Mechanism Of Cleavage And Polyadenylation Specificity Factor 100 On Determining Phenotypes And Ability Of Environmental Response Of Arabidopsis

Polyadenylation is an essential step for most of mRNA biogenesis in eukaryotic cells.This process includes recognizing poly(A)signals,cleavaging at the 3' of pre-RNA and adding a poly(A)tail.More than 75%of genes contain at least 2 poly(A)sites,and alternatively selection of an appropriate site can be used to adjusting gene expression.Thus,alternative polyadenylation(APA)is one of the key pathways to regulating gene expression in eukaryotes.Cleavage and Polyadenylation Specificity Factor 100(CPSF100)is a key protein of the polyadenylation machinery.It regulates transcription read-through,and coordinates gene silencing,plant flowering time and embryo development.Biochemistry studies revealed that CPSF100 probably serves as a skeleton protein to assemble the crucial complex,CPSF complex,for polyadenylation.It resides at the hub of protein-protein interaction network of poly(A)factors.CPSF 100 was also found necessary for the function of polyadenylation cleavage factor,CPSF73 and mRNA export factor THOC,which indicated that CPSF 100 is important for accurately polyadenylation in eukaryotic cells.It was also found can be co-purified with RNA splicing factors,suggesting its potential role in RNA splicing.However,how CPSF 100 control polyadenylation including poly(A)signal recognition is still unknown,and its other roles in transcription are also a mystery.Herein,we used Arabidopsis as a model,and employed Poly(A)Tags Sequencing(PAT-seq)technology and bioinformatics methods to study the functions of AtCPSF10O in polyadenylation and RNA biogenesis.The main results are showed as following:(1)AtCPSF100 is involved in several plant morphologies regulation,such as seed setting rate,seed weight,primary root length,and root hair density and length;It also regulates plant resistance for bacterial infection.(2)PAT-seq results revealed that AtCPSF100 positively regulates the number of poly(A)tag clusters(PAC)and poly(A)tags(PAT)in 3'UTR regions.However,it suppresses the number of PACs and PATs in intergenic and promoter regions.(3)Polyadenylation of both coding genes and non-coding genes is coordinated by AtCPSF 100,which indicates the genome-wide regulation role of AtCPSF 100.(4)Poly(A)signal of intronic polyadenylation events are controlled by AtCPSF 100.This indicates the connection of polyadenylation and RNA splicing;Moreover,AtCPSF 100 significantly affects the far up stream element(FUE)poly(A)signal usage,which is preferred to use G-rich FUE poly(A)signals.(5)Differential expression(DE)analysis uncovered 1385 genes with DE poly(A)cluster(DE-PAC)and 507 genes with differential total expression level(DE gene).This two gene clusters overlap for 398 genes,indicating that most of DE genes are induced by DE-PAC.Gene Ontology(GO)analysis shows that DE-PAC genes can be significantly enriched in 14 GO terms which are involved in plant development and resistance.However,DE genes cannot be significantly enriched.These results suggest that genes involve in plant development and defense are much more common to be regulated by alternative polyadenylation.However,the role of AtCPSF 100 in regulating gene expression does not have preference.(6)AtCPSF 100 mainly regulates the polyadenylation of genes encoding binding proteins.These binding proteins may be employed to boost the function of AtCPSF100.(7)The expression level of CTD PHOSPHATASE-LIKE 3(CPL3)is significantly regulated by AtCPSF 100.AtCPSF100 adjusts the expression of CPL3 to dephosphorylate Ser2 on C-terminal domain(CTD)of RNA polymerase II(Pol II).Thus,Pol ? can determine an appropriate site for transcription termination.This ensures genes escape from gene silencing induced by read-through.(8)AtCPSF100 is essential for the expression of DRM1,which encodes de novo DNA methylase.It also regulates the poly(A)profile of itself and another poly(A)factor gene,PCFS4.Moreover,AtPCSF100 affects the poly(A)profile of genes encoding RNA splicing factors.The poly(A)profiles of most of these genes are shifted to distal sites.These indicate that AtCPSF100 probably participates in other pathway of RNA biogenesis;However,the mutation of AtCPSF100 in mutant esp5 does not change the miRNAtargets a lot.Collectively,this study elaborated the role of AtCPSF100 in accurate polyadenylation.Here,we firstly reported the polyadenylation of both coding genes and non-coding genes were regulated by the same poly(A)factor in plants;And we firstly reported a poly(A)factor involved in FUE poly(A)signal selection and transcription termination.Our findings enhanced the understanding of molecular mechanisms of polyadenylation in plants.These results also are beneficial for us to understanding the mechanism of RNA 3' processing and how it helps plants response to environment.