TIA-1 and TIAR Activate Splicing of Alternative Exons with Weak 5′ Splice Sites followed by a U-rich Stretch on Their Own Pre-mRNAs
Caroline Le GuinerFabrice LejeuneDelphine GalianaLiliane KisterRichard BreathnachJames StéveninFabienne Del Gatto–Konczak
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TIA-1 has recently been shown to activate splicing of specific pre-mRNAs transcribed from transiently transfected minigenes, and of some 5' splice sites in vitro, but has not been shown to activate splicing of any endogenous pre-mRNA. We show here that overexpression of TIA-1 or the related protein TIAR has little effect on splicing of several endogenous pre-mRNAs containing alternative exons, but markedly activates splicing of some normally rarely used alternative exons on the TIA-1 and TIAR pre-mRNAs. These exons have weak 5' splice sites followed by U-rich stretches. When the U-rich stretch following the 5' splice site of a TIA-1 alternative exon was deleted, TIAR overexpression induced use of a cryptic 5' splice site also followed by a U-rich stretch in place of the original splice site. Using in vitro splicing assays, we have shown that TIA-1 is directly involved in activating the 5' splice sites of the TIAR alternative exons. Activation requires a downstream U-rich stretch of at least 10 residues. Our results confirm that TIA-1 activates 5' splice sites followed by U-rich sequences and show that TIAR exerts a similar activity. They suggest that both proteins may autoregulate their expression at the level of splicing.Keywords:
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Transcriptional isoforms are not just random combinations of exons. What has caused exons to be differentially spliced and whether exons with different splicing frequencies are subjected to divergent regulation by potential elements or splicing signals? Beyond the conventional classification for alternatively spliced exons (ASEs) and constitutively spliced exons (CSEs), we have classified exons from alternatively spliced human genes and their mouse orthologs (12,314 and 5,464, respectively) into four types based on their splicing frequencies. Analysis has indicated that different groups of exons presented divergent compositional and regulatory properties. Interestingly, with the decrease of splicing frequency, exons tend to have greater lengths, higher GC content, and contain more splicing elements and repetitive elements, which seem to imply that the splicing frequency is influenced by such factors. Comparison of non-alternatively spliced (NAS) mouse genes with alternatively spliced human orthologs also suggested that exons with lower splicing frequencies may be newly evolved ones which gained functions with splicing frequencies altered through the evolution. Our findings have revealed for the first time that certain factors may have critical influence on the splicing frequency, suggesting that exons with lower splicing frequencies may originate from old repetitive sequences, with splicing sites altered by mutation, gaining novel functions and become more frequently spliced.
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Voltage-gated sodium channels are the primary molecules responsible for the rising phase of action potentials in electrically excitable cells. There are 10 distinct sodium channel isoforms Nav 1.1-1.8 (SCNIA-SCN5A and SCN8A-SCNIIA) and the majority of these undergo tissue and developmentally regulated alternative splicing. Two such examples are those of the SCN8A (Nav 1.6) and SCN9A (Nav 1.7) genes. SCN8A gene contains two mutually exclusive exons, 18N and 18A. Transcripts with exon 18N have a conserved inframe stop codon that predicts the synthesis of a truncated, non functional sodium channel. This protein is expressed in fetal brain and non-neuronal tissues. Once the exon 18A is included, the resulted protein will be a functional channel, that is expressed in adult neurons ofCNS and PNS. The SCN9A exon 5N is preferentially expressed in the PNS and CNS of adult tissues and significant usage of exon 5A was found only in DRG. These two isoforms differ in one amino acid in the S3 domain I (exons 5A and 5N). This change of one amino acid induced a small shift of activation to more hyperpolarized potentials forexon SA compared with exon SN. Analysis of SeNSA pre-mRNA splicing supports a model in which exon 18A exclusion in non-neuronal tissue is regulated primarily by the presence in the cell types of several hnRNPs proteins that function through an exonic splicing silencer (ESS) found in this exon together with the absence of neuron specific Fox-I protein. In neuronal cells the absence of these hnRNPs together with the presence of neuron specific Fox-l cause the exon to be included. The SeNSA exon 18N is included innon neuronal cells due to the SR proteins that function through an exonic splicing enhancer(ESE) found in this exon. In neuronal cells the lower levels of these SR proteins cause the exon 18N to be skipped. This type of control of mutually exclusive splicing through the proteome make-up of a cell type would appear to be influential in the temporal and tissue specific splicing of SeN8A, another member of the voltage gated sodium channels and may indeed represent a more general mechanism.
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Abstract Altered splicing of transcripts, including the insulin receptor (IR) and the cardiac troponin (cTNT), is a key feature of myotonic dystrophy type I (DM1). CELF and MBNL splicing factor members regulate the splicing of those transcripts. We have previously described an alteration of Tau exon 2 splicing in DM1 brain, resulting in the favored exclusion of exon 2. However, the factors required for alternative splicing of Tau exon 2 remain undetermined. Here we report a decreased expression of CELF family member and MBNL transcripts in DM1 brains as assessed by RT‐PCR. By using cellular models with a control‐ or DM1‐like splicing pattern of Tau transcripts, we demonstrate that ETR‐3 promotes selectively the exclusion of Tau exon 2. These results together with the analysis of Tau exon 6 and IR exon 11 splicing in brain, muscle, and cell models suggest that DM1 splicing alteration of several transcripts involves various factors. © 2006 Wiley‐Liss, Inc.
