Modulation of alternative splicing of trafficking genes by genome editing reveals functional consequences in muscle biology

Abstract

Alternative splicing is a regulatory mechanism by which multiple mRNA isoforms are generated from single genes. Numerous genes that encode membrane trafficking proteins are alternatively spliced. However, there is limited information about the functional consequences that result from these splicing transitions. Here, we developed appropriate tools to study the functional impact of alternative splicing in development within the most in vivo context. Secondly, we provided evidence of the physiological implications of splicing regulation during muscle development. Our previous work in mouse heart development identified three trafficking genes that are regulated by alternative splicing between birth and adulthood: the clathrin heavy chain, the clathrin light chain-a, and the trafficking kinesin binding protein-1. Here, we demonstrated that alternative splicing regulation of these three genes is tissue- and developmental stage-specific. To identify the functional consequences of splicing regulation in vivo, we used genome editing to block the neonatal-to-adult splicing transitions. We characterized the phenotype of one of these mouse lines and demonstrated that when splicing regulation of the clathrin heavy chain gene is prevented mice exhibit an increase in body and muscle weights which is due to an enlargement in myofiber size. The significance of this work has two components. First, we revealed novel roles of the clathrin heavy chain in muscle growth and showed that its regulation by alternative splicing contributes to muscle development. Second, the new mouse lines will provide a useful tool to study how splicing regulation of three trafficking genes affects tissue identity acquisition and maturation in vivo.

Alternative splicing of clathrin heavy chain contributes to the switch from coated pits to plaques

“…To further confirm the contribution of CLTC -ex31 in the formation of clathrin plaques, we included CLTC -ex31 in cells that mainly displayed CCPs. Because CELF1 splicing factor has been shown to regulate CLTC -ex31 alternative splicing during striated muscle development ( Blue et al, 2018 ), we took advantage of this regulation to promote CLTC -ex31 inclusion in myoblasts following siRNA-mediated CELF depletion ( Fig. 3 A ).…”

Clathrin’s life beyond 40: Connecting biochemistry with physiology and disease

“…As might be expected, there appear to be multiple routes to curvature, some involving growth of structures with constant curvature from the outset and others involving flat lattice rearrangement. Alternative splicing of CHC17 in skeletal muscle, introducing seven residues near the trimerisation domain, promotes flat lattice formation and loss of this splicing event is associated with myotonic dystrophy in humans [33] and muscle hypertrophy in mice [34]. In tissues without this splicing event, flat lattices may be actively maintained by accessory proteins, cargo interaction, membrane tension and cytoskeletal interactions.…”

“…Phenotypes for clathrin-associated defects have, so far, been detected mainly in specialized cells with highly differentiated membrane traffic such as neurons, muscle cells, lymphocytes and epithelia. In addition to neuronal and lymphocyte phenotypes for CLCa or CLCb depletion [15,62] and the muscle phenotypes for defective CHC splicing [33,34], mutation in the AAGAB protein affecting AP2 assembly manifests as the human skin disease punctate palmoplantar keratoderma type 1 [40] and AP1 mutations are associated with congenital enteropathy [63]. Deficiency of the CME-nucleating protein FCHO1 causes a T-lymphocyte defect [64], and loss of the assembly regulator AP180 affects murine cochlear inner hair cells [65] in addition to neuronal function, and the latter is seen for many other adaptor and assembly factor defects [66].…”

A Clathrin light chain A reporter mouse for in vivo imaging of endocytosis

“…An additional band of about 40 to 50 kDa was detected in tissue lysates of small intestine and kidney. This additional band could be a result of tissue specific alternative splicing in these organs, but also exon-skipping due to the artificial introduced eGFP would be possible [51]. Partial degradation to a slightly smaller band, as observed in intestine, however, cannot fully be excluded.…”