Aging is a major risk factor for chronic diseases, and calorie restriction (CR) is a robust non-pharmacological intervention that can extend health span in multiple species. Alternative splicing (AS) generates multiple RNA isoforms from a single pre-mRNA and becomes dysregulated with age; intriguingly, prior work suggests that CR can attenuate age-associated splicing noise. What has remained unclear is whether AS responses to CR are coordinated across tissues and whether they scale with the level of restriction.

In a new study researchers at the University of Aberdeen, Scotland analyzed multi-tissue RNA sequencing (RNA-seq) data from mice undergoing graded CR and show that a 3-month CR intervention induces a largely tissue-specific, yet functionally convergent, AS program that is largely independent of transcriptional (gene expression) changes.

Male C57BL/6J mice were maintained for 3 months on graded CR (10%, 20%, 30%, or 40% restriction) and compared with a 12-h ad libitum feeding control. The authors analyzed RNA-seq data from six tissues, including the epididymal white adipose tissue (eWAT), liver, hypothalamus, gastrocnemius muscle, testes, and stomach, quantifying differential gene expression (DE) and differential alternative splicing/differential transcript usage (DAS/DTU) at isoform resolution (Figure 1).

The gene-expression response to CR was strongly tissue-dependent, with the eWAT showing the greatest number of differentially expressed genes (DEGs), followed by the muscle and the liver. At 40% CR, only two genes (H2-Aa and H2-Eb1; both being MHC class II components) were consistently down-regulated across all six tissues, consistent with a systemic shift away from inflammatory antigen-presentation programs. In contrast, the AS response scaled with CR level across tissues but involved largely distinct loci in each tissue: approximately 94% of loci showing DTU were not differentially expressed, highlighting largely independent regulation of splicing versus transcription. Notably, the testes displayed a pronounced AS response despite relatively modest DE changes. Despite limited overlap of specific DAS loci between tissues, functional enrichment of DAS/DTU genes converged on shared processes—including the mitochondria and oxidative phosphorylation, ribosome/translation, and RNA and protein quality-control pathways—supporting the idea of a functionally integrated, cross-tissue program. A small subset of loci showed cross-tissue isoform switches, including Gna13 and Nfe2l2, as well as genes linked to endosomal sorting and extracellular vesicle biology (e.g., Arrdc4 and Pdcd6ip).

Differential gene expression analysis of male C57BL/6 mice exposed to graded levels (10%−40%) of short-term (3-month) CR compared to mice fed a control diet of AL feeding for 12 h (AL12) each day.

(a) Bar plots showing the number of DEGs at each CR level compared to AL12 in each of the six tested tissues. DEGs of the eWAT, liver, hypothalamus, gastrocnemius muscle, testes, and stomach are shown. Colored bar represents up-regulated gene number following CR and black bar represents down-regulated gene number following CR. (b) Combination matrix of DEGs common between CR levels within each tissue irrespective of direction. Numbers indicate the number of common DEGs and their position indicates the CR levels which share that number of DEGs. Colored points indicate the other CR conditions which are being ignored. (c) Aligned volcano plots showing DE results (log2(FC) in expression and adjusted P value) for each tissue at each CR level. (d) Enrichment map showing MetaScape functional enrichment results of up-regulated DEGs from each tissue combining all CR levels. (e) Enrichment map showing MetaScape functional enrichment results of down-regulated DEGs from each tissue combining all CR levels. Nodes are scaled by the maximum gene count per functional category and subset by counts per tissue. Edges represent genes shared by different functional nodes.

Overall, the study supports a model in which AS is a dose-responsive component of the CR adaptation, potentially intersecting with hallmarks of aging such as RNA/protein homeostasis. The authors note several important limitations: short and heterogeneous sequencing read lengths and depths across tissues, relatively small group sizes, a male-only cohort, a short (3-month) intervention, and the lack of functional validation. Future work leveraging long-read sequencing, both sexes, and mechanistic perturbations will be needed to determine which CR-responsive isoform changes are causal versus correlative.

Source – Higher Education Press