HnRNP A1/A2 Proteins Assemble onto 7SK snRNA via Context Dependent Interactions.
HnRNP A1/A2 Proteins Assemble onto 7SK snRNA via Context Dependent Interactions.
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HNRNP A1/A2蛋白通过上下文依赖性相互作用聚集在7SK snRNA上。
DOI：
10.1016/j.jmb.2021.166885
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发表时间：
2021-04-30
影响因子：
5.6
通讯作者：
Tolbert BS
中科院分区：
文献类型：
--
作者：
Luo L;Chiu LY;Sugarman A;Gupta P;Rouskin S;Tolbert BS
7SK小核RNA（7SK small nuclear RNA，snRNA）是一种广泛表达的非编码RNA，通过稳定不同的7SK蛋白复合物来调节RNA聚合酶II（RNAPII）的活性。流行的模型表明，7SK的二级结构是动态重塑其替代RNA蛋白池，使其架构差异调节同源结合伴侣的交换。核hnRNP A1/A2蛋白通过需要完整的茎环（SL）3结构域的过程影响7SK snRNA的生物学;然而，hnRNP组装到7SK snRNA上的分子细节尚未被描述。在这里，我们已经采取了综合的方法来呈现7SK-hnRNP A1复合物的详细描述。我们发现，未结合的7SK snRNA在溶液中采用至少两种主要构象，具有显着的结构差异定位于SL 2 -3接头和SL 3的碱基。系统发育分析表明，该区域是7SK snRNA遗传保守性最低的特征。通过进行DMS修饰与过量的蛋白质的存在下，我们揭示，hnRNP A1通过机制，增加了邻近推定的结合位点的RNA的灵活性与SL 3的选择性结合。量热滴定进一步验证了hnRNPA 1-SL 3组装与由周围RNA结构调节的离散结合事件的亲和力是复杂的。为了解释这种上下文依赖性结合现象，我们确定了SL 3的3D模型，以显示它折叠以在不同的局部环境中定位最小的hnRNP A1/A2结合位点（5 '-Y/RAG-3'）。通过SEC-MALS-SAXS解析的SL 3-蛋白复合物证实，多达四个hnRNP A1蛋白通过相互作用沿着SL 3的整个表面结合沿着，从而保持该结构域的整体结构完整性。总之，这里呈现的集体结果揭示了折叠的SL 3结构域通过由周围RNA结构调节的机制来支撑hnRNP A1/A2-7SK组装的特定作用。
7SK small nuclear RNA (snRNA) is an abundant and ubiquitously expressed noncoding RNA that functions to modulate the activity of RNA Polymerase II (RNAPII) in part by stabilizing distinct pools of 7SK-protein complexes. Prevailing models suggest that the secondary structure of 7SK is dynamically remodeled within its alternative RNA-protein pools such that its architecture differentially regulates the exchange of cognate binding partners. The nuclear hnRNP A1/A2 proteins influence the biology of 7SK snRNA via processes that require an intact stem loop (SL) 3 domain; however, the molecular details by which hnRNPs assemble onto 7SK snRNA are yet to be described. Here, we have taken an integrated approach to present a detailed description of the 7SK-hnRNP A1 complex. We show that unbound 7SK snRNA adopts at least two major conformations in solution, with significant structural differences localizing to the SL2–3 linker and the base of SL3. Phylogenetic analysis indicates that this same region is the least genetically conserved feature of 7SK snRNA. By performing DMS modifications with the presence of excess protein, we reveal that hnRNP A1 binds with selectivity to SL3 through mechanisms that increase the flexibility of the RNA adjacent to putative binding sites. Calorimetric titrations further validate that hnRNP A1-SL3 assembly is complex with the affinity of discrete binding events modulated by the surrounding RNA structure. To interpret this context-dependent binding phenomenon, we determined a 3D model of SL3 to show that it folds to position minimal hnRNP A1/A2 binding sites (5’-Y/RAG-3’) within different local environments. SL3-protein complexes resolved by SEC-MALS-SAXS confirm that up to four hnRNP A1 proteins bind along the entire surface of SL3 via interactions that preserve the overall structural integrity of this domain. In sum, the collective results presented here reveal a specific role for a folded SL3 domain to scaffold hnRNP A1/A2–7SK assembly via mechanisms modulated by the surrounding RNA structure.
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