Alternative splicing produces structural and functional changes in CUGBP2
Hitoshi SuzukiMakoto TakeuchiAyumu SugiyamaAHM Khurshid AlamLuyen Thi VuYoshiharu SekiyamaDam HieuShinya OhkiToshifumi Tsukahara
17
Citation
39
Reference
10
Related Paper
Citation Trend
Abstract:
CELF/Bruno-like proteins play multiple roles, including the regulation of alternative splicing and translation. These RNA-binding proteins contain two RNA recognition motif (RRM) domains at the N-terminus and another RRM at the C-terminus. CUGBP2 is a member of this family of proteins that possesses several alternatively spliced exons. The present study investigated the expression of exon 14, which is an alternatively spliced exon and encodes the first half of the third RRM of CUGBP2. The ratio of exon 14 skipping product (R3δ) to its inclusion was reduced in neuronal cells induced from P19 cells and in the brain. Although full length CUGBP2 and the CUGBP2 R3δ isoforms showed a similar effect on the inclusion of the smooth muscle (SM) exon of the ACTN1 gene, these isoforms showed an opposite effect on the skipping of exon 11 in the insulin receptor gene. In addition, examination of structural changes in these isoforms by molecular dynamics simulation and NMR spectrometry suggested that the third RRM of R3δ isoform was flexible and did not form an RRM structure. Our results suggest that CUGBP2 regulates the splicing of ACTN1 and insulin receptor by different mechanisms. Alternative splicing of CUGBP2 exon 14 contributes to the regulation of the splicing of the insulin receptor. The present findings specifically show how alternative splicing events that result in three-dimensional structural changes in CUGBP2 can lead to changes in its biological activity.Keywords:
RNA recognition motif
Exon skipping
Exonic splicing enhancer
Splicing factor
RBM10, originally called S1‐1, is a nuclear RNA‐binding protein with domains characteristic of RNA processing proteins. It has been reported that RBM10 constitutes spliceosome complexes and that RBM5, a close homologue of RBM10, regulates alternative splicing of apoptosis‐related genes, Fas and cFLIP . In this study, we examined whether RBM10 has a regulatory function in splicing similar to RBM5, and determined that it indeed regulates alternative splicing of Fas and Bcl‐x genes. RBM10 promotes exon skipping of Fas pre‐mRNA as well as selection of an internal 5′‐splice site in Bcl‐x pre‐mRNA. We propose a consensus RBM10‐binding sequence at 5′‐splice sites of target exons and a mechanistic model of RBM10 action in the alternative splicing.
Spliceosome
Splicing factor
Exonic splicing enhancer
Minigene
SR protein
Cite
Citations (69)
Splicing factor RBM10 and its close homologues RBM5 and RBM6 govern the splicing of oncogenes such as Fas, NUMB, and Bcl-X. The molecular architecture of these proteins includes zinc fingers (ZnFs) and RNA recognition motifs (RRMs). Three of these domains in RBM10 that constitute the RNA binding part of this splicing factor were found to individually bind RNAs with micromolar affinities. It was thus of interest to further investigate the structural basis of the well-documented high-affinity RNA recognition by RMB10. Here, we investigated RNA binding by combinations of two or three of these domains and discovered that a polypeptide containing RRM1, ZnF1, and RRM2 connected by their natural linkers recognizes a specific sequence of the Fas exon 6 mRNA with an affinity of 20 nM. Nuclear magnetic resonance structures of the RBM10 domains RRM1 and ZnF1 and the natural V354del isoform of RRM2 further confirmed that the interactions with RNA are driven by canonical RNA recognition elements. The well-known high-fidelity RNA splice site recognition by RBM10, and probably by RBM5 and RBM6, can thus be largely rationalized by a cooperative binding action of RRM and ZnF domains.
Splicing factor
SR protein
Exonic splicing enhancer
RNA recognition motif
Cite
Citations (9)
The human IGF-I gene has six exons, four of which are alternatively spliced.Variations in splicing involving exon 5 may occur, depending on the tissue type and hormonal environment.To study the regulation of splicing to IGF-I exon 5, we established an in vitro splicing assay, using a model pre-mRNA containing IGF-I exons 4 and 5 and part of the intervening intron.Using a series of deletion mutants, we identified an 18-nucleotide purine-rich splicing enhancer in exon 5 that increases the splicing efficiency of the upstream intron from 6 to 35%.We show that the serinearginine protein splicing factor-2/alternative splicing factor specifically promotes splicing in cultured cells and in vitro and is recruited to the spliceosome in an enhancerspecific manner.Our findings are consistent with a role for splicing factor-2/alternative splicing factor in the regulation of splicing of IGF-I alternative exon 5 via a purine-rich exonic splicing enhancer.
Exonic splicing enhancer
Minigene
Splicing factor
SR protein
Spliceosome
Polypyrimidine tract
Protein splicing
Exon skipping
Cite
Citations (14)
Q Sun, A Mayeda, R K Hampson, A R Krainer, and F M Rottman Department of Molecular Biology and Microbiology, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106-4960.
