端粒酶核糖核酸在老鼠胚胎幹細胞中的交互作用體

Abstract

端粒是細胞核裡染色體末端的重複序列結構，它可以保護本身序列的完整性並免於和其他染色體融合。當細胞進行分裂時，染色體末端因為沒有辦法複製而造成端粒長度減短，細胞經過多次複製後會因為沒有足夠的端粒保護引發凋亡機制。在細胞中有一種核糖核蛋白複合體叫做端粒酶（telomerase）可以幫助維持端粒長度，其中最重要的兩個結構分別為TERC和TERT，TERC可以當作模板去辨認端粒的結構，並使用TERT的活性去延長端粒長度。在分裂旺盛的細胞例如胚胎幹細胞或生殖細胞中端粒酶的活性普遍比體細胞高，因此這些細胞可以利用端粒酶的延長機制來穩定染色體長度並進行完整的複製。在先前的研究已經指出TERC除了調控端粒的長度之外，也發現它參與其他的調節機制，但是它所扮演的角色仍需要進一步探討。因此我們首先利用iDRiP的方法去看TERC會和哪些蛋白質有交互作用，結果顯示許多蛋白質都參與核糖體合成或是轉錄調控機制，同時也有蛋白質是參與細胞生長與凋零的過程。此外我們也用ChIRP技術去看TERC會和哪些DNA片段鍵結，結果也顯示TERC和核糖體DNA片段以及特定基因上的啟動子或內含子有交互作用，這些結果顯示TERC在核糖體的合成與基因調控機制上扮演一定程度的角色。另外我們從UV-RIP實驗確定RNA聚合酶和TERC有交互作用，並從DNA定序結果分析發現TERC會在基因的啟動子附近以及從染色結果看到TERC有在真染色體上的現象，這些結果都顯示TERC扮演基因調控的角色。最後這些這些實驗結果讓我們發現TERC除了在延長端粒上的機制之外也擔任其他調控機制的角色。

Telomeres are repetitive sequences at the end of the chromosomes, and these structures can protect them from deterioration or fusion with neighboring chromosomes. The ribonucleoprotein complex, telomerase, can prevent end replication problem by its reverse transcriptase activity. Telomerase RNA component (TERC) is one of the key components in the telomerase complex, and it serves as a template to extend telomere structure. Recent studies have shown that TERC is also involved in other cell processes in addition to telomere elongation, but how these mechanisms work is poorly understood. Here we used identification of direct RNA interacting protein (iDRiP) assay to reveal TERC RNA binding proteins and TERC’s potential regulating pathways. The result showed that TERC interacts with many ribonucleoproteins, which are involved in rRNA synthesis, transcription regulation, cell cycle, and telomere maintenance. By chromatin isolation by RNA purification (ChIRP) assay and DNA-seq analyses, we found that TERC also interacts with rDNA locus and DNA binding sites where the products are involved in ribosome synthesis, and apoptosis pathway, suggesting that TERC may regulate ribosome biogenesis and cell proliferation. TERC also binds to promoters and introns, suggesting that TERC participates in gene expression. Furthermore, UV-RNA immunoprecipitation (UV-RIP) experiment shows that RNA polymerase I/II, H3.3 histone variant are associated with TERC in vivo. These data suggest that TERC plays a role in ribosome biogenesis via two distinct mechanisms: contacting ribosomal proteins and transcriptional regulation by binding to RNA polymerase I. TERC also regulates gene expression by targeting to promoters and proteins such as RNA polymerase II and Wdr82. In sum, our study uncovers diverse functions of TERC that is not limited to telomere lengthening.