Department of Chemistry, Case Western Reserve University, Cleveland, OH 44106, United States.
Whitehead Institute for Biomedical Research, Cambridge, MA, United States.
J Mol Biol. 2021 Apr 30;433(9):166885. doi: 10.1016/j.jmb.2021.166885. Epub 2021 Mar 5.
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.
7SK 小核 RNA(snRNA) 是一种丰富且广泛表达的非编码 RNA，它通过稳定不同的 7SK-蛋白复合物池来调节 RNA 聚合酶 II(RNAPII) 的活性。流行的模型表明，7SK 的二级结构在其替代 RNA-蛋白池中动态重塑，使得其结构差异调节同源结合伴侣的交换。核 hnRNP A1/A2 蛋白通过需要完整茎环 (SL) 3 结构域的过程影响 7SK snRNA 的生物学功能；然而，hnRNPs 组装到 7SK snRNA 上的分子细节尚未被描述。在这里，我们采用了综合方法来详细描述 7SK-hnRNP A1 复合物。我们表明，未结合的 7SK snRNA 在溶液中至少采用两种主要构象，其结构差异主要定位于 SL2-3 连接区和 SL3 的底部。系统发育分析表明，该区域是 7SK snRNA 中遗传上最保守的特征。通过在存在过量蛋白质的情况下进行 DMS 修饰，我们揭示 hnRNP A1 通过增加邻近潜在结合位点的 RNA 灵活性的机制，选择性地结合到 SL3。量热滴定进一步验证 hnRNP A1-SL3 组装是复杂的，离散结合事件的亲和力受周围 RNA 结构的调节。为了解释这种依赖于上下文的结合现象，我们确定了 SL3 的 3D 模型，以表明它折叠到不同局部环境中最小化 hnRNP A1/A2 结合位点 (5'-Y/RAG-3') 的位置。通过 SEC-MALS-SAXS 解析的 SL3-蛋白复合物证实，多达四个 hnRNP A1 蛋白通过保留该结构域整体结构完整性的相互作用，沿着 SL3 的整个表面结合。总之，这里呈现的综合结果揭示了折叠的 SL3 结构域通过受周围 RNA 结构调节的机制来支架 hnRNP A1/A2-7SK 组装的特定作用。
