Nonsense-mediated mRNA decay and nonsense-mediated mRNA decay factors

 描述（由申请人提供）： 这种竞争性的更新建议继续我们的工作对哺乳动物蛋白质编码基因的调节，重点是转录后过程，特别是无义介导的mRNA衰变（NMD）。NMD通常在翻译的先锋轮期间在外显子连接复合物（EJC）的上游足够终止时发生。我们将先驱翻译起始复合物定义为由帽结合蛋白异二聚体CBP 80-CBP 20结合，并且如果mRNA来源于前体mRNA剪接，则由一个或多个EJC结合，并且我们已经表明CBP 80和EJC都促进NMD。在过去的~4年中，我们继续描述NMD过程中代表先驱翻译启动复杂性的变化。例如，我们已经表明，关键的NMD因子，ATP依赖性RNA解旋酶称为UPF 1，结合大多数，如果不是所有的细胞转录在一个低水平非特异性。然而，一系列精心设计的步骤导致NMD靶标结合的UPF 1通过磷酸化活化。我们已经发现磷酸化的UPF 1特异性地结合到NMD靶向3 '非翻译区（3' UTR），并且与稳态UPF 1（其在很大程度上是低磷酸化的）不同，提供了天然细胞NMD的第一个可靠标识符 目标的我们还证明了常用化疗药物引起的细胞DNA损伤触发了半胱天冬酶介导的UPF 1切割，从而产生显性负C端切割产物;然后该产物下调了NMD的效率，从而上调了我们使用转录组深度测序发现的一系列天然NMD靶标，包括编码促肿瘤蛋白的靶标。事实上，细胞可以在暴露于NMD抑制剂后进行更快和更有效的细胞凋亡，然后在化疗前将其撤出。通过与辛格实验室在她和我一起工作的最后一年，佐藤哈娜使用了新的辛格实验室技术，跟踪完整细胞中的单个转录本，以表明含有提前终止密码子（PTC）的β-珠蛋白mRNA的NMD发生在核膜的细胞质侧，而不是在核质中或释放到细胞质中后。在其他研究中，我们合作研究了在培养细胞和大鼠中人类UPF 1表达如何分别克服由肌萎缩侧索硬化症相关蛋白引起的细胞毒性或瘫痪。在这项提议中，AIM 1将进行全基因组清查，以使用单倍体遗传学来定义新的NMD因子。这是对我们正在进行的NMD机制研究的补充。目的2是根据我们未发表的数据确定NMD因素的新作用。我们的目的是研究（i）UPF 1与Tudor-SN在microRNA衰变中的假定作用，（ii）CBP 80在PGC 1 β介导的雌激素受体和孤儿雌激素相关受体激活基因的上调中的作用，以及（iii）UPF 1与FMRP、FRX 1和FXR 2在脆性X智力低下综合征中对mRNA代谢的作用。目的3是开发治疗许多不同的显性遗传性疾病的药物，这些疾病是由于PTC不能触发NMD并产生有毒的截短蛋白。

 DESCRIPTION (provided by applicant): This competitive renewal proposes to continue our work on the regulation of mammalian protein-encoding genes with a focus on post-transcriptional processes and, in particular, nonsense-mediated mRNA decay (NMD). NMD generally occurs when translation terminates sufficiently upstream of an exon-junction complex (EJC) during a pioneer round of translation. We define the pioneer translation initiation complex as bound by the cap-binding protein heterodimer CBP80-CBP20 and, if the mRNA derived from pre-mRNA splicing, one or more EJCs, and we have shown that both CBP80 and EJCs promote NMD. Over the last ~4 years we have continued to characterize changes that typify the pioneer translation initiation complex during the process of NMD. For example, we have shown that the key NMD factor, the ATP-dependent RNA helicase called UPF1, binds to most if not all cellular transcripts at a low level nonspecifically. However, a series of carefully choreographed steps leads to the activation of NMD target-bound UPF1 by phosphorylation. We have found that phosphorylated UPF1 binds specifically to NMD-target 3'-untranslated regions (3'UTRs) and, unlike steady-state UPF1, which is largely hypophosphorylated, provides the first reliable identifier of natural cellular NMD targets. We have also demonstrated that cellular DNA damage by commonly used chemotherapeutics triggers the caspase-mediated cleavage of UPF1 so as to generate a dominant-negative C-terminal cleavage product; this product then downregulates the efficiency of NMD so as to upregulate a battery of natural NMD targets that we have found using transcriptome deep-sequencing include those encoding apoptotic-promoting proteins. In fact, cells can be primed to undergo faster and more efficient apoptosis upon exposure to an NMD inhibitor followed by its withdrawal prior to chemotherapy. By working with the Singer lab during her last year with me, Hana Sato used new Singer-lab technologies that track individual transcripts in intact cells to show that the NMD of premature termination codon (PTC)-containing ß-globin mRNA occurs on the cytoplasmic-side of the nuclear envelop and not in the nucleoplasm or after release into the cytoplasm. In other studies, we have collaborated to examine in cultured cells and in rats how human UPF1 expression overcomes cytotoxicity or paralysis, respectively, caused by proteins implicated in amyotrophic lateral sclerosis. In this proposal, AIM 1 is to conduct a genome-wide inventory to define new NMD factors using haploid genetics. This AIM will supplement our on-going studies of the mechanism of NMD. AIM 2 is to identify new roles for NMD factors based on our unpublished data. We aim to investigate putative roles for (i) UPF1 together with Tudor-SN in microRNA decay, (ii) CBP80 in the PGC1ß-mediated upregulation of estrogen receptor- and orphan estrogen- related receptor-activated genes, and (iii) UPF1 together with FMRP, FRX1 and FXR2 on mRNA metabolism in fragile X mental retardation syndrome. AIM 3 is to develop therapeutics for the many and diverse dominantly inherited diseases that are due to PTCs that fail to trigger NMD and generate toxic truncated proteins.