Fig. 2.

Alternative splicing is increased in the hypothalamus of mice undergoing CR. a Alternative splicing events in mice on caloric restriction (CR) for 5 or 15 months (CR5 or CR15), as exhibited by the differential usage of cassette exons (CE; dark blue bars) or by intron retention (IR; dark brown bars). Spliced-in and spliced-out events are considered together to represent the total number of differential AS events (CE and IR only) during CR. The genes that these events correspond to are indicated in the lighter shades. Statistical test: Irwin–Hall P value summarization was used on each individual splicing event (details in the Methods section). Data are from five (for CR5 or AL5) or six (for CR15 or AL15) biological replicates. b Distribution of the number of AS events (both CE and IR considered together) per gene in CR5 and CR15. c Significant overlap between genes undergoing AS during early (CR5) and late (CR15) phases of CR. P value determined by hypergeometric test. d KEGG pathway, Gene Ontology (GO) term enrichment and protein domains were determined by DAVID Functional Annotation tool using differentially AS genes in mice under CR diet for 5 months. The listed pathways have a P value ≤ 0.05 (Fisher Exact Test) and FDR<10.0. The numbers within the blue bars represent genes detected in the category. e Representative images of AS involving cassette exons. For this, four terms were considered, reads mapping to the cassette exon (Exon), the flanking junctions (JUN1, JUN2) and that of the skipping junction when the exon is not included (JUN3). Each box represents five (for CR5 or AL5) or six (for CR15 or AL15) biological replicates in the RNA-seq experiments. In case of Exon 250310, the read counts mapping to the exon or the JUN3 were reciprocal in AL5 vs. CR5 as well as in AL15 vs. CR 15. On the other hand, the reciprocal relationship was observed either in case of early (Exon 186313) or late (Exon 265662) CR. cpm counts per million