Jun-ichi Takeda

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

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.

Competitive regulation of alternative splicing and alternative polyadenylation by hnRNP H and CstF64 determines acetylcholinesterase isoforms

Abstract Acetylcholinesterase (AChE), encoded by the ACHE gene, hydrolyzes the neurotransmitter acetylcholine to terminate synaptic transmission. Alternative splicing close to the 3΄ end generates three distinct isoforms of AChET, AChEH and AChER. We found that hnRNP H binds to two specific G-runs in exon 5a of human ACHE and activates the distal alternative 3΄ splice site (ss) between exons 5a and 5b to generate AChET. Specific effect of hnRNP H was corroborated by siRNA-mediated knockdown and artificial tethering of hnRNP H. Furthermore, hnRNP H competes for binding of CstF64 to the overlapping binding sites in exon 5a, and suppresses the selection of a cryptic polyadenylation site (PAS), which additionally ensures transcription of the distal 3΄ ss required for the generation of AChET. Expression levels of hnRNP H were positively correlated with the proportions of the AChET isoform in three different cell lines. HnRNP H thus critically generates AChET by enhancing the distal 3΄ ss and by suppressing the cryptic PAS. Global analysis of CLIP-seq and RNA-seq also revealed that hnRNP H competitively regulates alternative 3΄ ss and alternative PAS in other genes. We propose that hnRNP H is an essential factor that competitively regulates alternative splicing and alternative polyadenylation.

FUS-mediated regulation of alternative RNA processing in neurons: insights from global transcriptome analysis.

Fused in sarcoma (FUS) is an RNA‐binding protein that is causally associated with oncogenesis and neurodegeneration. Recently, the role of FUS in neurodegeneration has been extensively studied, because mutations in FUS are associated with amyotrophic lateral sclerosis (ALS), and the FUS protein has been identified as a major component of intracellular inclusions in neurodegenerative disorders including ALS and frontotemporal lobar degeneration. FUS is a key molecule in transcriptional regulation and RNA processing including processes such as pre‐messenger RNA (mRNA) splicing and polyadenylation. Interaction of FUS with various components of the transcription machinery, spliceosome, and the 3′‐end processing machinery has been identified. Furthermore, recent advances in high‐throughput transcriptomic profiling approaches have enabled us to determine the mechanisms of FUS‐dependent RNA processing networks at a cellular level. These analyses have revealed that depletion of FUS in neuronal cells affects alternative splicing and alternative polyadenylation of thousands of mRNAs. Gene ontology analysis has suggested that FUS‐modulated genes are implicated in neuronal functions and development. CLIP‐seq of FUS has shown that FUS is frequently clustered around these alternative sites of nascent RNA. ChIP‐seq of RNA polymerase II (RNAP II) has demonstrated that an interaction between FUS and nascent RNA downregulates local transcriptional activity of RNAP II, which is critically involved in RNA processing. Both alternative splicing and alternative polyadenylation are fundamental processes by which cells expand their transcriptomic diversity, and are particularly essential in the nervous system. Dependence of transcriptomic diversity on FUS makes the nervous system vulnerable to neurodegeneration, when FUS is functionally compromised. WIREs RNA 2016, 7:330–340. doi: 10.1002/wrna.1338

