Conserved N-terminal motifs of telomerase reverse transcriptase required for ribonucleoprotein assembly in vivo
Conserved N-terminal motifs of telomerase reverse transcriptase required for ribonucleoprotein assembly in vivo
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DOI：
10.1074/jbc.m210645200
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发表时间：
2003-02-07
影响因子：
4.8
通讯作者：
Lue, NF
中科院分区：
文献类型：
--
作者：
Bosoy, D;Peng, Y;Lue, NF
端粒酶是一种核糖核蛋白（RNP）逆转录酶，负责维持端粒末端重复序列的一条链。端粒酶复合物的关键蛋白亚基（称为 TERT）具有直接介导核苷酸添加的逆转录酶 (RT) 样基序。 RT 基序位于多肽的 C 末端区域。序列比对还揭示了 TERT N 末端区域存在四个保守基序（称为 GQ、CP、QFP 和 T）。先前已证明酵母 TERT 的 GQ 基序对于端粒酶催化至关重要，并且可能参与 RNP 的形成。在本报告中，我们表明酵母 TERT 的 CP、QFP 和 T 基序中保守残基的替换也会损害端粒维持和端粒酶活性，从而证实了序列比对的有效性。端粒缩短的程度与端粒酶活性、TERT 蛋白和 TERT 相关 TLC1 RNA 水平的降低程度相关。突变蛋白的过度表达不会导致端粒缩短，这意味着组装功能而不是催化功能受到影响。通过比较野生型和过表达菌株中 RNP 形成的效率进一步支持了这一观点。总而言之，我们的结果表明，四个 N 端基序中的三个是体内有效端粒酶 RNP 形成所必需的，但不是端粒酶的酶促功能所必需的。我们还表明，大多数端粒酶相关的 TLC1 RNA 具有比之前报道的更上游的 3' 末端，这与 RNP 成熟过程中的其他加工事件一致。
Telomerase is a ribonucleoprotein (RNP) reverse transcriptase responsible for the maintenance of one strand of the telomere terminal repeats. The key protein subunit of the telomerase complex, known as TERT, possesses reverse transcriptase (RT)-like motifs that directly mediate nucleotide addition. The RT motifs are located in the C-terminal region of the polypeptide. Sequence alignments also revealed the existence of four conserved motifs (named GQ, CP, QFP, and T) in the N-terminal region of TERT. The GQ motif of yeast TERT has been demonstrated previously to be essential for telomerase catalysis and may participate in RNP formation. In this report, we show that substitution of conserved residues in the CP, QFP, and T motifs of yeast TERT also impairs both telomere maintenance and telomerase activity, thus confirming the validity of the sequence alignment. The extent of telomere shortening correlates with the extent of reduction in the level of telomerase activity, TERT protein, and TERT-associated TLC1 RNA. Overexpression of the mutant proteins does not result in telomere shortening, implying that assembly rather than catalytic function was affected. This notion was further supported by comparing the efficiency of RNP formation in the wild type and the overexpression strains. Taken together, our results show that three of the four N-terminal motifs are required for efficient telomerase RNP formation in vivo but not for the enzymatic function of telomerase. We also show that the majority of telomerase-associated TLC1 RNA has a more upstream 3' end than previously reported, consistent with additional processing events during RNP maturation.