探討酵母菌中長鏈非編碼核醣核酸TERRA影響端粒重組之機制

Mechanistic analysis of long non-coding RNA TERRA affects telomere recombination in Saccharomyces cerevisiae

詹炘儒(Hsin-Ru Chan)

指導教授 ： 林敬哲

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2017年)

https://doi.org/10.6342/NTU201703424

摘要

端粒是位於染色體末端特殊的核蛋白結構，其功能在於阻止染色體末端遭受核酸酶降解、避免染色體末端融合，並且防止染色體不正常的修復和重組。在酵母菌Saccharomyces cerevisiae中，其端粒是由長度250至300鹼基對、TG1-3/C1-3A的重複性序列所組成，在正常狀況下，酵母菌藉由端粒酶維持端粒的長度，端粒酶是由一條 RNA與蛋白質次單元組成的複合體，其利用RNA作為合成端粒序列的模板，再用反轉錄酶的活性合成新的端粒DNA序列，然而在失去端粒酶活性的酵母菌中，端粒會隨著複製次數增加而逐漸縮短，接著進入老化階段(senescence)，此時有一群的酵母菌能夠藉由一種稱為端粒重組(telomere recombination)的方式維持端粒長度，撐過senescence而存活下來，這與人類癌細胞使用ALT (alternative lengthening of telomeres)維持端粒長度的方式非常相似，然而目前對於端粒重組的機制仍不甚清楚。在我們先前的研究發現一種稱為TERRA的telomeric long noncoding RNA會參與在端粒重組的過程中，TERRA RNA和端粒DNA形成RNA:DNA hybrid，在端粒酶缺失的酵母菌中TERRA會促使端粒重組的發生，因此我們想近一步探討TERRA到底是如何引發端粒重組的發生。利用基因剔除的技術我們發現參與在nucleotide excision repair、RNA polymerase regulation和chromatin remodeling pathway的基因對於端粒重組的發生影響不大，然而當剔除5' flap endonuclease RAD27基因後發現端粒重組提早發生，而且過量表現RNase H能夠抑制端粒重組提早發生的現象，顯示rad27 deletion所造成的端粒重組異常可能和TERRA有關。接著我們發現短片段端粒DNA會大量累積在rad27 deletion的酵母菌中，而過量表現5'-3' exonuclease/flap-endonuclease EXO1能抑制rad27 deletion所造成的端粒重組異常。接著利用偵測total TERRA level和DNA-IP的方式觀察到rad27 deletion會造成TERRA累積在端粒，顯示Rad27可能參與在處理TERRA結構的過程中，於是我們利用生化分析的方式將Rad27蛋白和R-loop結構在in vitro條件下進行反應，從初步結果發現Rad27可以切R-loop結構，但Rad27D179A endonuclease dead mutants無法切R-loop結構。這些結果顯示Rad27可能具有切除TERRA並調控端粒重組的新功能。結合以上結果和目前已知rad27 mutants導致人類細胞癌化以及遺傳性疾病的發生，我們的研究賦予Rad27在端粒重組甚至疾病致病機轉一個全新的角色。

關鍵字

端粒重組

並列摘要

Telomeres are nucleoprotein structures located at both ends of eukaryotic linear chromosomes, which protect chromosome ends from nucleolytic degradation, inter-chromosomal fusion, and unnecessary repair-recombination. In Saccharomyces cerevisiae, the telomeric DNA are 250-300 base pairs TG1-3/C1-3A repetitive sequences. In general condition, the telomere length of S. cerevisiae is maintained by a ribonucleoprotein (RNP) called telomerase, which uses its RNA component to synthesize new telomeric DNA. However, in S. cerevisiae lacking telomerase, a recombination-based mechanism is used to maintain telomere length, which is similar to alternative lengthening of telomeres (ALT) adapted by some cancer cells. Although the mechanism of how telomerase extends telomere length is well characterized, the mechanism of recombination-based ALT is still unclear. A telomeric long noncoding RNA termed telomeric repeat-containing RNA (TERRA) was identified in both mammalian cells and yeast. Using yeast S. cerevisiae as a model, our previous results show that TERRA was involved in telomere recombination, suggesting that TERRA formed RNA:DNA hybrid with telomeric DNA to stimulate telomere recombination in cells lacking telomerase. Here we focus on dissecting the mechanism of how TERRA stimulates telomere recombination. Analysis by genetic approaches showed that genes involved in nucleotide excision repair, RNA polymerase regulation and chromatin remodeling pathways play less significant roles in TERRA-mediated recombination. Surprisingly, we found deletion of RAD27, a 5' flap endonuclease required for Okazaki fragment processing and several repair pathways, induced early telomere recombination in a RNase H-dependent manner. Accumulation of short telomeric DNA fragments, a characteristic signature of ALT cells, was also observed in this deletion. Overexpression of EXO1 (encodes flap-endonuclease) and RNH1 (encodes RNase H) can rescue rad27 deletion defects. Further analysis showed that both total TERRA level and RNA:DNA hybrids accumulated in rad27 deletions. Preliminary biochemical analysis proved that Rad27 seemed to have the ability to cut R-loop substrates, but Rad27D179A endonuclease dead mutants failed to process R-loop substrates. These results reveal a new mechanism of how Rad27 processes and modulates long non-coding RNA TERRA to mediate telomere recombination. Since yeasts and human have very similar telomere structures, and rad27 mutants had been shown to occur in human tumors and inherited human diseases, our analyses highlight a possible pathway of telomere recombination and pathogenesis for the rad27 mutant diseases.

並列關鍵字

TERRA ； telomere recombination

參考文獻

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