Nuclear Speckles Regulate Splicing During Muscle Stem Cell Activation and Aging

Abstract Skeletal muscle contains a population of adult stem cells called satellite cells or muscle stem cells (MuSCs) that are responsible for regeneration after injury. MuSCs utilize gene expression programs to maintain quiescence and differentiate after injury and a key regulator of gene expression is splicing, which uniquely changes when transcripts interact with nuclear speckles (NS). NS are membrane-less biomolecular condensates that phase separate proteins, RNAs and chromatin, but how these organelles regulate molecular processes in MuSCs remains unknown. Herein, we build a comprehensive and systems-level understanding of NS influence on alternative splicing, transcriptional regulation and stem cell function before and after injury and in aging. We establish that NS increased in size and number in MuSCs following injury and influence MuSC activation dynamics. We generated a catalog of isoform-resolved splicing events and linked how RNA interactions with NS amplify splicing completion during the injury response. In old age, MuSCs lose NS, yet shifted towards longer, more completely spliced isoforms enriched for RNA binding protein motifs and multivalency. Our studies unveil evidence that RNA interactions with NS shape stem cell state and regenerative responses but are attenuated in old age. HIGHLIGHTS 3D super-resolution imaging of nuclear speckles in muscle stem cells before and after muscle injury shows intricate relationship with activation Isoform-resolved profiling of muscle stem cells shows increases in gene expression and splicing during injury response Mapping RNA interactions with nuclear speckles shows RNAs undergo strongest splicing when proximal to nuclear speckles Old aged muscle stem cells lose nuclear speckles and display aberrant splicing, with longer transcripts, more exons, and increased RNA binding protein motifs Full Text Availability The license terms selected by the author(s) for this preprint version do not permit archiving in PMC. The full text is available from the preprint server.