Controlling toxic RNA with rapamycin

描述（由申请人提供）：本提案的目标是探索雷帕霉素/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过早终止密码子（ptc）的异常mrna。在Aim 1中，我们提出了雷帕霉素可能增强NMD的四种不同的非排他性假设。这个探索性R21提案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mTORC1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提出雷帕霉素可能影响先驱和稳定轮翻译之间的平衡。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pre-mRNA剪接决定来调节NMD效率。综上所述，目的1将揭示雷帕霉素/mTOR调节NMD从而降解毒性mrna的新机制。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和特定亚型的翻译。利用我们实验室开发的创新测序方法和生物信息学，我们将确定在雷帕霉素/mTOR剪接、mRNA稳定性和翻译水平上受到调控的基因家族。Aim 2将揭示新的候选物，用于未来基于rna的治疗，利用反义寡核苷酸来调节特定的基因同工型加工，促进抗肿瘤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.