Splicing factor
Exonic splicing enhancer
Minigene
SR protein
Protein splicing
Precursor mRNA
Cite
Citations (286)
Many splicing factors in vertebrate nuclei belong to a class of evolutionarily conserved proteins containing arginine/serine (RS) or serine/arginine (SR) domains. Previously, we demonstrated the existence of SR splicing factors in plants. In this article, we report on a novel member of this splicing factor family from Arabidopsis designated atRSp31. It has one N-terminal RNA recognition motif and a C-terminal RS domain highly enriched in arginines. The RNA recognition motif shows significant homology to all animal SR proteins identified to date, but the intermediate region does not show any homology to any other known protein. Subsequently, we characterized two cDNAs from Arabidopsis that are highly homologous to atRSp31 (designated atRSp35 and atRSp41). Their deduced amino acid sequences indicate that these proteins constitute a new family of RS domain splicing factors. Purified recombinant atRSp31 is able to restore splicing in SR protein-deficient human S100 extracts. This indicates that atRSp31 is a true plant splicing factor and plays a crucial role in splicing, similar to that of other RS splicing factors. All of the three genes are differentially expressed in a tissue-specific manner. The isolation of this new plant splicing factor family enlarges the essential group of RS domain splicing factors. Furthermore, because no animal equivalent to this protein family has been identified to date, our results suggest that these proteins play key roles in constitutive and alternative splicing in plants.
Splicing factor
SR protein
RNA recognition motif
Exonic splicing enhancer
Homology
Protein splicing
Cite
Citations (81)
Npl3 is an SR‐like protein found in Saccharomyces cerevisiae that functions in co‐transcriptional pre‐mRNA splicing by promoting the recruitment of splicing factors to the pre‐mRNA during transcription. It contains two RNA recognition motifs (RRMs), an RS/RGG domain rich in arginine and serine repeats, and an uncharacterized N‐terminus. While the RRM domains are known to be required for Npl3 binding to RNA, the role of each of these functional domains in splicing is currently unknown. Here we have generated a panel of flag‐tagged Npl3 proteins lacking one or more of the protein domains. We identified genetic interactions between Npl3 mutations lacking the RS/RGG domain and mutations in genes that encode RNA splicing factors, suggesting that the RS/RGG domain is important for RNA splicing. Indeed, we show that deletion of the RS/RGG domain of Npl3 causes a significant decrease in RNA splicing efficiency of Npl3‐dependent pre‐mRNAs. In addition, deletion of the RS/RGG domain abolishes the ability of Npl3 to interact with splicing factors and proteins important for transcription, which strongly suggest that the RS domain is required to recruit the splicing machinery during transcription. This work demonstrates for the first time that the RS/RGG domain is required for the activity of Npl3 in RNA splicing.
SR protein
Splicing factor
Exonic splicing enhancer
Transcription
RNA recognition motif
Cite
Citations (1)
Serine/arginine (SR) proteins, one of the major families of alternative-splicing regulators in Eukarya, have two types of RNA-recognition motifs (RRMs): a canonical RRM and a pseudo-RRM. Although pseudo-RRMs are crucial for activity of SR proteins, their mode of action was unknown. By solving the structure of the human SRSF1 pseudo-RRM bound to RNA, we discovered a very unusual and sequence-specific RNA-binding mode that is centered on one α-helix and does not involve the β-sheet surface, which typically mediates RNA binding by RRMs. Remarkably, this mode of binding is conserved in all pseudo-RRMs tested. Furthermore, the isolated pseudo-RRM is sufficient to regulate splicing of about half of the SRSF1 target genes tested, and the bound α-helix is a pivotal element for this function. Our results strongly suggest that SR proteins with a pseudo-RRM frequently regulate splicing by competing with, rather than recruiting, spliceosome components, using solely this unusual RRM.
RNA recognition motif
SR protein
Spliceosome
Splicing factor
Cite
Citations (119)
SR protein
Splicing factor
Exonic splicing enhancer
Spliceosome
RNA recognition motif
Polypyrimidine tract
Conserved sequence
Cite
Citations (33)
The human IGF-I gene has six exons, four of which are alternatively spliced. Variations in splicing involving exon 5 may occur, depending on the tissue type and hormonal environment. To study the regulation of splicing to IGF-I exon 5, we established an in vitro splicing assay, using a model pre-mRNA containing IGF-I exons 4 and 5 and part of the intervening intron. Using a series of deletion mutants, we identified an 18-nucleotide purine-rich splicing enhancer in exon 5 that increases the splicing efficiency of the upstream intron from 6 to 35%. We show that the serine-arginine protein splicing factor-2/alternative splicing factor specifically promotes splicing in cultured cells and in vitro and is recruited to the spliceosome in an enhancer-specific manner. Our findings are consistent with a role for splicing factor-2/alternative splicing factor in the regulation of splicing of IGF-I alternative exon 5 via a purine-rich exonic splicing enhancer.
Exonic splicing enhancer
Minigene
Splicing factor
SR protein
Polypyrimidine tract
Protein splicing
Spliceosome
Exon skipping
Cite
Citations (46)
Splicing factor
SR protein
RNA recognition motif
Exonic splicing enhancer
Structural motif
Protein splicing
Cite
Citations (82)