| Background:Gene expression and alternative splicing(AS)at transcriptional and posttranscriptional regulations are essential for mammalian development,aging and circadian rhythm.Alternative splicing,whereby multiple distinct functional transcripts are produced from a single gene,is a widespread process in eukaryotic genomes.AS increases transcriptomic diversity and is an important mechanism of functional innovation that may underlie cell differentiation and lineage commitment,cellular function,and organismal complexity.At the transcriptional level,previous studies indicated that 79%-91% of protein-coding genes and 16-38% of lnc RNAs are dynamically expressed during vertebrate development,whereas more than 2,599 genes are aging-related in human and 81.7% of the protein-coding genes show rhythmical expression in monkey.Compared to our understanding of the gene expression and regulation in these processes,the dynamics and regulation of AS are largely unknown.Here,we explored the dynamic changes,function implications,and potential regulations at AS level using time-series RNA-seq data of development,aging,and rhythm in multiple species and organs.Methods:First,using transcriptomic data of 18 representative metazoan species with multiple developmental stages(2232 poly A+ RNA-seq sampled from Caenorhabditis elegans to Homo sapiens),we identified and quantified the AS during development,and then constructed a metazoan developmental alternative splicing database.Second,based on the developmental AS of 7 main organs(forebrain,hindbrain,heart,liver,kidney,testis and ovary)from 7 vertebrates(Homo sapiens,Macaca mulatta,Mus musculus,Rattus rattus,Oryctolagus cuniculus,Monodelphis domestica and Gallus gallus),we identified developmentally dynamic alternative splicing(DDAS)through correlation analysis,explored the potential function of DDAS by using functional enrichment,evaluated the occurrence and difference of AS during the evolution through comparative analysis and correlation analysis,inferred the AS effect on protein level by using the protein information from Uni Prot,and explored splicing regulation by integrating the information from e CLIP signals of 223 RNA binding proteins(RBPs)and gene knockdown samples of 448 RBPs in K562 and Hep G2 cell lines.Finally,based on the AS information derived from aging-related and circadian samples of mouse organs,we identified aging-related dynamic alternative splicing(ADAS)and rhythmically dynamic alternative splicing(RDAS)by correlation and Meta Cycle.We explored the potential function of ADAS and RDAS using gene ontology(GO)and other enrichment methods,and inferred the AS effect on protein level using Uni Prot,and established regulatory relationships between RBPs or splicing factors(SFs)and AS through correlation analysis.Results:(1)A metazoan developmental alternative splicing database—MeDASWe established a metazoan developmental alternative splicing database(Me DAS).Me DAS can be accessed through clearly interactive browsing or batch download of quantitative data for free at https://das.chenlulab.com.Interactive browsing allows users to search by species,tissue or genomic features(gene,transcript,exon or sequence),and to visualize expression at gene or transcript level and percent of spliced-in(PSI)at the exon level.The quantitative data is free for users for facilitating personalized analysis,such as customized screening and local visualization via genome browser.In short,we constructed a practical and easy-to-use developmentrelated AS database,which can provide AS information with spatial-temporal and evolutionary information for developmental research.(2)Dynamics and regulation of alternative splicing during mammalian developmentUsing transcriptomic data of multiple organs from 7 vertebrates,we evaluated the prevalence and dynamics of AS during development.The number of AS identified in species is decreased gradually as its evolutionary distance increased from human,with the highest AS in primates(such as human and rhesus)and the lowest AS in chicken.For the number of detected AS during development,brain(ectoderm)has the highest while liver(endoderm)has the lowest.Largest dynamic changes of AS were observed in testis,forebrain and hindbrain during development.With the development of mammalian organs,the difference in the number of detected AS among organs is increased gradually,and the number of detected AS in heart,liver and kidney is decreased.The number of differential alternative splicing(DAS)between adjacent developmental stages varies across organs during development,and the stages with the largest number of DAS tended to be the periods of important or functional transition stages.The trends of DAS changes tended to occur around corresponding stages across species,indicating that the occurrence and expression of AS are regulated and some are conserved across species,which may play functional roles in organ development.Around 50% of AS are dynamically regulated during development across species,and these DDAS tended to be organ-specific,with the fractions of DDAS in forebrain,hindbrain,and testis are significantly higher than those in other organs.Host genes of DDAS are enriched in organ development.For example,the genes in brain are from pathways of neuron development,axon development,development and regulation of central nervous system,synaptic signaling,and synaptic assembly.Host genes of DDAS in heart are significantly enriched in the biological processes of cardiovascular system development,development