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

We humans have evolved by acquiring diversity of alternative RNA metabolisms including alternative means of splicing and transcribing non‐coding genes, and not by acquiring new coding genes. Tissue‐specific and developmental stage‐specific alternative RNA splicing is achieved by tightly regulated spatiotemporal regulation of expressions and activations of RNA‐binding proteins that recognize their cognate splicing cis‐elements on nascent RNA transcripts. Genes expressed at the neuromuscular junction are also alternatively spliced. In addition, germline mutations provoke aberrant splicing by compromising binding of RNA‐binding proteins, and cause congenital myasthenic syndromes (CMS). We present physiological splicing mechanisms of genes for agrin (AGRN), acetylcholinesterase (ACHE), MuSK (MUSK), acetylcholine receptor (AChR) α1 subunit (CHRNA1), and collagen Q (COLQ) in human, and their aberration in diseases. Splicing isoforms of AChET, AChEH, and AChER are generated by hnRNP H/F. Skipping of MUSK exon 10 makes a Wnt‐insensitive MuSK isoform, which is unique to human. Skipping of exon 10 is achieved by coordinated binding of hnRNP C, YB‐1, and hnRNP L to exon 10. Exon P3A of CHRNA1 is alternatively included to generate a non‐functional AChR α1 subunit in human. Molecular dissection of splicing mutations in patients with CMS reveals that exon P3A is alternatively skipped by hnRNP H, polypyrimidine tract‐binding protein 1, and hnRNP L. Similarly, analysis of an exonic mutation in COLQ exon 16 in a CMS patient discloses that constitutive splicing of exon 16 requires binding of serine arginine‐rich splicing factor 1. Intronic and exonic splicing mutations in CMS enable us to dissect molecular mechanisms underlying alternative and constitutive splicing of genes expressed at the neuromuscular junction.

Rules and tools to predict the splicing effects of exonic and intronic mutations.

Development of next generation sequencing technologies has enabled detection of extensive arrays of germline and somatic single nucleotide variations (SNVs) in human diseases. SNVs affecting intronic GT‐AG dinucleotides invariably compromise pre‐mRNA splicing. Most exonic SNVs introduce missense/nonsense codons, but some affect auxiliary splicing cis‐elements or generate cryptic GT‐AG dinucleotides. Similarly, most intronic SNVs are silent, but some affect canonical and auxiliary splicing cis‐elements or generate cryptic GT‐AG dinucleotides. However, prediction of the splicing effects of SNVs is challenging. The splicing effects of SNVs generating cryptic AG or disrupting canonical AG can be inferred from the AG‐scanning model. Similarly, the splicing effects of SNVs affecting the first nucleotide G of an exon can be inferred from AG‐dependence of the 3′ splice site (ss). A variety of tools have been developed for predicting the splicing effects of SNVs affecting the 5′ ss, as well as exonic and intronic splicing enhancers/silencers. In contrast, only two tools, the Human Splicing Finder and the SVM‐BP finder, are available for predicting the position of the branch point sequence. Similarly, IntSplice and Splicing based Analysis of Variants (SPANR) are the only tools to predict the splicing effects of intronic SNVs. The rules and tools introduced in this review are mostly based on observations of a limited number of genes, and no rule or tool can ensure 100% accuracy. Experimental validation is always required before any clinically relevant conclusions are drawn. Development of efficient tools to predict aberrant splicing, however, will facilitate our understanding of splicing pathomechanisms in human diseases. WIREs RNA 2018, 9:e1451. doi: 10.1002/wrna.1451

A Palindromic Motif in the -2084 to -2078 Upstream Region is Essential for ABCA12 Promoter Function in Cultured Human Keratinocytes

ATP-binding cassette transporter family A member 12 (ABCA12) is a keratinocyte transmembrane lipid transporter that plays a critical role in preserving the skin permeability barrier. Biallelic loss of function of the ABCA12 gene is causative of some forms of recessive congenital ichthyosis, an intractable disease marked by dry, thickened and scaly skin on the whole body. Genetic diagnosis is essential, although the results may occasionally be inconclusive, because some patients with low ABCA12 expression have one mutant allele and one apparently intact allele. Aside from aberrant splicing or deletion mutations, one possible explanation for such discrepancy is loss of promoter function. This study aims to elucidate the promoter region of ABCA12 and to locate the essential elements therein, thus providing the necessary information for genetic diagnostic screening of congenital ichthyosis. Close examination of the 2980-bp upstream regions of the ABCA12 gene revealed that a palindromic motif (tgagtca) at −2084 to −2078 is essential for the promoter function, and a short fragment of −2200/−1934 alone has potent promoter activity. Identification of the key promoter element of ABCA12 in this study may provide relevant information for genetic diagnosis of recessive congenital ichthyosis.

