TOE1调控端粒酶活性的生物学功能和分子机制研究

The stability and length of telomere are closely related to human cell senescence. The telomerase is composed of catalytic TERT and non-coding RNA template TERC, responsible for telomere lengthening. The regulatory mechanisms of TERC 3’ processing remain largely unknown. For the first time our group identified a protein called TOE1 (Target of Early Growth Response 1) located in the Cajal Body which interacts with telomerase associated protein complex. Cajal Body is the place where TERC maturates and telomerase assembly. TOE1 was reported as a deadenylase while its target specificity was unclear yet. We hypothesized that TOE1 may regulate telomerase activity via TERC 3’ processing. Our preliminary data found TOE1 is required for the maintainance of telomerase activity in telomerase positive cells. Loss of TOE1 downregulates telomerase activity and decreases the ratio of mature TERC. We will use multiple techniques such as molecular cell biology, biochemistry, protein-RNA interaction genomics as well as high throughput sequencing and bioinformatics, to systemically illustrate the role of TOE1 in regulating telomerase activity as well as the detail molecular mechanism in modulating 3’ processing within the telomerase positive tumor cells and other cells, as well as within the clinical tumor samples. Our study will provide novel mechanism and target for the ageing related disorders such as tumor biogenesis.

人体细胞衰老与端粒的稳定性和长度密切相关。端粒的合成由端粒酶非编码RNA TERC为模板, TERT亚基催化，其中TERC的3’加工机制还不清楚。申请人团队首次鉴定出定位于卡哈尔小体的早期生长应答因子靶蛋白TOE1能够结合端粒酶装配复合物，卡哈尔小体是TERC成熟和端粒酶装配之处。已有证据显示TOE1具脱腺苷酸酶活性，但体内底物特异性仍不清楚。我们假设，TOE1能否通过调控TERC的加工进而影响端粒酶活性？我们初步研究显示TOE1对维持端粒酶阳性细胞的端粒酶活性是必需的。TOE1的缺失下调端粒酶活性，降低成熟TERC的比例。本项目拟用分子细胞生物学、生物化学、蛋白质-RNA组学、高通量测序生物信息学分析等技术，利用端粒酶阳性的肿瘤细胞和其他细胞及临床样品为研究体系，系统阐明TOE1对端粒酶活性调控的生物学功能和影响TERC 3’加工的分子机制，为衰老相关疾病如恶性肿瘤提供新的机制和靶点。

人体细胞衰老与端粒的稳定性和长度密切相关。哺乳动物细胞中，以RNA TERC为模板，端粒酶催化亚基TERT延伸端粒。TERC的3’加工是保证其功能的关键，例如poly(A)特异的核酸酶PARN通过切除poly(A)尾巴而对TERC前体进行加工，避免TERC被降解。然而，TERC的3’端加工成熟的详细机制还不清楚。卡哈尔小体是TERC成熟和端粒酶组装的位置，申请人团队首次鉴定出定位于卡哈尔小体的TOE1能够结合端粒酶复合物。TOE1作为脱腺苷酸酶，其底物特异性还不清楚。敲除细胞实验表明TOE1对维持端粒酶阳性细胞的端粒酶活性和端粒长度是必需的。3’RACE及测序表明TOE1缺失的细胞中含有oligoA以及3’端延伸碱基的TERC前体显著积累。生化实验表明TOE1作为脱腺苷酸酶能直接切割带有OligoA的TERC前体，进一步可以作为3’-5’外切酶修剪带3’端延伸碱基的TERC前体，如果TOE1不能正常工作，滞留的TERC前体会显著引起端粒酶的活性下降。我们系统阐明了TOE1能够作为一个脱腺苷酸酶和3’-5’外切酶参与TERC RNA的精细加工，补充了TERC的3’端加工成熟的详细机制，并为人类7型脑桥小脑发育不良疾病的机制提供了重要思路，TOE1可能作为该疾病诊断和治疗的新靶点。在本项目资助下，我们还进行了TOE1的邻近蛋白组学研究，鉴定出TOE1和端粒酶的新结合蛋白ZC3H15；鉴定出靶向端粒体POT1促进衰老的非编码miR-185；发现了新端粒结合蛋白TRIM28；构建了端粒酶报告细胞系，筛选鉴定出天然产物血根氯铵能够通过抑制端粒酶促进肿瘤细胞衰老；阐明了端粒体蛋白Tin2参与干细胞多能性和分化的作用与机制。共发表7篇论文，申请和授权专利各1项，培养博士研究生3人，硕士研究生1人。