and differentiation of muscle cells,and the organization and regulation of actin filament-related processes.Based on the annotation of the alternative exon,we compared the difference between DDAS and non-developmentally dynamic alternative splicing(non-DDAS),and found that DDAS are more likely to have higher percent of the DNA binding domain(DBD),intrinsically disordered region(IDR)and phosphorylation(PP),but with lower percent of activation and catalytic sites,acetylation(AC)and ubiquitylation(Ubiq).These results indicated that DDAS may play important roles in organ development through altering the availability of functional regions(DBD and IDR)and post-translational modification(PTM)regions at the protein level.At the both gene and exon level,old genes are more likely to be with AS and be spliced dynamically during development,indicating the conservation of AS and DDAS.In the principal component analysis(PCA),we found that samples were clustered according to germ layers,developmental stages,and evolutionary relationships on PC1,PC2 and PC3,respectively.Through the hierarchical clustering of sample correlation,samples were formed in a species-clustered pattern to an organclustered pattern as the number of orthologous exons with AS increased,and species with closely evolutionary relationship and organs with similar function tended to cluster together.Higher similarities of AS profile were found at corresponding developmental stages of same organ between species in an organ-specific manner.As evolutionary distance increased,the consistency of the developmental expression pattern of orthologous AS is decreased.Interestingly,we recapitulated von Bear’s divergence for individual organs at AS level,that is,the AS profile correlations between species to consistently decline with developmental time.These results indicated that DDAS tended to be from old genes.Given the closer species are,the more similar AS patterns were observed,the AS may play functional roles and the organs may gains novel functions via AS.AS is co-regulated by cis-acting elements and trans-acting factors.To investigate the AS regulation during development,we compared the conservation score of the cisacting sequence around splice sites in DDAS and that of non-DDAS and found a higher conservation in DDAS.Next,we evaluated the developmental expression of transacting factors(RBPs or SFs),and found that 87.6% in human and 92.3% in mouse of them are dynamically expressed with high organ specificities.Using e CLIP data,we found that the binding sites of RBPs,such as PTBP1,RBFOX2,U2(U2AF1 and U2AF2)and QKI,were enriched in the regulatory region of broad AS(orthologous exons are alternative in all species),and the binding sites of 13 RBPs(including QKI,PTBP1 and RBFOX2)were enriched in the regulatory region of DDAS.Next,we correlated the expression of trans-acting factors and PSI of AS,and found that the negative correlation between PSI of PSD-95 exon 18,which is essential for synaptic maturity and plasticity in brain,and its regulator—PTBP1.Similarly,the PSI of 61nt exon of NIN,which controls the transition from neural progenitor cells(NPC)to neurons,is positively correlated with the RBFOX2.Finally,we validated that the PSI of PSD-95 exon 18 was increased after PTBP1 knockdown in K562 and Hep G2,in consistent with the reported regulatory effect of PTBP1.Therefore,these results suggested that RBPs,such as PTBP1 and RBFOX2,may regulation DDAS.(3)Dynamic alternative splicing during aging and circadian rhythmUsing the transcriptomic data of aging and circadian,we explored the dynamic changes and regulation of AS.Host genes of aging-related dynamic AS(ADAS)of human dermal fibroblasts were enriched in the processes of injury response,cellular organization,apoptosis,autophagy and cellular aging.In mouse,host genes of ADAS in brain are neural function related,and host genes of ADAS in heart are muscle function related.We explored the difference between DDAS and ADAS of mouse,and found that PSI of AS from genes involved in heart contraction(Tpm2,Dlg1 and Cacna1c)and genes with neural function(Ubr4,Abi2,and Madd)in brain have opposite trends between development and aging.Therefore,we assumed that dynamic AS is important to both development and aging.During circadian rhythm,we found that rhythmically dynamic alternative splicing(RDAS)is organ-specific,and RDAS may play important roles in the rhythmic activities of organs,such as PSI changing rhythmically for the AS from genes related to insulin signaling pathway in liver and hypothalamus.ADAS and RDAS tend to regulate cellular activities by changing the functional regions(such as DBD and IDR)and phosphorylation regions dynamically at the protein level.Finally,we established potentially regulatory relationships through the correlation between the expression of trans-acting factors and the PSI of AS,and found that trans-acting factors correlated with the dynamic changes of AS during circadian rhythm(HNRNPC and FUS)and aging(ZC3HAV1 and HSPD1).In summary,we integrated a large number of time-series transcriptomic data during development,aging and circadian rhythm,and constructed a multi-species development-related AS database—Me DAS.We discovered highly dynamic patterns of AS during all these biological processes.Furthermore,we explored their conservation and potential splicing regulation.Our results provide candidates for studying the functional roles of these AS events and their potential splicing regulators during development,aging and circadian rhythm. |
