Identification of specific let-7 microRNA binding complexes in Caenorhabditis elegans
Identification of specific let-7 microRNA binding complexes in Caenorhabditis elegans
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DOI：
10.1261/rna.551208
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发表时间：
2008-10-01
期刊：
影响因子：
4.5
通讯作者：
Slack, Frank J.
中科院分区：
文献类型：
--
作者：
Chan, Shih-Peng;Ramaswamy, Gopalakrishna;Slack, Frank J.
人们对microRNA的形成和功能所需的蛋白质复合体知之甚少。在这里，我们使用天然凝胶电泳法来鉴定秀丽线虫的miRNA核糖核蛋白复合体(MiRNPs)。我们的数据显示，在体外，有多个不同的miRNP组装在let-7 miRNA上。这些复合体的形成会受到alg-1或alg-2零突变的影响，但不会被消除。最大的络合物(M*)估计分子质量为>；669 kDa，与已知的RISC因子ALG-1、VIG-1和TSN-1共分。M*复合体和两个分子量类似的复合体M3和M4也组装在我们实验中使用的所有其他miRNAs上。两个较小的复合体M1(类似于160 kDa)和M2(；250 kDa)组装在let-7 miRNAs家族的成员上，而不是Lin-4或mir-234，它们的形成高度依赖于let-7 59种子区域的特定序列。此外，通过紫外光触发的交联检测到一种未知的蛋白质p40，它只出现在M1和M2复合体中，而不是LIN-4。P40与let-7的交联性也依赖于let-7序列。在所有LET-7结合复合体和LIN-4交联产物中都检测到另一种未知蛋白质p13。我们的数据表明，除了存在于某些大小类似于RISC的大小的miRNP中外，let-7 miRNA还与特定的结合蛋白组装，形成不同的小复合体。
Little is known about the protein complexes required for microRNA formation and function. Here we used native gel electrophoresis to identify miRNA ribonucleoprotein complexes (miRNPs) in Caenorhabditis elegans. Our data reveal multiple distinct miRNPs that assemble on the let-7 miRNA in vitro. The formation of these complexes is affected but not abolished by alg-1 or alg-2 null mutations. The largest complex (M*) with an estimated molecular mass of >669 kDa cofractionates with the known RISC factors ALG-1, VIG-1, and TSN-1. The M* complex and two complexes, M3 and M4, with similar molecular weights of similar to 500 kDa, also assemble on all other miRNAs used in our experiments. Two smaller complexes, M1 (similar to 160 kDa) and M2 (; 250 kDa), assemble on the members of the let-7 miRNAs family but not lin-4 or mir-234, and their formation is highly dependent on specific sequences in the 59 seed region of let-7. Moreover, an unidentified protein, p40, which only appears in the M1 and M2 complexes, was detected by UV triggered cross-linking to let-7 but not to lin-4. The cross-linking of p40 to let-7 is also dependent on the let-7 sequence. Another unidentified protein, p13, is detected in all let-7 binding complexes and lin-4 cross-linked products. Our data suggest that besides being present in certain large miRNPs with sizes similar to reported RISC, the let-7 miRNA also assembles with specific binding proteins and forms distinct small complexes.