Encoded Conformational Dynamics of the HIV Splice Site A3 Regulatory Locus: Implications for Differential Binding of hnRNP Splicing Auxiliary Factors.
Encoded Conformational Dynamics of the HIV Splice Site A3 Regulatory Locus: Implications for Differential Binding of hnRNP Splicing Auxiliary Factors.
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HIV剪接位点A3调控位点的编码构象动力学：hnRNP剪接辅助因子差异结合的意义。
DOI：
10.1016/j.jmb.2022.167728
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发表时间：
2022-09-30
影响因子：
5.6
通讯作者：
Tolbert, Blanton S.
中科院分区：
文献类型：
--
作者：
Chiu, Liang-Yuan;Emery, Ann;Jain, Niyati;Sugarman, Andrew;Kendrick, Nashea;Luo, Le;Ford, William;Swanstrom, Ronald;Tolbert, Blanton S.
关键词：
HIV转录组的选择性剪接是通过顺式调控元件作为增强子或沉默子来控制的，这取决于它们的环境和它们招募的宿主RNA结合蛋白的类型。剪接位点受体A3 （ssA3）是HIV转录组中使用最少的受体位点之一，其活性决定了该mRNA的水平。剪接受体3受顺式调控序列、辅助剪接因子和可能的RNA结构的组合调控。这些多种调控成分协调决定ssA3使用频率的机制尚不清楚。通过核磁共振波谱和系统发育分析，我们发现ssA3调控位点在构象上是异质的，并且包含该位点的序列在大多数HIV分离株中是保守的。尽管存在构象异质性，但在体外观察到的主要茎环（A3SL1）折叠成碱基对，将聚吡啶通道（PPyT）与外显子剪接沉默者2p （ESS2p）元件和一个保守的下游连接子配对。通过核磁共振光谱确定的三维结构进一步表明，A3共识切割位点嵌入在顶端环内独特的立体化学环境中，在那里它被交替的碱基相互作用所包围。尽管ESS2p元件被描述为hnRNP H的受体，但它通过碱基配对被隔离在PPyT的3 '端，在这种情况下，它不能与hnRNP H形成稳定的复合物。相比之下，hnRNP A1直接结合位于顶端环内的A3共识切割位点，这表明它可以直接调节U2AF的组装。旨在破坏PPyT:ESS2p螺旋结构稳定的序列突变导致hiv感染细胞内ssA3的使用增加，这与PPyT更容易被U2AF识别一致。引入下游ESS2元件的其他突变与ESS2p协同作用，导致ssA3的使用进一步增加。综上所述，我们的工作提供了一个统一的画面，通过顺式调控序列，剪接辅助因子和RNA结构合作提供对ssA3的严格控制。我们将其描述为限制PPyT进入的配对锁定机制，并假设它的作用是调节围绕ssA3调控位点的异质结构的一个子集。
Alternative splicing of the HIV transcriptome is controlled through cis regulatory elements functioning as enhancers or silencers depending on their context and the type of host RNA binding proteins they recruit. Splice site acceptor A3 (ssA3) is one of the least used acceptor sites in the HIV transcriptome and its activity determines the levels of tat mRNA. Splice acceptor 3 is regulated by a combination of cis regulatory sequences, auxiliary splicing factors, and presumably RNA structure. The mechanisms by which these multiple regulatory components coordinate to determine the frequency in which ssA3 is utilized is poorly understood. By NMR spectroscopy and phylogenetic analysis, we show that the ssA3 regulatory locus is conformationally heterogeneous and that the sequences that encompass the locus are conserved across most HIV isolates. Despite the conformational heterogeneity, the major stem loop (A3SL1) observed in vitro folds to base pair the Polypyrimdine Tract (PPyT) to the Exon Splicing Silencer 2p (ESS2p) element and to a conserved downstream linker. The 3D structure as determined by NMR spectroscopy further reveals that the A3 consensus cleavage site is embedded within a unique stereochemical environment within the apical loop, where it is surrounded by alternating base-base interactions. Despite being described as a receptor for hnRNP H, the ESS2p element is sequestered by base pairing to the 3’ end of the PPyT and within this context it cannot form a stable complex with hnRNP H. By comparison, hnRNP A1 directly binds to the A3 consensus cleavage site located within the apical loop, suggesting that it can directly modulate U2AF assembly. Sequence mutations designed to destabilize the PPyT:ESS2p helix results in an increase usage of ssA3 within HIV-infected cells, consistent with the PPyT becoming more accessible for U2AF recognition. Additional mutations introduced into the downstream ESS2 element synergize with ESS2p to cause further increases in ssA3 usage. When taken together, our work provides a unifying picture by which cis regulatory sequences, splicing auxiliary factors and RNA structure cooperate to provide stringent control over ssA3. We describe this as the pair-and-lock mechanism to restrict access of the PPyT, and posit that it operates to regulate a subset of the heterogenous structures encompassing the ssA3 regulatory locus.
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