Analyses of the stepwise maturation of the yeast telomerase

端粒酶是一种酶促核糖核蛋白复合物，通过合成重复的DNA序列来对抗真核生物染色体末端在复制时的持续损失。它们由一种RNA（酿酒葡萄球菌中的TLC1）和几种蛋白质组成。TLC1在输出到细胞质中招募额外的蛋白质进行完整组装之前经历核成熟。成熟的端粒酶随后被重新输入细胞核，在那里完成它的功能。我们最近的研究表明，TLC1需要mRNA输出机制（例如，Mex67）以及ran依赖的核丝蛋白Crm1/Xpo1才能从细胞核输出（Wu et al. 2014, Cell Reports）。此外，我们未发表的研究结果进一步表明，与Mex67相互作用的三种sr蛋白Npl3、Gbp2和Hrb1也与TLC1相互作用。事实上，它们的基因缺失会导致严重的端粒缩短缺陷。引人注目的是，他们的基因缺失都没有显示出TLC1输出缺陷；NPL3缺失反而显示出核再进口缺陷。这些发现表明这些基因在TLC1的活周期中起着至关重要的作用，并强调了其核细胞质穿梭的必要性。在本项目中，我们将通过遗传、生化和细胞定位研究来分析sr -蛋白在TLC1成熟和转运中的功能。此外，我们将从野生型细胞和将该RNA捕获在细胞核（例如mex67-5）或细胞质（删除MTR10）中的菌株中纯化TLC1，以丰富其成熟不同状态的核糖核文章。相关蛋白将在质谱法中纯化和鉴定。

Telomerases are enzymatic ribonucleoprotein complexes that antagonize the constant loss of the eukaryotic chromosome ends upon replication by the synthesis of repetitive DNA sequences. They are composed of an RNA (TLC1 in S. cerevisiae) and several proteins. TLC1 undergoes nuclear maturation before it is exported into the cytoplasm to recruit additional proteins for complete assembly. The mature telomerase is subsequently re-imported into the nucleus where it fulfills its function.We have recently shown that TLC1 requires the mRNA export machinery (e.g. Mex67) in addition to the Ran-dependent karyopherin Crm1/Xpo1 for its export from the nucleus (Wu et al. 2014, Cell Reports). Moreover, our unpublished results further reveal that the three SR-proteins Npl3, Gbp2 and Hrb1, which interact with Mex67, also interact with TLC1. In fact, their gene deletions result in severe telomere shortening defects. Strikingly, none of their gene deletions shows TLC1 export defects; Deletion of NPL3 rather shows nuclear re-import defects. These findings indicate crucial functions for these genes in the live cycle of TLC1 and underline the necessity for its nucleo-cytoplasmic shuttling. In this project we will analyze the functions of the SR-proteins on TLC1 maturation and transport with genetic, biochemical and cellular localization studies. Moreover, we will purify TLC1 from wild type cells and from strains that trap this RNA in the nucleus (e.g. mex67-5) or in the cytoplasm (Deletion of MTR10) to enrich the ribonucleoparticle at different states of its maturation. The associated proteins will then be purified and identified in mass spectrometry.