Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly.
Structural requirements for protein-catalyzed annealing of U4 and U6 RNAs during di-snRNP assembly.
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DOI：
10.1093/nar/gkv1374
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发表时间：
2016-02-18
影响因子：
14.9
通讯作者：
Butcher SE
中科院分区：
文献类型：
--
作者：
Didychuk AL;Montemayor EJ;Brow DA;Butcher SE
在di-snRNP组装期间U4和U6 snRNA的碱基配对需要由U6 snRNP蛋白Prp 24陪伴的RNA结构的大规模重塑。我们研究了U4/U6退火的机制，在体外使用的测定，使可视化的核糖核蛋白复合物和忠实地概括已知的体内决定因素的过程。我们发现，退火，而不是U6 RNA结合，是高度依赖于一个20毫米宽的凹槽表面上的Prp 24的正电性字符。在退火过程中，我们观察到U4和U6 RNA与Prp 24之间形成稳定的三元复合物，表明Prp 24在体内的置换需要额外的因素。稳定U6 '端粒'螺旋的突变使退火速率增加高达15倍，表明端粒形成是U4/U6配对的限速因素。Lsm 2 -8复合物与U6的3′端的微管结合，提供了相当的速率增强。总的来说，这些数据确定了U6 snRNP的结构域，这些结构域对于megaDalton U4/U6.U5 tri-snRNP复合物组装的第一步之一至关重要，并导致了U4/U6配对的动态模型，该模型涉及蛋白质和RNA之间惊人程度的进化协同性。
Base-pairing of U4 and U6 snRNAs during di-snRNP assembly requires large-scale remodeling of RNA structure that is chaperoned by the U6 snRNP protein Prp24. We investigated the mechanism of U4/U6 annealing in vitro using an assay that enables visualization of ribonucleoprotein complexes and faithfully recapitulates known in vivo determinants for the process. We find that annealing, but not U6 RNA binding, is highly dependent on the electropositive character of a 20 Å-wide groove on the surface of Prp24. During annealing, we observe the formation of a stable ternary complex between U4 and U6 RNAs and Prp24, indicating that displacement of Prp24 in vivo requires additional factors. Mutations that stabilize the U6 ‘telestem’ helix increase annealing rates by up to 15-fold, suggesting that telestem formation is rate-limiting for U4/U6 pairing. The Lsm2–8 complex, which binds adjacent to the telestem at the 3′ end of U6, provides a comparable rate enhancement. Collectively, these data identify domains of the U6 snRNP that are critical for one of the first steps in assembly of the megaDalton U4/U6.U5 tri-snRNP complex, and lead to a dynamic model for U4/U6 pairing that involves a striking degree of evolved cooperativity between protein and RNA.