Stoichiometry of a regulatory splicing complex revealed by single-molecule analyses.
Stoichiometry of a regulatory splicing complex revealed by single-molecule analyses.
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DOI：
10.1038/emboj.2010.103
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发表时间：
2010-07-07
期刊：
影响因子：
11.4
通讯作者：
Eperon, Ian C.
中科院分区：
文献类型：
--
作者：
Cherny, Dmitry;Gooding, Clare;Eperon, Giles E.;Coelho, Miguel B.;Bagshaw, Clive R.;Smith, Christopher W. J.;Eperon, Ian C.
剪接受许多RNA结合蛋白的复杂相互作用调节。所涉及的分子机制仍然难以捉摸，在很大程度上是因为对调控复合物中蛋白质数量的无知。多聚嘧啶片段结合蛋白（PTB）调节组织特异性剪接，通过侧翼内含子中的富含嘧啶的片段抑制α-原肌球蛋白的外显子3。目前的抑制模型涉及PTB介导的成环或复合物在纤维束之间的传播。为了测试这些模型，我们使用单分子方法来计数结合的PTB分子的数量，通过计数复合物内与PTB连接的GFP分子的漂白步骤的数量和通过分析它们的总排放。这两种方法都表明，五个或六个PTB分子组装。鉴于域结构，这表明分子主要占据多个重叠的潜在网站中的聚嘧啶道，不包括传播模型。作为直接循环的替代方案，我们提出抑制涉及一个多步骤的过程，其中PTB结合形成小的局部循环，为其他蛋白质的招募创造一个平台，使这些循环非常接近。
Splicing is regulated by complex interactions of numerous RNA-binding proteins. The molecular mechanisms involved remain elusive, in large part because of ignorance regarding the numbers of proteins in regulatory complexes. Polypyrimidine tract-binding protein (PTB), which regulates tissue-specific splicing, represses exon 3 of α-tropomyosin through distant pyrimidine-rich tracts in the flanking introns. Current models for repression involve either PTB-mediated looping or the propagation of complexes between tracts. To test these models, we used single-molecule approaches to count the number of bound PTB molecules both by counting the number of bleaching steps of GFP molecules linked to PTB within complexes and by analysing their total emissions. Both approaches showed that five or six PTB molecules assemble. Given the domain structures, this suggests that the molecules occupy primarily multiple overlapping potential sites in the polypyrimidine tracts, excluding propagation models. As an alternative to direct looping, we propose that repression involves a multistep process in which PTB binding forms small local loops, creating a platform for recruitment of other proteins that bring these loops into close proximity.
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DOI：
10.1007/s00249-008-0383-z
发表时间：
2009-04-01
影响因子：
2
作者：
Cherny, Dmitry I.;Eperon, Ian C.;Bagshaw, Clive R.
通讯作者：
Bagshaw, Clive R.
影响因子：
10.5
作者：
LAVIGUEUR, A;LABRANCHE, H;CHABOT, B
通讯作者：
CHABOT, B
影响因子：
4.5
作者：
Clerte, C;Hall, KB
通讯作者：
Hall, KB
影响因子：
4.5
作者：
Amir-Ahmady, B;Boutz, PL;Black, DL
通讯作者：
Black, DL
影响因子：
64.8
作者：
Leake, Mark C.;Chandler, Jennifer H.;Armitage, Judith P.
通讯作者：
Armitage, Judith P.