Molecular Mechanisms Of Aging In C.elegans

The mRNA alternative splicing-mediated post-transcriptional regulation of gene expression plays important roles in neuronal development and plasticity.However,little is known about whether and how neuronal mRNA alternative splicing affects aging.Here we show that UNC-75,a highly conserved CELF family RNA binding protein and a key regulator of neuronal mRNA alternative splicing,serves as a novel modulator of aging in C.elegans.Deletion of unc-75 delays aging as indicated by significant lifespan extension and healthspan improvement.Extensive genetic epistasis studies demonstrate that the lifespan extension produced by the unc-75 deletion cannot be suppressed by mutations in positive regulators of longevity,whereas the unc-75 mutation has synergistic effects on longevity when combined with long-lived mutants from various agingrelated pathways.These results indicate that UNC-75 is a novel and potentially a fundamental regulator of longevity.Cas9-based cell-specific knockouts of unc-75 and transgenic rescue experiments demonstrate that the glutamatergic neurons,more specifically a pair of sensory neurons(ASHs),are the major sites in which UNC-75 function to regulate lifespan.Moreover,mRNA-Seq and bioinformatic analyses have been performed to identify genes that show differential mRNA or alternative splicing levels in the unc-75 mutant.Functional analyses reveal that two lipase genes,lipl-1 and lipl-2,function downstream of unc-75 to regulate lifespan.The dual-color splicing fluorescence reporter and d Cas13 system have been constructed,which will help to identify the direct splicing targets of UNC-75 and characterize their roles in aging in future studies.Collectively,our results reveal a novel role of the RNA binding protein UNC-75 in aging.Further studies on the targets of UNC-75 will elucidate the molecular connections among mRNA alternative splicing,neuronal functions and aging.Tissue-specific genetic manipulations are indispensable and critical in complex biological studies.Tissue-specific promoters-driving transgene rescue of mutations of the RNAi machinery such as RDE-1,an Argonaute protein functions cell autonomously to ensure RNAi capacity,has been performed to achieve tissue-specific RNAi knockdown in C.elegans.To facilitate genetic analysis of the germline,an important tissue with multiple regulatory roles,we rescued the rde-1 null mutant created via CRISPR/Cas9 with a single copy of rde-1 transgene driven by the germline-specific sun-1 promoter.The germline RNAi efficiency and specificity were validated by RNAi phenotypes examination,tissue-specific GFP reporter fluorescence knockdown via gfp RNAi,as well as measuring mRNA levels of tissue-specific genes upon corresponding RNAi treatment.Furthermore,the germline-specific RNAi strain shows normal reproduction profile,lipid accumulation,thermo-tolerance ability and lifespan compared to wild-type animals.An RNAi screen using a library with RNAi clones against all phosphatase genes was performed in the germline-specific RNAi strain for enhanced thermo-tolerance,and four phosphatase genes were identified as novel regulators of stress resistance.The germline expression of these genes was confirmed by RT-PCR using micro-dissected gonadal tissues.Together,we have created a useful RNAi tool that will help to study germlinespecific functions and regulation in C.elegans.