Controlling toxic RNA with rapamycin

描述(申请人提供)：这项建议的目标是探索雷帕霉素/mTOR途径、无义介导的衰退(NMD)和基因表达的转录后控制之间的新联系。我们认为，通过增强NMD，雷帕霉素有助于降解含有提前终止密码子(PTCs)的异常mRNAs。在目标1中，我们提出了四种不同的、非排他性的假说，通过这些假说，雷帕霉素可以增强NMD。这个探索性的R21提案的目标是至少使这些假设中的一个无效，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，我们认为雷帕霉素/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先行轮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可能通过直接调控细胞核中的前mRNA剪接决定来调节NMD的效率。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，从而调节有毒mRNAs的衰退。在目标2中，我们将使用高通量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可以协调特定异构体的AS和翻译。利用我们实验室开发的一种创新的测序方法，以及生物信息学，我们将识别在剪接、mRNA稳定性和翻译水平上调控的基因家族--雷帕霉素/mTOR。目的2将揭示未来在基于RNA的治疗中使用反义寡核苷酸来调节特定基因异构体加工并促进抗癌基因和促长寿基因的剪接和翻译的新候选者。

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.