Assembly of the U1 snRNP involves interactions with the backbone of the terminal stem of U1 snRNA
Assembly of the U1 snRNP involves interactions with the backbone of the terminal stem of U1 snRNA
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DOI：
10.1261/rna.2136103
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发表时间：
2003-02-01
期刊：
影响因子：
4.5
通讯作者：
Steitz, JA
中科院分区：
文献类型：
--
作者：
McConnell, TS;Lokken, RP;Steitz, JA
核苷酸类似干扰作图(NAIM)是识别蛋白质-RNA相互作用中的RNA功能基团的有效方法。我们在体外检测了组装在修饰的U1小核RNA(SnRNAs)上的颗粒，并检测到两类干扰。第一类影响两个高阶复合体的稳定性，包括环区两个腺苷A65和A70的变化，该环区以前被确定为U1小核核糖核蛋白(SnRNP)特异性U1A蛋白的结合位点。将外环胺添加到A65的第2位会强烈干扰蛋白质的结合，而去除或修饰第6位的外环胺几乎没有影响。A70的修饰表现出相反的效果：允许在位置2添加，但在位置6的外环胺的修饰显著抑制蛋白质结合。这些相互作用在体外SnRNP组装的背景下对U1A-U1SnRNA的识别至关重要，这与先前对分离蛋白的结构研究一致，该分离蛋白的RNA发夹包含U1A结合位点。第二类干扰通过降低Sm核心蛋白结合的稳定性来影响所有部分组装的U1-蛋白质复合体。有趣的是，大多数强干扰发生在U1末端茎环区域的磷酸盐上，而不是在Sm结合部位。这些数据表明，与末端茎环的磷酸骨架的相互作用对于Sm核心蛋白与U1 SnRNA的稳定结合是必不可少的。我们认为所有Sm单链RNA的茎环可能起到夹持Sm蛋白环的作用。
Nucleotide analog interference mapping (NAIM) is a powerful method for identifying RNA functional groups involved in protein-RNA interactions. We examined particles assembled on modified U1 small nuclear RNAs (snRNAs) in vitro and detected two categories of interferences. The first class affects the stability of two higher-order complexes and comprises changes in two adenosines, A65 and A70, in the loop region previously identified as the binding site for the U1 small nuclear ribonucleoprotein (snRNP)-specific U1A protein. Addition of an exocyclic amine to position 2 of A65 interferes strongly with protein binding, whereas removal or modification of the exocyclic amine at position 6 makes little difference. Modifications of A70 exhibit the opposite effects: Additions at position 2 are permitted, but modification of the exocyclic amine at position 6 significantly inhibits protein binding. These interactions, critical for U1A-U1 snRNA recognition in the context of in vitro snRNP assembly, are consistent with previous structural studies of the isolated protein with the RNA hairpin containing the U1 A binding site. The second category of interferences affects all partially assembled U1-protein complexes by decreasing the stability of Sm core protein associations. Interestingly, most strong interferences occur at phosphates in the terminal stem-loop region of U1, rather than in the Sm binding site. These data argue that interactions with the phosphate backbone of the terminal stem loop are essential for the stable association of Sm core proteins with the U1 snRNA. We suggest that the stem loop of all Sm snRNAs may act as a clamp to hold the ring of Sm proteins in place.