ABSTRACT
Fragile-X mental retardation autosomal homologue-1 (FXR1) is a muscle-enriched RNA-binding protein. FXR1 depletion is perinatally lethal in mice, Xenopus, and zebrafish; however, the mechanisms driving these phenotypes remain unclear. The FXR1 gene undergoes alternative splicing, producing multiple protein isoforms and mis-splicing has been implicated in disease. Furthermore, mutations that cause frameshifts in muscle-specific isoforms result in congenital multi-minicore myopathy. We observed that FXR1 alternative splicing is pronounced in the serine- and arginine-rich intrinsically disordered domain; these domains are known to promote biomolecular condensation. Here, we show that tissue-specific splicing of fxr1 is required for Xenopus development and alters the disordered domain of FXR1. FXR1 isoforms vary in the formation of RNA-dependent biomolecular condensates in cells and in vitro. This work shows that regulation of tissue-specific splicing can influence FXR1 condensates in muscle development and how mis-splicing promotes disease.
Subject(s)
Alternative Splicing/genetics , Muscle Cells/metabolism , RNA-Binding Proteins/genetics , Xenopus Proteins/genetics , Adult , Aged , Animals , Cells, Cultured , Female , Humans , Infant , Male , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Middle Aged , Muscle Development , Muscles/metabolism , RNA-Binding Proteins/metabolism , Xenopus , Xenopus Proteins/metabolism , Young AdultABSTRACT
BACKGROUND & AIMS: The enteroendocrine cell (EEC) lineage is important for intestinal homeostasis. It was recently shown that EEC progenitors contribute to intestinal epithelial growth and renewal, but the underlying mechanisms remain poorly understood. MicroRNAs are under-explored along the entire EEC lineage trajectory, and comparatively little is known about their contributions to intestinal homeostasis. METHODS: We leverage unbiased sequencing and eight different mouse models and sorting methods to identify microRNAs enriched along the EEC lineage trajectory. We further characterize the functional role of EEC progenitor-enriched miRNA, miR-7, by in vivo dietary study as well as ex vivo enteroid in mice. RESULTS: First, we demonstrate that miR-7 is highly enriched across the entire EEC lineage trajectory and is the most enriched miRNA in EEC progenitors relative to Lgr5+ intestinal stem cells. Next, we show in vivo that in EEC progenitors miR-7 is dramatically suppressed under dietary conditions that favor crypt division and suppress EEC abundance. We then demonstrate by functional assays in mouse enteroids that miR-7 exerts robust control of growth, as determined by budding (proxy for crypt division), EdU and PH3 staining, and likely regulates EEC abundance also. Finally, we show by single-cell RNA sequencing analysis that miR-7 regulates Xiap in progenitor/stem cells and we demonstrate in enteroids that the effects of miR-7 on mouse enteroid growth depend in part on Xiap and Egfr signaling. CONCLUSIONS: This study demonstrates for the first time that EEC progenitor cell-enriched miR-7 is altered by dietary perturbations and that it regulates growth in enteroids via intact Xiap and Egfr signaling.
Subject(s)
Enteroendocrine Cells/physiology , Inhibitor of Apoptosis Proteins/genetics , Intestinal Mucosa/physiology , MicroRNAs/metabolism , Stem Cells/physiology , Animals , Cell Lineage/genetics , Cell Proliferation/genetics , Cells, Cultured , Computational Biology , ErbB Receptors/metabolism , Feeding Behavior/physiology , Female , Inhibitor of Apoptosis Proteins/metabolism , Intestinal Mucosa/cytology , Male , Mice , Mice, Transgenic , Models, Animal , Organoids , Primary Cell Culture , RNA-Seq , Signal Transduction/genetics , Single-Cell AnalysisABSTRACT
The cell biology field has outstanding working knowledge of the fundamentals of membrane-trafficking pathways, which are of critical importance in health and disease. Current challenges include understanding how trafficking pathways are fine-tuned for specialized tissue functions in vivo and during development. In parallel, the ENCODE project and numerous genetic studies have revealed that alternative splicing regulates gene expression in tissues and throughout development at a post-transcriptional level. This Review summarizes recent discoveries demonstrating that alternative splicing affects tissue specialization and membrane-trafficking proteins during development, and examines how this regulation is altered in human disease. We first discuss how alternative splicing of clathrin, SNAREs and BAR-domain proteins influences endocytosis, secretion and membrane dynamics, respectively. We then focus on the role of RNA-binding proteins in the regulation of splicing of membrane-trafficking proteins in health and disease. Overall, our aim is to comprehensively summarize how trafficking is molecularly influenced by alternative splicing and identify future directions centered on its physiological relevance.
