Schizosaccharomyces pombe Ccq1 and TER1 bind the 14-3-3-like domain of Est1, which promotes and stabilizes telomerase-telomere association
Schizosaccharomyces pombe Ccq1 and TER1 bind the 14-3-3-like domain of Est1, which promotes and stabilizes telomerase-telomere association
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DOI：
10.1101/gad.181826.111
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发表时间：
2012-01-01
影响因子：
10.5
通讯作者：
Zakian, Virginia A.
中科院分区：
文献类型：
--
作者：
Webb, Christopher J.;Zakian, Virginia A.
端粒酶蛋白 Est1 存在于多种生物体中，包括粟酒裂殖酵母、人类和酿酒酵母，但其功能仅在酿酒酵母中得到了仔细研究，它是端粒酶的招募剂/激活剂。在这里，我们证明了粟酒裂殖酵母 Est1 是端粒酶全酶的端粒关联所必需的，这表明它也具有招募作用。它与端粒的关联依赖于催化亚基 Trt1 和端粒蛋白 Ccq1。令人惊讶的是，Est1 端粒结合仅部分依赖于 TER1（端粒酶 RNA），尽管 Est1 结合了 TER1 的 415-507 位核苷酸。 ter1-Delta 415-507菌株在S期后期具有短端粒和非常低的Est1和Trt1端粒关联，但没有衰老。对减少 Est1-TER1 相互作用的突变的公正搜索发现，仅在 Est1 14-3-3 样结构域（一种磷酸丝氨酸结合基序）中存在突变，这是具有 RNA 结合活性的 14-3-3 样结构域的第一个例子。这些突变还减少了 Est1-Ccq1 结合。其中一种突变体阻止 Est1 端粒关联并导致端粒丢失和缓慢衰老，与 ccq1 Delta 类似。我们认为 Est1-Ccq1 相互作用对于端粒酶招募至关重要，而 Est1-TER1 相互作用在 Ccq1 介导的招募下游发挥作用，以稳定端粒上的全酶。
The telomerase protein Est1 exists in multiple organisms, including Schizosaccharomyces pombe, humans, and Saccharomyces cerevisiae, but its function has only been closely examined in S. cerevisiae, where it is a recruiter/activator of telomerase. Here, we demonstrate that S. pombe Est1 was required for the telomere association of the telomerase holoenzyme, suggesting that it too has a recruitment role. Its association with telomeres was dependent on Trt1, the catalytic subunit, and Ccq1, a telomeric protein. Surprisingly, Est1 telomere binding was only partially dependent on TER1, the telomerase RNA, even though Est1 bound nucleotides 415-507 of TER1. A ter1-Delta 415-507 strain had short telomeres and very low Est1 and Trt1 telomere association in late S phase but did not senesce. An unbiased search for mutations that reduced Est1-TER1 interaction identified mutations only in the Est1 14-3-3-like domain, a phosphoserine-binding motif, the first example of a 14-3-3-like domain with RNA-binding activity. These mutations also reduced Est1-Ccq1 binding. One such mutant prevented Est1 telomere association and caused telomere loss and slow senescence, similar to ccq1 Delta. We propose that the Est1-Ccq1 interaction is critical for telomerase recruitment, while the Est1-TER1 interaction acts downstream from Ccq1-mediated recruitment to stabilize the holoenzyme at the telomere.