Role of Alternative Splicing and Polyadenylation in Regulation of Spleen Development

Alternative splicing (AS) and alternative polyadenylation (APA), as post-transcriptional regulatory mechanisms, are involved in various biological processes through the generation of transcript variants. However, genome-wide studies of AS and APA during spleen development are scarce. This study aimed to characterize transcript diversity and changes in transcript isoforms in the spleen at two developmental stages using full-length isoform sequencing integrated with short-read RNA sequencing. We revealed widespread transcript diversity and identified 17,294 unannotated transcripts, most of which originated from known genes in the current pig genome annotation. The top 500 genes with the highest isoform diversity were mainly associated with disease occurrence and immune function, as revealed by Kyoto Encyclopedia of Genes and Genomes enrichment analysis. We also observed changes in major transcript usage and polyadenylation site selection during spleen development. Our results indicated that genes regulated immunological development mainly by switching dominant transcript isoforms rather than altering overall expression levels. In addition, genes exhibited a tendency of age-dependent preference for distal polyadenylation sites. Furthermore, transcription factors important for spleen development were identified, and the regulatory axes MYBL2/WEE1 and E2F1/WEE1 were constructed for the first time using molecular biology techniques. These findings not only refined the current pig genome annotation, but also provided a foundation for exploring the molecular mechanisms responsible for spleen development.