligonukleotidom. Identifitsirovana dlinnaia nekodiruiushchaia RNK (1157 nukleotidov), iz kotoroĭ obrazuetsia aktivnyĭ 47-chlennyĭ RNK-oligonukleotid. Predlozhen sleduiushchiĭ mekhanizm splaĭsinga mRNK hTERT: c kodiruiushcheĭ tsepi gena hTERT sinteziruetsia pre-mRNK, a s matrichnoĭ – dlinnaia nekodiruiushchaia RNK; EndoG vyrezaet iz dlinnoĭ nekodiruiushcheĭ RNK 47-chlennyĭ oligonukleotid, komplementarnyĭ pre-mRNK hTERT, v meste soedineniia ékzona 8 i introna 8. Vzaimodeĭstvie étogo oligonukleotida s pre-mRNK hTERT indutsiruet al'ternativnyĭ splaĭsing.
Keywords: CaCo-2; EndoG; alternative splicing; hTERT; telomerase.