A group II intron-encoded maturase functions preferentially In cis and requires both the reverse transcriptase and X domains to promote RNA splicing
A group II intron-encoded maturase functions preferentially In cis and requires both the reverse transcriptase and X domains to promote RNA splicing
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DOI：
10.1016/j.jmb.2004.05.004
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发表时间：
2004-07-02
影响因子：
5.6
通讯作者：
Lambowitz, AM
中科院分区：
文献类型：
--
作者：
Cui, XX;Matsuura, M;Lambowitz, AM
移动的组11内含子编码具有逆转录酶活性和成熟酶活性的蛋白质，逆转录酶活性在内含子移动中起作用，成熟酶活性通过稳定内含子RNA的催化活性结构促进RNA剪接。先前对乳酸乳球菌L1.LtrB内含子的研究提出了一种模型，其中内含子编码的蛋白质首先结合到内含子亚结构域DIVa中的高亲和力结合位点，这是其自身编码区开始处的特异质结构，然后与保守的催化核心区进行额外的接触以稳定活性RNA结构。在这里，我们开发了一种大肠杆菌的遗传分析，链接的剪接的L1.LtrB内含子的表达的绿色荧光蛋白和使用，它来研究在体内剪接的野生型和突变体内含子和划定区域的成熟酶所需的剪接。我们的研究结果表明，成熟酶的功能最有效地表达时，顺式从相同的转录作为内含子RNA。与先前的体外测定一致，我们发现DIVa中的高亲和力结合位点是体内L1.LtrB内含子有效剪接所需的，但在不存在DIVa的情况下，通过成熟酶与催化核心的直接结合发生6-10%的残余剪接。通过统计分析从诱变PCR（“单基因进化”）生成的库中分离的功能性LtrA变体中的错义突变与沉默突变的比率，鉴定了成熟酶的关键区域。该分析表明，逆转录酶结构域和结构域X（可能对应于逆转录酶拇指）都是RNA剪接所需的，而C-末端DNA结合和DNA内切酶结构域则不是必需的。在逆转录酶结构域中，成熟酶活性的最关键区域包括在HIV-1逆转录酶中的模板和引物结合中起作用的手指和手掌的部分，但逆转录酶活性位点的完整性不是必需的。LtrA突变体的生化分析表明，反转录酶结构域的N末端是必需的内含子RNA的高亲和力结合，可能通过直接与DIVa的相互作用，而结构域X的部分与催化核心的保守区域相互作用。我们的研究结果支持这一假设，即内含子编码的蛋白质通过使用，至少部分地，最初用于识别作为逆转录模板的内含子RNA的相互作用来适应剪接功能。(C)2004爱思唯尔有限公司保留所有权利。
Mobile group 11 introns encode proteins with both reverse transcriptase activity which functions in intron mobility, and maturase activity, which promotes RNA splicing by stabilizing the catalytically active structure of the intron RNA. Previous studies with the Lactococcus lactis L1.LtrB intron suggested a model in which the intron-encoded protein binds first to a high-affinity binding site in intron subdomain DIVa, an idiosyncratic structure at the beginning of its own coding region, and then makes additional contacts with conserved catalytic core regions to stabilize the active RNA structure. Here, we developed an Escherichia coli genetic assay that links the splicing of the L1.LtrB intron to the expression of green fluorescent protein and used, it to study the in vivo splicing of wild-type and mutant introns and to delineate regions of the maturase required for splicing. Our results show that the maturase functions most efficiently when expressed in cis from the same transcript as the intron RNA. In agreement with previous in vitro assays, we find that the high-affinity binding site in DIVa is required for efficient splicing of the L1.LtrB intron in vivo, but in the absence of DIVa, 6-10% residual splicing occurs by the direct binding of the maturase to the catalytic core. Critical regions of the maturase were identified by statistically analyzing ratios of missense to silent mutations in functional LtrA variants isolated from a library generated by mutagenic PCR ("unigenic evolution"). This analysis shows that both the reverse transcriptase domain and domain X, which likely corresponds to the reverse transcriptase thumb, are required for RNA splicing, while the C-terminal DNA-binding and DNA endonuclease domains are not required. Within the reverse transcriptase domain, the most critical regions for maturase activity include parts of the fingers and palm that function in template and primer binding in HIV-1 reverse transcriptase, but the integrity of the reverse transcriptase active site is not required. Biochemical analysis of LtrA mutants indicates that the N terminus of the reverse transcriptase domain is required for high-affinity binding of the intron RNA, possibly via direct interaction with DIVa, while parts of domain X interact with conserved regions of the catalytic core. Our results support the hypothesis that the intron-encoded protein adapted to function in splicing by using, at least in part, interactions used initially to recognize the intron RNA as a template for reverse transcription. (C) 2004 Elsevier Ltd. All rights reserved.