Structure And Function Of PUF-8 In C.elegans

Lifespan and aging are important processes in the development of organisms.Among the continuous progress of modern medicine,the average lifespan of human beings has been greatly extended over the past century.In order to further explore the lifespan and aging,especially aging-related human diseases.It is necessary to further investigate the molecular mechanisms and cellular processes of lifespan and aging.Caenorhabditis elegans(C.elegans),usually called worms,is an ideal model organism for investigating the aging process due to its short and reproducible lifespan(around 18 days at 20 ?).Knock-out of specific genes in worms can significantly prolong or shorten lifespan.Nutritional restriction or reproductive system intervention will also affect the lifespan.Many of the well-known signaling pathways involved in longevity regulation were first discovered in nematodes.The transcription factor,DAF16 controls lifespan by regulating genes at the transcriptional level through the Insulinlike signaling pathway.In addition to transcriptional regulation,RNA binding proteins(RBPs)can also modify and regulate mRNA at the post-transcriptional level.RBPs play important roles in aging process.RBPs interact with RNAs in both specific and nonspecific ways to form protein-RNA complexes,which subsequently regulate gene expression of target mRNAs by degradation,activation,and silencing.PUF-8 is a classical RBP in nematodes,playing important roles in regulating the development and differentiation of germline stem cells and the switch of sperm/oocyte cells.Recent studies have suggested a potential role for PUF-8 in longevity regulation.PUF-8 has a classical PUF(Pumilio and FBF)domain consisting of eight PUF repeats,each repeat consists of three a helices.Generally,each PUF repeat can bind one RNA nucleotide and form a zip-like pairing pattern.In order tounderstand the molecular mechanism of PUF-8 protein interacting with RNA,we determined the crystal structure of the complex of the PUF domain of PUF-8 with single-stranded target RNA.In the structure,we found that PUF-8 shows similar structural conformation compared to the orthology PUF protein,like HsPUM2.However,on the interface of RNA binding pocket,REP-4(repeat-4)and REP-8 have unique recognition mechanisms to the RNA bases.To verify that these two unique mechanisms contribute to RNA binding affinity,we explored the RNA binding affinity between the mutants with substrate RNAs by mutation experiments and fluorescence polarization experiment.By using of X-ray crystallography,we determined the crystal structures of the wild-type PUF domain and the MUT-4A mutant PUF domain together with different RNA sequences.Our results showed that K513 of PUF-8 protein involved in the recognition of REP-8 to the RNA base,and affect the ability of the entire domain to bind to RNA.On the other hand,the structure of the MUT-4A mutant changed the conformation of the PUF-8 protein recognition base in the REP-4 region,yet the REP-4 mutant showed no significant changes in the RNA binding affinity.In vivo experiments confirmed that the REP-4 mutation had an impact on the lifespan and fertility.We determined the role of PUF-8 in the regulation of lifespan in C.elegans.PUF8 mutant prolonged the lifespan and reduced the brood size.We then performed the mRNA deep sequencing and identified six genes differentially expressed that related to lifespan regulation:pqm-1,hlh-30,pha-4,blmp-1,vhp-1 and ctl-2.The mRNAs of these six genes had the PUF binding element(PBE).We verified that PUF-8 interacts with these mRNAs by in vitro RNA Pull down and in vivo RNA CLIP assay.We used RNA interference(RNAi)and knocked down these six genes on N2 and PUF-8(ok302),respectively.The results showed that knock down of pqm-1 gene could restore the lifespan extension induced by puf-8(ok302),suggesting that PUF-8 affect the lifespan by regulating pqm-1 mRNA.