HnRNP C, YB-1 and hnRNP L coordinately enhance skipping of human MUSK exon 10 to generate a Wnt-insensitive MuSK isoform

Abstract

Muscle specific receptor tyrosine kinase (MuSK) is an essential postsynaptic transmembrane molecule that mediates clustering of acetylcholine receptors (AChR). MUSK exon 10 is alternatively skipped in human, but not in mouse. Skipping of this exon disrupts a cysteine-rich region (Fz-CRD), which is essential for Wnt-mediated AChR clustering. To investigate the underlying mechanisms of alternative splicing, we exploited block-scanning mutagenesis with human minigene and identified a 20-nucleotide block that contained exonic splicing silencers. Using RNA-affinity purification, mass spectrometry, and Western blotting, we identified that hnRNP C, YB-1 and hnRNP L are bound to MUSK exon 10. siRNA-mediated knockdown and cDNA overexpression confirmed the additive, as well as the independent, splicing suppressing effects of hnRNP C, YB-1 and hnRNP L. Antibody-mediated in vitro protein depletion and scanning mutagenesis additionally revealed that binding of hnRNP C to RNA subsequently promotes binding of YB-1 and hnRNP L to the immediate downstream sites and enhances exon skipping. Simultaneous tethering of two splicing trans-factors to the target confirmed the cooperative effect of YB-1 and hnRNP L on hnRNP C-mediated exon skipping. Search for a similar motif in the human genome revealed nine alternative exons that were individually or coordinately regulated by hnRNP C and YB-1.

Anomalous Structural Transition and Electrical Transport Behaviors in Compressed Zn2SnO4: Effect of Interface

“…Zn 2 SnO 4 (ZTO) is an important n-type transparent conducting oxide, which has been attracting intense interest due to its outstanding performance and potential use in applications such as optoelectronic devices, anode materials for dye-sensitized solar cells, and photocatalysts for the degradation of organic pollutants8910. At ambient conditions, ZTO crystallizes into a typical inverse spinel structure with the Fd 3 m space group111213.…”

Splicing Aberrations in Congenital Myasthenic Syndromes

“…Dissection of p.E415G in exon 16 of COLQ revealed that SRSF1 is essential for inclusion of constitutive exon 16 [12]. Dissection of IVS3-8G>A in intron 3 of CHRNA1 [21] and P3A23'G>A in exon P3A of CHRNA1 [22] disclosed that alternative skipping of exon P3A is mediated by hnRNP H, PTBP1, and hnRNP L. Although the analysis was not based on CMS mutations, we found that HnRNP C, YB-1, and hnRNP L cooperatively enhance skipping of MUSK exon 10 and make a Wnt-insensitive MuSK isoform [28]. Splicing mutations prompted us to dissect the underlying mechanisms associated with the mutations.…”

“…MUSK exon 10 is alternatively spliced in human but not in mouse [28]. MuSK mediates AChR clustering at the motor endplate and exon 10 encodes a frizzled-like cysteine-rich domain (Fz-CRD), which is essential for Wnt-mediated AChR clustering.…”

“…MuSK mediates AChR clustering at the motor endplate and exon 10 encodes a frizzled-like cysteine-rich domain (Fz-CRD), which is essential for Wnt-mediated AChR clustering. We characterized splicing cis-elements in MUSK exon 10, where binding of hnRNP C promotes binding of YB-1 and hnRNP L to the immediate downstream sites and these three molecules cooperatively induce skipping of exon 10 [28]. Therefore, mutations in the identified splicing cis-elements in exon 10 potentially affect the function of MuSK by changing alternative splicing of exon 10, and can subsequently impede neuromuscular signal transmission.…”

Splicing regulation and dysregulation of cholinergic genes expressed at the neuromuscular junction

“…(b) Binding of hn RNP C to RNA enhances binding of YB ‐1 and hn RNP L to the immediate downstream sites and enhances skipping of exon 10 (Nasrin et al . ). The indicated cis ‐element starts from 81 nucleotides downstream of the intron 9/exon 10 junction, and ends at 165 nucleotides upstream of the exon 10/intron 10 junction.…”