Screening For Trans-acting Factors In Alternative Polyadenylation Process And Development Of A Novel Apa Perturbation Method

Cells function is mainly determined by gene expression which is precisely controlled at space and time for every step,transcription,RNA 5' capping,splicing,RNA polyadenylation,and translation.Alternative polyadenylation is an RNAprocessing mechanism that produces different 3'ends on RNA polymerase ?transcripts,so that a gene can produce different lengths of 3'UTR or even different coding sequence.This mechanism is particularly useful for complex organisms because it provides a way to express the same protein but in different amounts and locations.About 70%of human genes contain multiple polyadenylation sites.Different polyadenylation sites are selected under different conditions,the fine control of alternative polyadenylation is important for normal function of the human body and unstable and incorrect regulation will cause diseases.In alternative polyadenylation process,a variety of cis-regulatory elements in pre-mRNA,a large number of transacting factors are required to participate and are affected by a variety of cellular microenvironmental factors.However,cis-regulatory elements,trans-acting factors and mechanisms that regulate alternative polyadenylation are still not very clear.Here,we identified several enhancers for alternative polyadenylation site usage including TAGCGCCA,AGAACCCG,GCGCCACA,AACTCCCA and showed their effects for RBM12 polyadenylation site in pRiG reporter.Then,we performed CRISPR/Cas9 based screen on AACTCCCA motif for its associated trans-acting factors.NR2E3,MGST2,and HOXC12 genes showed significant enrichment in screened library.Moreover,numerous studies have shown that differences in mRNA expression caused by the selection of different poly(A)sites can affect the process of extracellular signal stress,growth and development,and the occurrence and development of various diseases.Thus,methods regulating alternative polyadenylation is important for researches on alternative polyadenylation functions.Here,we tested dCasRx in alternative polyadenylation reporter genes and verified its ability to block 3' poly(A)process.We also applied dCasRx on endogenous alternative polyadenylation sites,and successfully manipulated alternative polyadenylation in tandem alternative polyadenylation(PHB,CD47,GNAI2)and alternative last exon(AK2,LMNA).Other than these,we interfered trans-acting factors'binding at different locus of alternative polyadenylation site in GNA11 gene,which is a non-canonical alternative polyadenylation site,by dCasRx.Alternative polyadenylation site usage significantly changed while targeted by dCasRx which showed dCasRx's ability in cis-regulatory elements screen.