Molecular Mechanism Of Polyadenylation Factor FY On Determining Poly(A) Site Usage And Affect Environmental Responses In Arabidopsis

Polyadenylation of eukaryotic mRNA is an essential posttranscriptional process achieved by poly(A)signal recognition,cleavage and addition of a poly(A)tail.The recognition of poly(A)signals is a crucial step for the determination of poly(A)sites in eukaryotic pre-mRNAs.Recently,in mammals,the CPSF30-WDR33-CPSF160 complex has high affinity for the poly(A)signal AAUAAA,and CPSF30 and WDR33 directly bind the AAUAAA signal.The deficiency of Arabidopsis CPSF30(AtCPSF30)leads to a shift from A-rich poly(A)signals to U-rich ones,indicating that AtCPSF30 involves in poly(A)signal recognition.However,genetic evidence for the role of the WDR33 homolog FY in Arabidopsis in recognizing plant NUE signals has never been reported.The relationship between FY and AtCPSF30 is also rarely reported.FY is a flowering time regulator in the autonomic flowering pathway.The PPLPP domains of FY can interact with the WW domain of the nuclear RNA-binding protein FCA.FCA/FY interaction regulates FCA and FLC poly(A)site usage and affects flowering time.In addition,FY also regulates alternative polyadenylation(APA)of key dormancy factor DOG1.But,the role of FY in genome-wide poly(A)site selection remains unclear.In this paper,a poly(A)tag sequencing(PAT-seq)approach has been adopted to study the poly(A)profiles in a set of defective FY mutants and FY/CPSF30 double mutants.We assessed the roles of FY in poly(A)site usage and investigated the genetic mechanism of FY and AtCPSF30 in signal recognition.In addition,we also uncovered the mechanism that how FY and AtCPSF30 are involved in plant growth,development and response to environmental stresses by APA.The results will provide genetic evidence for revealing the mechanism of poly(A)signal recognition and the molecular mechanism of polyadenylation in plants.This research lays the foundation for understanding the mechanism of plant growth,development and plant response to environment stresses regulated by APA.The main results are(1)More than 60%of poly(A)site usage has changed,indicating that FY widely involve in poly(A)site choice.Interestingly,WD40 domain mutation of FY has a more significant influence than PPPLPP deficiency on poly(A)site choice at the genomic level.(2)WD40 domain of FY mutation decreases nucleotide A usage,from 45%to 40%.However,the deficiency in PPLPP domains increases A usage and decreases U usage.The trend of nucleotide usage in the fy2/oxt6 and fy3/oxt6 double mutants is similar to that of oxt6,decreasing A usage and increasing U and C usage,indicating that the WD40 mutation or PPLPP mutation in FY do not enhance the variation in the single nucleotide profile of oxt6.In addition,FY overexpression may partially complement AtCPSF30 function,resulting in fy6/oxt6 with only 72 specific poly(A)cluster(PAC)(3)WD40 mutation decreases AAUAAA and lnt variation signal usage(about 10%).However,PPLPP deficiency increases these signals usage(about 10%).The trend of signal usage in the fy2/oxt6 and fy3/oxt6 double mutants is similar to that of oxt6,decreasing these signals usage(about 30%).The results indicate that FY and AtCPSF30 involve in recognition A-rich signal.But,AtCPSF30 has a greater role on signal selection.(4)Co-expression and differential analysis of transcripts further show that the WD40 domain mutation of FY has a more influence on poly(A)site selection.Poly(A)site choice of thousands of transcripts are significantly altered in WD40 mutant and double mutants.However,fy6/oxt6 affects fewer transcripts than fy6 and oxt6 single mutants alone,suggesting that FY overexpression may partially complement AtCPSF30 function.On the other hand,differentially expressed PAC(DE-PAC)in fy and in oxt6 only occupy a small proportion,suggesting that FY and AtCPSF30 play different roles in modulating transcript expression(5)The fraction of PAT and PAC in the 3'UTR significantly increases in WD40 mutant,coupled with dramatically reduces PATs in the intergenic region.However,the distribution of PAT reads and PACs in the 3'UTR and intergenic regions is opposite to that of PPLPP deficiency mutants,indicating that WD40 mutation and PPLPP deficiency have a different effect on full-length transcript expression.The PAT and PAC distribution of full-length transcript in double mutants is similar to that of oxt6,suggesting that AtCPSF30 plays an important role in regulating expression of full-length transcript(6)WD40 domain mutation in FY show a preference for using proximal poly(A)sites in the 3'UTR.However,PPLPP domain deficiency in FY resultes in a preference for using distal poly(A)sites in the 3'UTR.Genes also prefer to use proximal poly(A)sites in the 3'UTR in oxt6 and double mutants,indicating that FY and AtCPSF30 may co-regulate 3'UTR APA.(7)WD40 domain mutation in FY(fy3)affects primary root growth based on the observations that the primary root length of fy3 is shorter than that of the WT.A similar trend also exists in oxt6 and its double mutants.In addition,the distal poly(A)transcript expression and gene expression of root growth-related SAHH and ATHB13 are altered in the mutants,but the variation in expression levels is not the same in different mutants.The results indicate that FY and AtCPSF30 involve in the growth of primary root by mediating related gene APA.But the mechanism of root growth development may be different among the mutants.(8)The primary root length of fy3 is significantly reduced compared to WT after drought stress,showing that fy3 is sensitive to drought stress.The trend of primary root length in fy3/oxt6 and oxt6 is similar to fy3.The distal poly(A)transcript expression and gene expression of drought-related RD26 and ERD10 are changed in the mutants,but it is not the same in different mutants.The results show that FY and AtCPSF30 regulate drought-related genes APA,affecting gene expression.But the mechanism of drought stress response may be different among the mutants.