Structural Studies of Messenger RNA Maturation and Decay

描述(申请人提供)：真核信使RNA(MRNA)的稳定性部分由共转录和转录后修饰调节，包括封端、剪接和多聚腺苷化。5‘m7GpppN帽是转录过程中第一次共转录的mRNA修饰，是有效的前mRNA剪接、输出、稳定性和翻译所必需的。封顶由RNA三磷酸酶、RNA鸟苷酸转移酶和RNA(鸟嘌呤-N7)甲基转移酶催化，封顶装置通过与RNA聚合酶II最大亚单位(RNAPII)的磷酸化C-末端结构域直接相互作用而被招募到转录复合体中。封顶装置和RNAPII之间的相互作用也参与了转录起始和延伸的激活和抑制，其细节尚不清楚。我们建议通过以下方面来阐明真核mRNA加工和转录的重要决定因素和调控元件：AIM 1)在相互作用的、与RNAPII的复合体中、在与磷酸化的RNAPII CTD的复合体中的帽子形成酶的结构和遗传分析；AIM 2)CTD相互作用蛋白的结构、遗传和生化特征，包括哺乳动物的CAP酶、酵母CTD磷酸酶和甲基转移酶；AIM 3)Spt4/Spt5转录延伸复合体的特征。 RNA帽结构在这两条主要的RNA衰变途径中也起着关键作用。在5‘-3’衰变途径中多聚腺苷酸化的mRNA去烯化后，Dcp1/Dcp2去壳复合体水解mRNA帽，使5‘RNA端暴露于5’-3‘外切核糖核酸酶活性。在3‘-5’衰变途径中，外切体介导的RNA降解发生在去烯化后的3‘端，最终产生一个帽子结构，该结构被该途径中的酶所降解。残余帽结构的水解预计会消除m7G帽中间体，这些中间体可能是mRNA翻译、输出和通过5‘m7G帽识别信使RNA的加工因子的潜在抑制剂。我们在AIM 4)中建议阐明5‘-3’和3‘-5 RNA衰退途径中的结构、机制和调节基础--介导与5’m7GpppN帽或帽RNA的相互作用并降解这些酶。

DESCRIPTION (provided by applicant): The stability of eukaryotic messenger RNA (mRNA) is regulated in part by co-transcriptional and posttranscriptional modifications that include capping, splicing, and polyadenylation. The 5' m7GpppN cap is the first co-transcriptional mRNA modification made during transcription and is required for efficient premRNA splicing, export, stability, and translation. Capping is catalyzed in three enzymatic steps by RNA triphosphatase, RNA guanylyltransferase, and RNA (guanine-N7) methyltransferase and the capping appartus is recruited to the transcription complex by direct interaction with the phosphorylated C-terminal domain of the largest subunit of RNA polymerase II (RNAPII). Interactions between the capping apparatus and RNAPII are also involved in activation and repression of transcription initiation and elongation, the details of which are poorly understood. We propose to illuminate important determinants and regulatory elements for eukarytic mRNA processing and transcription through Aim 1) structural and genetic analysis of cap forming enzymes in complex with one another, in complex with RNAPII, in complex with the phosphorylated RNAPII CTD; Aim 2) the structural, genetic, and biochemical characterization of CTD interacting proteins that include mammalian capping enzymes and yeast CTD phosphatase and methyltransferase enzymes; Aim 3) characterization of the Spt4/Spt5 transcriptional elongation complex. The RNA cap structure also plays a critical role in both major RNA decay pathways. After deadenylation of polyadenylated mRNA in the 5'-3' decay pathway, the Dcp1/Dcp2 decapping complex hydrolyzes the mRNA cap to expose the 5' RNA end to 5'-3' exoribonuclease activities. In the 3'-5' decay pathway, exosome-mediated degradation of RNA occurs from the 3' end after deadenylation, ultimately generating a cap structure that is hydrolyzed by enzymes in this pathway. Hydrolysis of the residual cap structure is predicted to eliminate m7G cap intermediates that might serve as potential inhibitors of mRNA translation, export, and processing factors that recognize messenger RNA via the 5' m7G cap. We propose in Aim 4) to illuminate the structural, mechanistic, and regulatory basis-for decapping enzymes in both 5'-3' and 3'-5 RNA decay pathways that mediate interactions with and degrade the 5' m7GpppN cap or capped RNA.