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，它通过合成重复的DNA序列来拮抗真核生物复制时染色体末端的不断丢失。它们由RNA（S.酿酒酵母）和几种蛋白质。TLC 1在输出到细胞质中以募集额外的蛋白质用于完全组装之前经历核成熟。成熟的端粒酶随后被重新输入到细胞核中，在那里它实现其功能。我们最近发现，除了RAN依赖性核转运蛋白Crm 1/Xpo 1外，TLC 1还需要mRNA输出机制（例如Mex 67）才能从细胞核中输出（Wu et al. 2014，Cell Reports）。此外，我们未发表的结果进一步揭示了与Mex 67相互作用的三种SR蛋白Np 13、Gbp 2和Hrb 1也与TLC 1相互作用。事实上，它们的基因缺失导致严重的端粒缩短缺陷。引人注目的是，它们的基因缺失都没有显示TLC 1输出缺陷; NPL 3的缺失反而显示核再输入缺陷。这些发现表明这些基因在TLC 1的生命周期中具有重要功能，并强调了其核质穿梭的必要性。在本项目中，我们将分析SR蛋白在TLC 1成熟和转运中的功能，并进行遗传、生化和细胞定位研究。此外，我们将从野生型细胞和从将该RNA捕获在细胞核（例如mex 67 -5）或细胞质（MTR 10的缺失）中的菌株中纯化TLC 1，以在其成熟的不同状态下富集核糖核颗粒。然后将相关的蛋白质纯化并在质谱中鉴定。

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 Moreover, our unpublished results further reveal that the three SR-proteins Npl3, Gbp2 and Hrb1, which interact with Mex67, also interact with TLC1.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