Controlling toxic RNA with rapamycin

描述（由申请人提供）：本提案的目的是探索雷帕霉素/mTOR途径、无义介导的衰变（NMD）和基因表达的转录后控制之间的新联系。我们提出，通过增强NMD，雷帕霉素有助于降解含有提前终止密码子（PTC）的异常mRNA。在目标1中，我们提出了四个不同的，非排他性的假设，雷帕霉素可能会提高NMD。这个探索性的R21建议的目标是使这些假设中的至少一个无效，使我们能够专注于那些与衰老和转录后基因调控最相关的机制。在假设1中，我们将确定mTORC 1通路是否调节NMD。在假设2中，我们提出雷帕霉素/mTOR调节NMD机制的表达、磷酸化和/或定位。假设3提出，雷帕霉素可能会影响之间的平衡的先锋和稳定轮的翻译。最后，在假设4中，我们提出雷帕霉素/mTOR可能通过直接调节细胞核中的前体mRNA剪接决定来调节NMD效率。总的来说，目标1将揭示一种新的机制，雷帕霉素/mTOR通过该机制调节NMD，从而调节毒性mRNA的衰减。在目标2中，我们将采用高通量RNA-Seq来确定雷帕霉素/mTOR如何协调与细胞衰老相关的基因调控网络的表达。我们的初步结果表明，雷帕霉素/mTOR协调AS和翻译的特定亚型。采用我们实验室开发的创新测序方法和生物信息学，我们将识别在雷帕霉素/mTOR的剪接、mRNA稳定性和翻译水平上调节的基因家族，B。目的2将揭示新的候选人，为未来使用的RNA为基础的治疗，采用反义寡核苷酸来调节特定的基因异构体加工和促进抗癌和促长寿基因的剪接和翻译。

DESCRIPTION (provided by applicant): The goal of this proposal is to explore novel connections between the rapamycin/mTOR pathway, non-sense mediated decay (NMD) and post- transcriptional control of gene expression. We propose that by enhancing NMD, rapamycin contributes to degradation of aberrant mRNAs containing premature termination codons (PTCs). In Aim 1 we propose four different, non-exclusive hypotheses by which rapamycin may enhance NMD. The goal of this exploratory R21 proposal is to invalidate at least one of these hypotheses, enabling us to focus on those mechanisms most relevant to aging and post- transcriptional gene regulation. In hypothesis 1, we will determine if the mTORC1 pathway regulates NMD. In hypothesis 2 we propose that rapamycin/mTOR regulates the expression, phosphorylation and/or localization of the NMD machinery. Hypothesis 3 proposes that rapamycin may influence the equilibrium between the pioneer and steady rounds of translation. Finally, in hypothesis 4 we propose that rapamycin/mTOR may modulate NMD efficiency by directly regulating pre-mRNA splicing decisions in the nucleus. Collectively, aim 1 will reveal a novel mechanism by which rapamycin/mTOR modulates NMD and thereby the decay of toxic mRNAs. In Aim 2 we will employ high throughput RNA-Seq to determine how rapamycin/mTOR coordinates the expression of gene regulatory networks associated with cellular aging. Our preliminary results shown that rapamycin/mTOR coordinates AS and translation of specific isoforms. Employing an innovative sequencing method, developed in our lab, and bioinformatics we will identify gene families regulated at the level of splicing, mRNA stability and translation b rapamycin/mTOR. Aim 2 will reveal novel candidates for future use in RNA-based therapeutics employing antisense-oligonucleotides to modulate specific gene isoforms processing and promote splicing and translation of anti-oncogenic and pro-longevity genes.