Comprehensive Profiling Of Alternative Splicing (AS) And Alternative Polyadenylation (APA) In Bamboo

Moso bamboo(Phyllostachys edulis)represents one of the fastest-spreading plants in the world,due in part to its well-developed rhizome system.However,the post-transcriptional mechanism for the development of the rhizome system in bamboo has not been comprehensively studied.We therefore used a combination of single-molecule real time sequencing(SMRT)technology and polyadenylation site sequencing(PAS-seq)to re-annotate the bamboo genome,and identify genome-wide alternative splicing(AS)and alternative polyadenylation(APA)in the rhizome system.In total,145,522 mapped full-length non-chimeric(FLNC)reads were analyzed,resulting in the correction of 2,241 mis-annotated genes and the identification of 8,091 previously unannotated loci.Notably,more than 42,280 distinct splicing isoforms were derived from 128,667 intron-containing full-length FLNC reads,including a large number of AS events associated with rhizome systems.In addition,we characterized 25,069 polyadenylation sites from 11,450 genes,6,311 of which have APA sites.Further analysis of intronic polyadenylation revealed that LTR/Gypsy and LTR/Copia were two major transposable elements within the intronic polyadenylation region.Furthermore,this study provided a quantitative atlas of poly(A)usage.Several hundred differential poly(A)sites in the rhizome-root system were identified.Taken together,these results suggest that post-transcriptional regulation may potentially have a vital role in the underground rhizome-root system.Related data is released at the following link:http://www.forestrylab.org/db/PhePacBio.The single-molecule real time sequencing technology shows its unparalleled advantages in this project,but there is no comprehensive database to query the AS events detected by the single-molecule real time sequencing technology.Therefore,we integrated the latest single molecule real time isoform sequencing(Iso-Seq)and Illumina sequencing data to identify AS events.In addition to bamboo,we also include four perennial species of forest(Populus trichocarpa,Eucalyptus grandis,Pinus taeda,Picea abies),five economic crops(Oryza sativa,Zea mays,Sorghum bicolor,Solanum lycopersicum,Glycine max)and model plant Arabidopsis,identified a total of 333,241 AS events from linear RNA.It’s like the linear transcripts,circular RNA(circRNA)have the alternative splicing events too.But the miRNA target sites in the alternative back splicing region of circular RNA have not been reported.In this project,we have identified 63,561 AS events from the circular RNA.In addition to AS,microRNA(miRNA)mediated post-transcriptional regulation has been extensively studied in most eukaryotes.However,the interplay between AS and miRNA has not been explored in plants.Besides,the overall profile of miRNA target sites in circular RNAs generated by alternative back-splicing has never been reported previously.Among them,there were 55,850 and 26,751 miRNA target sites located in linear and circular AS region,respectively.All of the above data has been integrated into the database(ASmiR,www.forestrylab.org/bioinfor/db/ASmiR).Through the ASmiR friendly web search interface,users can view the distribution of AS events and potential target sites for miRNA.In addition,users can also submit user-defined data sets to the web server to search AS regions harboring small RNA target sites.In summary,this study not only analyzes the post-transcriptional regulation mechanism of bamboo,but also provides an unprecedented database to understand regulatory relationships between miRNA and AS in both gymnosperms and angiosperms.