Structural Studies of Messenger RNA Maturation and Decay

描述（由申请人提供）：真核生物信使RNA（mRNA）的稳定性部分受共转录和转录后修饰（包括加帽、剪接和聚腺苷酸化）的调节。5'm7 GpppN帽是在转录期间进行的第一个共转录mRNA修饰，并且是有效的前体mRNA剪接、输出、稳定性和翻译所需的。加帽在三个酶促步骤中由RNA三磷酸酶、RNA鸟苷酰转移酶和RNA（鸟嘌呤-N7）甲基转移酶催化，并且加帽装置通过与RNA聚合酶II（RNAPII）的最大亚基的磷酸化C末端结构域直接相互作用而被募集到转录复合物中。加帽装置和RNAPII之间的相互作用也参与转录起始和延伸的激活和抑制，其细节知之甚少。我们提出通过以下目的阐明真核细胞mRNA加工和转录的重要决定子和调控元件：1）帽形成酶彼此复合、与RNAPII复合、与磷酸化RNAPII CTD复合的结构和遗传分析;目的2）结构，遗传，和CTD相互作用蛋白的生物化学表征，所述CTD相互作用蛋白包括哺乳动物加帽酶和酵母CTD磷酸酶和甲基转移酶;目的3）研究Spt 4/Spt 5转录延伸复合体。 RNA帽结构在两种主要的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去腺苷酸化后，Dcp 1/Dcp 2脱帽复合物水解mRNA帽以使5' RNA末端暴露于5'-3 '核糖核酸外切酶活性。在3 '-5'衰变途径中，RNA的外泌体介导的降解在去腺苷化后从3'端发生，最终产生帽结构，其被该途径中的酶水解。预计剩余帽结构的水解消除m7 G帽中间体，其可能充当mRNA翻译、输出和加工因子的潜在抑制剂，所述mRNA翻译、输出和加工因子经由5'm7 G帽识别信使RNA。我们在目标4）中提出阐明5 '-3'和3 '-5 RNA衰变途径中的脱帽酶的结构、机制和调控基础，所述脱帽酶介导与5' m7 GpppN帽或加帽RNA的相互作用并降解所述5'm7 GpppN帽或加帽RNA。

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.