Dynamics Of Alternative Polyadenylation And Gene Expression Regulation By The Absence Of M6A On Plant Messenger RNA

Polyadenylation,known as the final step for mature messenger RNA biogenesis in eukaryotes,has alternative potency.Pre-mRNA use different sites to modulate the length of 3'-end,which increases the diversity of transcripts,thus control mRNA stability or translation effect without changing nucleotide sequences.Alternative polyadenylation(APA)regulates gene expression at post-transcriptional level and plays a crucial role in plant development and environmental responses.N6-methyl-adenosine(m6A),the most abundant modification on eukaryotic mRNA,which is widespread and highly evolutionarily conserved,has been reported to be a posttranscriptional regulator.The distribution of m6A caught our attention as it happens to be enriched around the stop codon and within 3'UTR before the poly(A)tail.To study the correlation between m6A and APA,we used the poly(A)Tags Sequencing(PAT-seq)technology to reveal poly(A)site choice and gene expression regulation by the absence of m6A on an Arabidopsis mutant that lacks m6A methylase.Moreover,bioinformatics analysis and gene function annotation suggest a potential role of m6A on alternative polyadenylation and gene expression.The main results are:1)APA events occurred in many genes.The absence of m6A increased the number of poly(A)sites in mRNA of A rabidopsis thaliana.The total number of poly(A)sites increased from 33,860 to 36,387 in leaf and increased from 50,816 to 52,337 in inflorescence tip.2)APA-gene ratio increased by the absence of m6A on mRNA.A total of 18,227 genes were expressed in wild-type leaves,including 9,105 APA-genes,accounting for 50.0%.A total of 18,802 genes were expressed in the mutant that lack of m6A,the number of APA-genes increased to 9,921,and the ratio of APA-genes increased to 52.8%.A total of 22,084 genes were expressed in the wild-type inflorescence tip,including 13,763 APA-genes,accounting for 62.3%.A total of 21,944 genes were expressed in the mutant that lack of m6A,the number of APA-genes increased to 14,000,and the ratio of APA-genes increased to 63.8%.3)The absence of m6A affects the recognition of poly(A)signals in Arabidopsis.Poly(A)signals determine poly(A)site usage.Significant change of poly(A)signals was found by comparing single nucleotide profile surrounds poly(A)sites between up-regulated PAC(poly(A)cluster)and down-regulated PAC.The usage of AAUAAA and its one nucleotide variants near upstream dropped significantly while the ratio of other signals increased in up-regulated PAC.4)M6A regulates transcript length so as to increase transcript abundance.We calculated the weighted distance between poly(A)sites to investigate the transcript length,the results showed that m6A level mediates the usage of both proximal and distal poly(A)sites.There is an early polyadenylation tendency since more genes have their weighted distance between poly(A)sites shortened than lengthened.5)Global gene expression patterns have been changed both in leaf and inflorescence tip of the rabidopsis m6A methylase mutant.We screened 261 up-regulated genes in leaf and 757 in inflorescence tip,155 down-regulated genes in leaf and 978 in inflorescence tip.6)APA plays an important role in gene expression regulation.About 75.4%of the differential gene expression in leaf are caused by differential PAC expression,and that number in inflorescence tip is 76.1%.