Isolation and characterization of post-splicing lariat-intron complexes.
Isolation and characterization of post-splicing lariat-intron complexes.
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DOI：
10.1093/nar/gkn1002
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发表时间：
2009-02
影响因子：
14.9
通讯作者：
Ohno M
中科院分区：
文献类型：
--
作者：
Yoshimoto R;Kataoka N;Okawa K;Ohno M
前mrna剪接发生在一个大的复杂剪接体中。对剪接体组装和剪接反应的步骤进行了广泛的分析，并确定了许多涉及的因素。然而，剪接后的内含子转换过程，特别是在脊椎动物中，仍有待研究。在本文中，我们开发了一种双标签亲和纯化方法，用于纯化体外剪接反应获得的lariat内含子rna -蛋白复合物。甘油梯度沉降分析显示，剪接后的内含子复合物至少有两种形式，我们将其命名为“大内含子（IL）”和“小内含子（IS）”复合物。IL复合体含有U2、U5和U6 snrna及其他蛋白质剪接因子，而IS复合体不含此类U snrna或蛋白质。我们还发现，酵母Ntr1的人类同源物TFIP11存在于IL复合物中，缺乏与hPrp43蛋白相互作用结构域的TFIP11突变蛋白在体外引起IL复合物的积累和is复合物形成的减少。综上所述，我们的研究结果强烈表明，TFIP11与hPrp43合作介导了从IL复合体到IS复合体的转变，导致了切除内含子的有效脱支和周转率。
Pre-mRNA splicing occurs in a large complex spliceosome. The steps of both spliceosome assembly and splicing reaction have been extensively analyzed, and many of the factors involved have been identified. However, the post-splicing intron turnover process, especially in vertebrates, remains to be examined. In this paper, we developed a two-tag affinity purification method for purifying lariat intron RNA–protein complexes obtained from an in vitro splicing reaction. Glycerol gradient sedimentation analyses revealed that there are at least two forms of post-splicing intron complexes, which we named the ‘Intron Large (IL)’ and the ‘Intron Small (IS)’ complexes. The IL complex contains U2, U5 and U6 snRNAs and other protein splicing factors, whereas the IS complex contains no such U snRNAs or proteins. We also showed that TFIP11, a human homolog of yeast Ntr1, is present in the IL complex and the TFIP11 mutant protein, which lacks the interaction domain with hPrp43 protein, caused accumulation of the IL complex and reduction of IS complex formation in vitro. Taken together, our results strongly suggest that TFIP11 in cooperation with hPrp43 mediates the transition from the IL complex to the IS complex, leading to efficient debranching and turnover of excised introns.
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影响因子：
5.3
作者：
Deckert, Jochen;Hartmuth, Maus;Luehrmann, Reinhard
通讯作者：
Luehrmann, Reinhard
影响因子：
16
作者：
Hirose, T;Shu, MD;Steitz, JA
通讯作者：
Steitz, JA
影响因子：
64.5
作者：
KONARSKA, MM;SHARP, PA
通讯作者：
SHARP, PA
DOI：
10.1073/pnas.94.22.11798
发表时间：
1997-10-28
影响因子：
11.1
作者：
Arenas, JE;Abelson, JN
通讯作者：
Abelson, JN
影响因子：
64.5
作者：
Denis, MM;Tolley, ND;Weyrich, AS
通讯作者：
Weyrich, AS