Alternative Splicing Of Adult Neural Stem Cells In The Mouse Subventricular Zone Based On Single-Cell Transcriptome Sequencing

Alternative splicing（AS）events of precursor m RNAs are widespread in eukaryotic organisms.AS events play diversified regulation roles such as neural cell differentiation,neuronal migration,synapse formation,and brain development in the central nervous system（CNS）^[1-5].Disruptions in the splicing machinery can result in neurodegeneration^[6].These findings emphasize the importance of characterizing AS events in CNS tissues and understanding its spatiotemporal and dynamic regulations.The CNS has enormously complex cellular diversity.Population-based AS analyses of bulk tissues with heterogeneous cellular composition reveal the average of all of the different cells in a particular tissue and does not truly reflect functions of any particular or rare but important cell types in these tissues.Single-cell-based alternative splicing analyses can circumvent such a problem.Alternative splicing features in single cells of important cell types at neurogenic regions are not well understood which hampered the understanding of the biological role of alternative splicing in these regions.The subventricular zone（SVZ）of the adult mouse forebrain,one of the known neurogenic regions where contains multiple distinct cell types related to adult NSC activities^[7].In this study,we analyzed the transcriptomic data of 28 single-cell samples and 4 pooled-cell samples from adult mouse ependymal and subependymal cells,and the specific work and conclusions are as follows:1、Identification and characterization of AS events（1）3611 AS events were identified from 1908 genes in 28 single-cell samples,and their detailed AS characterization was systematically described.RT-PCR with primers designed for randomly selected 10 AS events and Sanger sequencing were performed for confirming them.Results showed that all these detected AS events can be verified by Sanger sequencing,suggesting the single-cell RNA-seq is effective for AS analysis.（2）Compared the variance of different types and the quantity of AS events between 28 single-cell samples and 4 pooled-cell samples with mixed ten single-cell libraries in each sample.We found that there were no significant differences in the proportion of AS types detected between the single and pooled cells.However,more AS events were detected in 28 single-cell samples（3611）than in 4 ten-cell pooled samples（3379 in 40 cells）.Analysis showed that most of differentially expressed alternative splicing（DEAS）events detected only in single-cell samples are low-abundance and novel splicing isoforms,suggesting that single-cell RNA-seq has the advantage in uncovering rare splicing isoforms compared to bulk RNA-seq at the population level.2、Heterogeneity and diversity analysis of AS events（1）The variance of different types and the quantity of AS events of CD133+ependymal quiescent NSCs,activated NSCs,and neuroblast cells from the SVZ region were analyzed,with cluster analysis of cell type-specific and expression differences.Results showed that the proportion of AS types was consistent among the three types of cells.Only a few AS events were cell-type-specific and most of the AS events were highly heterogeneous in the same cell type.（2）AS events and the corresponding parent genes of four samples were analyzed,which were randomly selected from four cell types,including quiescent NSCs,activated NSCs,neuroblast cells,and oligodendrocyte cells,respectively.Results showed that simultaneous presence of multiple isoforms from the same gene in a single cell is prevalent.（3）New transcriptional fusions of adjacent genes and novel bicistronic transcripts were detected in single cells of SVZ.3、Identification of bicistronic transcripts and study on translation regulation mechanism.（1）The presence of the two novel bicistronic transcripts were verified by RT-PCR,cloning,and Sanger sequencing.（2）Two different types of bicistronic Ech1-Lgals4 transcripts were cloned respectively into p CMV-N-HA and then transfected into HEK293T cells,using empty p CMV-N-HA as control.We then examined the expression levels of Ech1 and Lgals4 by western blot in control cells and bicistronic transcript overexpressed HEK293T cells.We found that Ech1 and Lgals4 protein levels were higher only in the cells that overexpressed the bicistronic transcript with 77 nucleotides than in the control group.These results suggest that the sequences between the ORF of Ech1and Lgals4 in this bicistronic transcript can drive the expression of Lgals4.（3）5 short sequences complementing 18Sr RNA were found in the ORF intersequence of the two genes.7 RFP-ORF intersequence-pi RES2-EGFP recombinant DNA and 3 RFP-ORF intersequence-Lgals4 gene sequence-Pi RES2-EGFP were constructed,and RFP-PIRES2-EGFP was used as control.The expression of RFP and GFP in overexpressed HEK293T cells was detected.It was speculated that the intersequence of ORF had the partial function of IRES sequence,and 2 of the 5 short sequences were positively correlated with ribosome recruitment,and 3 were negatively correlated with ribosome recruitment.The downstream Lgals4gene sequence is not directly associated with translation regulation.