SRRM4-dependent neuron-specific alternative splicing of protrudin transcripts regulates neurite outgrowth

Abstract

Alternative splicing gives rise to diversity of the proteome, and it is especially prevalent in the mammalian nervous system. Indeed, many factors that control the splicing process govern nervous system development. Among such factors, SRRM4 is an important regulator of aspects of neural differentiation including neurite outgrowth. The mechanism by which SRRM4 regulates neurite outgrowth has remained poorly understood, however. We now show that SRRM4 regulates the splicing of protrudin gene (Zfyve27) transcripts in neuronal cells. SRRM4 was found to promote splicing of protrudin pre-mRNA so as to include a microexon (exon L) encoding seven amino acids in a neuron-specific manner. The resulting protein (protrudin-L) promotes neurite outgrowth during neurogenesis. Depletion of SRRM4 in Neuro2A cells impaired inclusion of exon L in protrudin mRNA, resulting in the generation of a shorter protein isoform (protrudin-S) that is less effective at promoting neurite extension. SRRM4 was found to recognize a UGC motif that is located immediately upstream of exon L and is necessary for inclusion of exon L in the mature transcript. Deletion of exon L in Neuro2A or embryonic stem cells inhibited neurite outgrowth. Our results suggest that SRRM4 controls neurite outgrowth through regulation of alternative splicing of protrudin transcripts.

The Current State-of-the Art of LRRK2-Based Biomarker Assay Development in Parkinson’s Disease

“…Interestingly, deregulation of transcripts linked to mitochondrial dysfunction is also observed by the above studies (Ohnishi et al, 2017). For instance, a significant upregulation of nine mitochondrial genes was noted, emphasizing the critical role of mitochondria in the disease process.…”

“…In addition, another analysis using single-cell transcriptional profiles of LRRK2 multipotent neural stem cells revealed neuronal lineages with signature similar to PD. Of note, among these genes, two regulate neurite extension upon down-regulation (NRSN1) or overexpression (SRRM4) (Ohnishi et al, 2017). The authors suggest that it could explain the discrepancies in the results obtained on neurite outgrowth assaying the LRRK2 role in neurite extension (Garcia-Miralles et al, 2015).…”

MicroExonator enables systematic discovery and quantification of microexons across mouse embryonic development

“…In agreement with previous reports that have highlighted the importance of microexons for axonal and neurite outgrowth (Ohnishi et al, 2017;Quesnel-Vallières et al, 2015) , we detected 17 alternative neuronal microexons that affects 14 proteins in the PPI network that are annotated as part of the "Axon guidance" Reactome pathway. These proteins are found in the center of the network and they are connected with the domains involved with membrane trafficking and transsynaptic protein-protein interactions (Fig 5A-E).…”

Protrudin and PDZD8 contribute to neuronal integrity by promoting lipid extraction required for endosome maturation

“…Immunostaining was performed as described previously 55 . HeLa cells or primary neurons cultured on glass cover slips were fixed for 10 min at room temperature with 3.7% formaldehyde in phosphate-buffered saline (PBS) and then incubated consecutively with primary antibodies and Alexa Fluor 488- or Alexa Fluor 546-labeled secondary antibodies in PBS containing 0.5% Triton X-100.…”