Analyses of the stepwise maturation of the yeast telomerase

端粒酶是一种酶促核糖核蛋白复合体，通过合成重复的DNA序列来对抗复制时真核细胞染色体末端的持续丢失。它们由一个RNA(酿酒酵母中的TLC1)和几个蛋白质组成。TLC1在被输出到细胞质以招募额外的蛋白质进行完整组装之前，经历了核成熟。成熟的端粒酶随后被重新输入到细胞核中，在那里它完成其功能。我们最近发现，TLC1除了需要依赖RAN的核粘蛋白CRM1/XPO1外，还需要mRNA输出机制(例如Mex67)(Wu等人)。2014年，《细胞报告》)。此外，我们未发表的结果进一步表明，与Mex67相互作用的三个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。值得注意的是，它们的基因缺失都没有显示出TLC1出口缺陷；而NPL3的缺失则显示了核再进口缺陷。这些发现表明这些基因在TLC1的生命周期中具有至关重要的功能，并强调了其核质穿梭的必要性。在这个项目中，我们将通过遗传学、生化和细胞定位研究来分析SR-蛋白在TLC1成熟和运输中的功能。此外，我们将从野生型细胞和将该RNA捕获到细胞核(例如mex67-5)或细胞质(MTR10缺失)的菌株中纯化TLC1，以在其不同的成熟状态下丰富核糖核文章。然后，相关蛋白质将被提纯，并在质谱学中进行鉴定。

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 Moreover, our unpublished results further reveal that the three SR-proteins Npl3, Gbp2 and Hrb1, which interact with Mex67, also interact with TLC1.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.