TERRA如何在哺乳類動物細胞中調控端粒的完整性

Abstract

端粒為真核生物染色體末段的特殊線性構造，能夠保護染色體末端的完整性。而端粒的組成包含:重複性序列(TTAGGG)n 與端粒結合蛋白，可以阻止DNA損傷反應的發生、核酸酶降解、以及染色體的融合。在經過每一次循環的DNA複製後，端粒將會變得越來越短，導致「染色體末端複製問題」。端粒酶能夠藉由本身的RNA模板和反轉錄酶(TERT)進行延長端粒區域重複性序列的反應。近期的研究顯示端粒附近的區域能夠轉錄出一段long non-coding RNA (lncRNA)，稱為Telomeric repeat- containing RNA (TERRA)，攜帶著(UUAGGG)n重複性序列在自身的3’-end。 我們的實驗發現到TERRA會與端粒結合蛋白交互作用並且能夠調控端粒的完整性。為了進一步了研究TERRA是如何調控端粒，我們利用antisense-oligo (ASO)的方式將老鼠胚胎幹細胞中的TERRA去除。藉由Immuno-DNA-FISH，我觀察到了在TERRA去除之後，端粒酶在端粒上的比例會下降，間接說明了TERRA可能會幫助端粒酶徵招到染色體尾端的作用。相反的，在ChIP-qPCR的資料中顯示POT1與CTC1在端粒的位置上比率在TERRA去除之後會有所提升，這說明了TERRA會阻止端粒結合蛋白與CST complex結合至端粒上。結合先前的研究發現到CST complex會終止端粒酶在端粒上延長的作用，這個結果推測TERRA幫助端粒酶結合到DNA上可能是藉由抑制CST complex到端粒上的徵招。我們在另外一項實驗中發現到TERRA與端粒酶在細胞週期中的S時期的中期時回提升，並且在細胞週期到G2時期時會下降。此項結果與端粒酶與端粒ＤＮＡ 結合的時間相吻合。總括上述，我們的研究發現TERRA可以調控端粒酶與端粒DNA的結合，並影響端粒蛋白招募到端粒ＤＮＡ上，進而維持端粒的完整性。

Telomeres are nucleoprotein complex composed of repetitive sequence ”TTAGGG” and shelterin proteins (TRF1/TRF2/POT1/TPP1/TIN2). Telomerase (TERT) extends telomeres through binding its RNA template to G-rich strand of telomeres. Recent studies have shown that telomeres transcribe a long non-coding RNA, TERRA, which carries “UUAGGG” repeats at 3’-end. Our previous study has shown that TERRA interacts with shelterin proteins and regulates telomere integrity (Chu et al., 2017). To investigate how TERRA regulates telomere maintenance, I depleted TERRA using anti-sense oligos in mouse embryonic stem cells (mESCs). By immuno-DNA FISH staining, I observed that the number of TERT foci at telomeres was decreased upon TERRA depletion, implying that TERRA promotes the recruitment of telomerase to telomere ends. In contrast, ChIP-qPCR showed that POT1 and CTC1 occupancy at telomeres were elevated upon TERRA depletion, indicating that TERRA prevents CTC1 and shelterin complex from binding to telomere ends. Combined with the previous study showing that CTC1 terminates telomerase, these data suggest that TERRA may assist telomerase loading to DNA by expelling CTC1 from binding to telomeres. In agreement, the levels of both TERRA and telomerase at telomeres are high in Mid-S phase and low in G2 phase in mESCs. Together, this study provides a mechanism in which TERRA regulates the telomere maintenance by balancing the recruitment of telomerase and telomeric DNA binding proteins to telomeres.