Functional Requirement Of Alternative Splicing During Stem Cell Development

Single-cell transcriptional profiling has greatly advanced our understanding of cellular identity and cellular state transitions.For the past decade,single-cell RNA sequencing(scRNAseq)technologies have been widely applied to identify subpopulations or delineate cell developmental trajectories.However,most of the single-cell transcriptomic studies are mainly based on the gene level,while only a few of them were based on the isoform level.Since eukaryotic genes generate functionally distinct isoforms after alternative splicing(AS),genecentered studies may obscure the function of isoforms.Therefore,isoform-centered studies may more accurately characterize cell identity and describe cell developmental states.Here,we performed full-length scRNA-seq on seven sequential cell populations during hematopoietic stem cell(HSC)development and constructed a dynamic splicing landscape during the entire HSC ontogeny.Besides,we also identified hundreds of stage-specific isoforms that potentially affect the HSC formation and validated their expression using single-cell Nanopore sequencing of full-length transcripts.Subsequently,in vitro knockdown experiments showed that knockdown of some hemogenic-specific isoforms indeed affects the generation of HSCs.Next,motif enrichment combined with knockout experiments confirmed that the RNAbinding protein(RBP),SRSF2,could affect the specialization of endothelial cells into hematopoietic stem cells by regulating the AS of key transcription factors such as Runx1 and Myb.In addition,to further explore the role of RBPs in regulating stem cell differentiation,we combined bioinformatic analyses with functional screening and identified seven RBPs potentially regulating the exit from pluripotency of embryonic stem cells(ESCs).Next,expression and alternative splicing analysis indicated that hnRNPLL can promote the exit of pluripotency in embryonic stem cells.Finally,further analysis revealed that hnRNPLL mediates the ESCs pluripotency by regulating the AS of two transcription factors,and Bptf,which is important for pluripotency exit.Moreover,we also explored the dynamic distribution of alternative splicing in different subcellular locations from the subcellular level.In summary,we used high-throughput transcriptome sequencing data to construct the dynamic splicing landscape of stem cells,and identify stage-stage-isoforms and AS events during stem cell development.In combination with in vivo and in vitro experiments,we successfully identified and validated the splicing factors that specifically regulate HSC and ESC development.This study redefines our understanding of the development of stem cells,especially hematopoietic stem cells,at the transcriptome level,and provides new insight into in vitro induction of stem cells and the application of stem cell therapy in regenerative medicine.