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.
Rapamycin and the mTOR pathway are intimately linked to human lifespan and disease, yet the molecular mechanisms regulated by this pathway remain enigmatic. Here we explore the role of rapamycin in surveillance of aberrant RNA transcripts and post- transcriptional gene regulation. We will employ a powerful combination of biochemical assays, RNA-Seq and bioinformatics employing human diploid cells to determine how rapamycin/mTOR modulates NMD efficiency and controls the expression of gene regulatory networks involved in aging and disease.
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