Ku can contribute to telomere lengthening in yeast at multiple positions in the telomerase RNP.
Ku can contribute to telomere lengthening in yeast at multiple positions in the telomerase RNP.
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DOI：
10.1261/rna.2483611
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发表时间：
2011-02
期刊：
影响因子：
4.5
通讯作者：
David C. Zappulla;Karen J. Goodrich;J. R. Arthur;Lisa A. Gurski;Elizabeth M Denham;A. Stellwagen;T. Cech
中科院分区：
文献类型：
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作者：
David C. Zappulla;Karen J. Goodrich;J. R. Arthur;Lisa A. Gurski;Elizabeth M Denham;A. Stellwagen;T. Cech
与核糖核蛋白复合物具有高度有序的整体结构（如核糖体）不同，酵母端粒酶似乎受到更松散的约束。在这里，我们调查的Ku亚基内的1157-nt的酵母端粒酶RNA（TLC 1）的定位的重要性。缺失TLC 1 RNA中的48-nt Ku结合发夹（tlc 1 Δ48）会缩短端粒长度，减少具有总染色体重排的细胞的存活，并在断裂的染色体末端重新添加端粒。为了测试Ku在端粒酶RNP中新位置的功能，我们将其结合位点重新引入tlc 1 Δ48 RNA的446或1029位。我们发现，Ku结合到这些重新定位的网站在体内和端粒长度略有增加，但统计学显着。端粒酶通过修复受损的染色体臂来促进具有总染色体重排的细胞存活的能力也部分恢复，而DNA添加到特定染色体断裂的动力学被延迟。在TLC 1中有两个Ku位点导致端粒的可变子集进行性超伸长，这与Ku在端粒酶募集到染色体末端中的作用一致。在TLC 1中的Ku结合位点的数量有助于体内端粒酶RNA丰度，但仅部分负责端粒长度表型。因此，端粒酶RNA水平和端粒长度调节可以通过端粒酶RNA中Ku位点的数量来调节。此外，Ku在端粒酶RNP中的相对定位对于天然端粒长度的维持有很大的灵活性，尽管没有必需的Est 1 p亚基那么大的灵活性。
Unlike ribonucleoprotein complexes that have a highly ordered overall architecture, such as the ribosome, yeast telomerase appears to be much more loosely constrained. Here, we investigate the importance of positioning of the Ku subunit within the 1157-nt yeast telomerase RNA (TLC1). Deletion of the 48-nt Ku-binding hairpin in TLC1 RNA (tlc1Δ48) reduces telomere length, survival of cells with gross chromosomal rearrangements, and de novo telomere addition at a broken chromosome end. To test the function of Ku at novel positions in the telomerase RNP, we reintroduced its binding site into tlc1Δ48 RNA at position 446 or 1029. We found that Ku bound to these repositioned sites in vivo and telomere length increased slightly, but statistically significantly. The ability of telomerase to promote survival of cells with gross chromosomal rearrangements by healing damaged chromosome arms was also partially restored, whereas the kinetics of DNA addition to a specific chromosome break was delayed. Having two Ku sites in TLC1 caused progressive hyperelongation of a variable subset of telomeres, consistent with Ku's role in telomerase recruitment to chromosome ends. The number of Ku-binding sites in TLC1 contributed to telomerase RNA abundance in vivo but was only partially responsible for telomere length phenotypes. Thus, telomerase RNA levels and telomere length regulation can be modulated by the number of Ku sites in telomerase RNA. Furthermore, there is substantial flexibility in the relative positioning of Ku in the telomerase RNP for native telomere length maintenance, although not as much flexibility as for the essential Est1p subunit.