Laboratoire de Biologie Moléculaire Eucaryote du CNRS, UMR5099, IFR109 CNRS, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex 9, France.
Nucleic Acids Res. 2013 Apr;41(8):4686-98. doi: 10.1093/nar/gkt159. Epub 2013 Mar 6.
Through controlling the nuclear level of active positive transcription elongation factor b (P-TEFb), the 7SK small nuclear RNA (snRNA) functions as a key regulator of RNA polymerase II transcription. Together with hexamethylene bisacetamide-inducible proteins 1/2 (HEXIM1/2), the 7SK snRNA sequesters P-TEFb into transcriptionally inactive ribonucleoprotein (RNP). In response to transcriptional stimulation, the 7SK/HEXIM/P-TEFb RNP releases P-TEFb to promote polymerase II-mediated messenger RNA synthesis. Besides transiently associating with HEXIM1/2 and P-TEFb, the 7SK snRNA stably interacts with the La-related protein 7 (Larp7) and the methylphosphate capping enzyme (MePCE). In this study, we used in vivo RNA-protein interaction assays to determine the sequence and structural elements of human 7SK snRNA directing assembly of the 7SK/MePCE/Larp7 core snRNP. MePCE interacts with the short 5'-terminal G1-U4/U106-G111 helix-tail motif and Larp7 binds to the 3'-terminal hairpin and the following U-rich tail of 7SK. The overall RNA structure and some particular nucleotides provide the information for specific binding of MePCE and Larp7. We also demonstrate that binding of Larp7 to 7SK is a prerequisite for in vivo recruitment of P-TEFb, indicating that besides providing stability for 7SK, Larp7 directly participates in P-TEFb regulation. Our results provide further explanation for the frequently observed link between Larp7 mutations and cancer development.
通过控制活跃的正转录延伸因子 b (P-TEFb) 的核水平，7SK 小核 RNA (snRNA) 作为 RNA 聚合酶 II 转录的关键调节剂发挥作用。7SK snRNA 与六亚甲基双乙酰胺诱导蛋白 1/2 (HEXIM1/2) 一起，将 P-TEFb 隔离到转录不活跃的核糖核蛋白 (RNP) 中。响应转录刺激时，7SK/HEXIM/P-TEFb RNP 释放 P-TEFb 以促进聚合酶 II 介导的信使 RNA 合成。除了与 HEXIM1/2 和 P-TEFb 短暂结合外，7SK snRNA 还与 La 相关蛋白 7 (Larp7) 和甲基磷酸封端酶 (MePCE) 稳定相互作用。在这项研究中，我们使用体内 RNA-蛋白质相互作用测定来确定指导人 7SK snRNA 组装 7SK/MePCE/Larp7 核心 snRNP 的序列和结构元件。MePCE 与短的 5'-末端 G1-U4/U106-G111 螺旋-尾基序相互作用，Larp7 结合 7SK 的 3'-末端发夹和随后的 U 丰富尾巴。RNA 整体结构和一些特定核苷酸为 MePCE 和 Larp7 的特异性结合提供了信息。我们还证明 Larp7 与 7SK 的结合是体内募集 P-TEFb 的先决条件，表明除了为 7SK 提供稳定性外，Larp7 还直接参与 P-TEFb 调节。我们的结果进一步解释了 Larp7 突变与癌症发展之间经常观察到的联系。
