Effects Of PIWIL1 And PABPC1 On Protein Translation During Mouse Spermatogenesis

Spermatogenesis is the biological process of gradual transformation and development of spermatozoa over an extended and complicated process with in the boundaries of the seminiferous tubules of testis.This process involves interplay between hundreds of gene products.In post-meiotic round spermatids,a lot of mRNAs were stored in a translational repressive state.Premature translation of these mRNAs in round spermatids caused abnormal head morphology and reduced sperm motility,leading to male infertility.Therefore,mouse spermatogenesis is a highly complex cell division and differentiation process which are regulated at both transcriptional and post-transcriptional levels.Study of the mechanisms that govern these would be important for exploring the biological event that occurr during mouse spermatogenesis.During the study of sperm-specific protein AKAP3(Protein Kinase A Anchoring Protein-3),we used AKAP3 antibody to perform coimmunoprecipitation from testis lysate,and we observed PABPC1 and PIWIL1 in the protein complex.PABPC1 is capable of binding poly(A)tails of mRNAs and plays critical roles in stabilization of mRNAs and translation initiation in eukaryotic cells.PIWIL1,a murine Argonaute family member,plays important roles in piRNA induced gene silencing events.Both proteins were considered to play important roles in post-translational regulation during spermatogenesis.In order to unveil the molecular mechanisms that regulate their functions during spermatogenesis,we conducted the following experiments.Through the study of the interaction between PABPC1 and PIWIL1,we found that PABPC1 and PIWIL1 bind each other both in vivo and in vivitro.RNAs stablized their interaction.In order to identify the protein domains that are responsible for their direct interaction,we truncated PABPC1 and PIWIL1 into three independent domains,PABPC1 were separated into RRM(N-terminal),PABPC1-L(middle region)and MLLE(C-terminal).PIWIL1 were separated into PIWI-N(N-terminal),PAZ(middle region)and PIWID(C-terminal).We found that PABPC1-L domain of PABPC1 had physical interaction with PIWIL1 in vitro and PIWI-N,PIWID domains of PIWIL1 had physical interactions with PABPC1 in vitro.In vivo assay showed that RRM domain of PABPC1 interacted with PIWIL1,PIWI-N and PIWID domain interacted with PABPC1.These interactions depended on RNAs.We observed that PABPC1,RRM.PABPC1-L.PIWIL1 and PIWI-N directly interact with the 3' UTR of mRNAs of Tnp1,Tnp2,Prm1,Prm2 and Akap3.Immunostaining further showed that PIWIL1,PABPC1 and Tnp2,Prm2.Akap3 mRNAs have the same patterns of distribution in spermatids.RRM domain of PABPC1,PIWI-N and PIWID domain of PIWIL1 interacted with the same target RNA,while PABPC1-L of PABPC1 directly interacted with PIWI-N and PIWID domain of PIWIL1.These suggested that PIWIL1,PABPC1 and the target RNA form complex to regulate mRNA translation.It is the first time that two domains from PABPC1 and PIWIL1(PABPC1-L and PIWI-N respectedly)were found to interact with mRNA directly.We further examined the functions of PIWIL1 and PABPC1 using pLuc-3'UTR dual luciferase reporter system.In 293T cells.PIWIL1 and PABPC1 co-expression dramatically increased the translational efficiency of the reporter gene,indicating that both PIWIL1 and PABPC1 have positive effects on translation.RRM,PABPCI-L domains of PABPC1,PAZ domain of PIWIL1 also showed positive effects on translation.This is the first time that PIWIL1,PAZ domain and PABPC1-L were found to promote protein translation.We extracted polyribosome,monoribosome.and RNP components from testes lysate,PIWIL1 and its truncated domains expressed in 293T cell lysates separately using sucrose density gradient centrifugation.We observed that PABPC1,PIWIL1,PIWI-N,PIWID were distributed in all of those components,including polyribosomal fractions.Our selected spermiogenic mRNAs were also enriched in polyribosomes and RNP components.These data suggested that there are two ways PABPC1 interacts with PIWIL1 in spermatogenic cells:in a form of PI WILI1 PABPC1.mRNA complex,or binding each other through domains PABPC1-L,PIWI-N,PIWID.In round spermatids,PIWIL1?PABPC1 and mRNAs of sperm-specific genes co-localized in cell cytoplasm and chromatoid body(CB).In 293T cells,PIWIL1 and PABPC1 promoted the translational efficiency of the report gene,and RRM?PABPC1-L domains of PABPC1,PAZ domain of PIWIL1 showed positive effects during the translation of reporter genes' mRNA.In summary,we deduced that RNA-dependent interaction between PABPC1 and PIWIL1;PABPC1,PIWIL1 and RRM,PABPC1-L,PIWI-N domains from the two proteins interact with mRNAs directly:The two proteins showed positive effects on the protein translation of mRNAs of the reporter gene.The mechanisms that governing the binding of the proteins and mRNA's untranslated regions,and how they function in regulating protein synthesis require further study.