Six GU-rich (6GUR) FUS-binding motifs detected by normalization of CLIP-seq by Nascent-seq

FUS, an RNA-binding protein (RBP), is mutated or abnormally regulated in neurodegenerative disorders. FUS regulates various aspects of RNA metabolisms. FUS-binding sites are rich in GU contents and are highly degenerative. FUS-binding motifs of GGU, GGUG, GUGGU and CGCGC have been previously reported. These motifs, however, are applicable to a small fraction of FUS-binding sites. As CLIP-seq tags are enriched in genes that are highly expressed, we normalized CLIP-seq tags by Nascent-seq tags or RNA-seq tags of mouse N2a cells. Nascent-seq identifies nascent transcripts before being processed for splicing and polyadenylation. We extracted frequently observed 4-nt motifs from Nascent-seq-normalized CLIP regions, RNA-seq-normalized CLIP regions, and native CLIP regions. Specific GU-rich motifs were best detected in Nascent-seq-normalized CLIP regions. Analysis of structural motifs using Nascent-seq-normalized CLIP regions also predicted GU-rich sequence forming a stem structure. Sensitivity and specificity were calculated by examining whether the extracted motifs were present at the cross-linking-induced mutation sites (CIMS), where FUS was directly bound. We found that a combination of six motifs (UGUG, CUGG, UGGU, GCUG, GUGG, and UUGG), which were extracted from Nascent-seq-normalized CLIP-regions, had a better discriminative power than (i) motifs extracted from RNA-seq-normalized CLIP regions, (ii) motifs extracted from native CLIP regions, (iii) previously reported individual motifs, or (iv) 15 motifs in SpliceAid 2. Validation of the 6 GU-rich (6GUR) motifs using CLIP-seq of the cerebrum and the whole brain showed that the 6GUR motifs were specifically enriched in CIMS. The number of the 6GUR motifs in an uninterrupted region was counted and multiplied by four to calculate the area, which was defined as the 6GUR-Score. The 6GUR-Score of 8 or more best discriminated CIMS from CIMS-flanking regions. We propose that the 6GUR motifs predict FUS-binding sites more efficiently than previously reported individual motifs or 15 motifs in SpliceAid 2.

SRSF1 suppresses selection of intron-distal 5' splice site of DOK7 intron 4 to generate functional full-length Dok-7 protein.

Dok-7 is a non-catalytic adaptor protein that facilitates agrin-induced clustering of acetylcholine receptors (AChR) at the neuromuscular junction. Alternative selection of 5′ splice sites (SSs) of DOK7 intron 4 generates canonical and frame-shifted transcripts. We found that the canonical full-length Dok-7 enhanced AChR clustering, whereas the truncated Dok-7 did not. We identified a splicing cis-element close to the 3′ end of exon 4 by block-scanning mutagenesis. RNA affinity purification and mass spectrometry revealed that SRSF1 binds to the cis-element. Knocking down of SRSF1 enhanced selection of the intron-distal 5′ SS of DOK7 intron 4, whereas MS2-mediated artificial tethering of SRSF1 to the identified cis-element suppressed it. Isolation of an early spliceosomal complex revealed that SRSF1 inhibited association of U1 snRNP to the intron-distal 5′ SS, and rather enhanced association of U1 snRNP to the intron-proximal 5′ SS, which led to upregulation of the canonical DOK7 transcript. Integrated global analysis of CLIP-seq and RNA-seq also indicated that binding of SRSF1 immediately upstream to two competing 5′ SSs suppresses selection of the intron-distal 5′ SS in hundreds of human genes. We demonstrate that SRSF1 critically regulates alternative selection of adjacently placed 5′ SSs by modulating binding of U1 snRNP.