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Mutations that affect mRNA splicing often produce multiple mRNA isoforms, resulting in complex molecular phenotypes. Definition of an exon and its inclusion in mature mRNA relies on joint recognition of both acceptor and donor splice sites. This study predicts cryptic and exon-skipping isoforms in mRNA produced by splicing mutations from the combined information contents (Ri, which measures binding-site strength, in bits) and distribution of the splice sites defining these exons. The total information content of an exon (Ri,total) is the sum of the Ri values of its acceptor and donor splice sites, adjusted for the self-information of the distance separating these sites, that is, the gap surprisal. Differences between total information contents of an exon (ΔRi,total) are predictive of the relative abundance of these exons in distinct processed mRNAs. Constraints on splice site and exon selection are used to eliminate nonconforming and poorly expressed isoforms. Molecular phenotypes are computed by the Automated Splice Site and Exon Definition Analysis (http://splice.uwo.ca) server. Predictions of splicing mutations were highly concordant (85.2%; n = 61) with published expression data. In silico exon definition analysis will contribute to streamlining assessment of abnormal and normal splice isoforms resulting from mutations.
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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.
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Using a transient expression system of mouse IgM mini-gene constructs in mouse B-cell lines and in fibroblast L cell, we investigated splicing of the IgM transcript. We observed that the efficiency of splicing between exons C4 and M1 (C4-to-M1 splicing), the splicing reaction leading to the production of membrane-bound form (microns) mRNA, was drastically affected by mutations in a specific portion of the downstream exon (M2). The results show that the specific exon M2 sequence activates the C4-to-M1 splicing. This activation was not observed when splicing between exons M1 and M2 was abolished by base substitutions at the splice sites. These results indicate that positioning of the downstream exon is crucial for efficient splicing of the preceding intron.
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Alternative splicing events that are conserved in orthologous genes in different species are commonly viewed as reliable evidence of authentic, functionally significant alternative splicing events. Several recent bioinformatic analyses have shown that conserved alternative exons possess several features that distinguish them from alternative exons that are species-specific. One of the most striking differences between conserved and species-specific alternative exons is the high percentage of exons that preserve the reading frame (exons whose length is an exact multiple of 3, termed symmetrical exons) among the conserved alternative exons. Here, we examined conserved alternative exons and found several features that differentiate between symmetrical and non-symmetrical alternative exons. We show that symmetrical alternative exons have a strong tendency not to disrupt protein domain structures, whereas the tendency of non-symmetrical alternative exons to overlap with different fractions of protein domains is similar to that of constitutive exons. Additionally, skipping isoforms of non-symmetrical alternative exons are strongly underrepresented, compared with their including isoforms, suggesting that skipping of a large fraction of non-symmetrical alternative exons produces transcripts that are degraded by the nonsense-mediated mRNA decay mechanism. Non-symmetrical alternative exons also show a tendency to reside in the 5′ half of the CDS. These findings suggest that alternative splicing of symmetrical and non-symmetrical exons is governed by different selective pressures and serves different purposes.
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The mis-splicing of Tau is instrumental to the development of neurofibrillary degeneration (NFD) naturopathies such as FTDP-17 in which MAPTmutations lead to a defective splicing of Tau. A mis-splicing of Tau is also observed in the brain and muscle of patients affected by myotonic dystrophy of type I (DM1). Moreover, this Tau mis-splicing is associated with the development of NFD. DM1 is a neuromuscular disease characterized by an unstable CTG expansion. This mutation is responsible for a toxic gain of RNA-function, leading to the gain or loss of function of several splicing factors, including the CELF and MBNL families, respectively. It ultimately leads to an indirect mis-splicing of numerous transcripts. In regards to Tau mis-splicing, exon-2 inclusion is highly decreased in DM1 brains. In contrast, a mis-splicing of Tau exon-10 is seldom observed in DM1 but the mechanisms triggering this mis-splicing event remain unknown. We herein aimed to gain further insights into these molecular mechanisms involved in normal and pathological splicing of Tau exons 2 and 10 using cell-based models. Two among the brains of 5 DM1 patients showed an exon-10 mis-splicing whereas exon-2 inclusion was reduced in all DM1 patients. The over-expression of long stretches of CTGrepeats promoted the exclusion of both Tau exon 2 and exon 10. Interestingly, MBNL1 loss of expression efficiently repressed Tau exon 2 inclusion but failed to modulate Tau exon-10 splicing. Analyzing the splicing functions of three major CELF splicing factors, we showed that CELF 4 acts as a repressor for exon-2 or as an enhancer for exon-10 inclusion. In sharp contrast, CELF2 repressed both exons 2 and 10 inclusions whereas CELF1 was inefficient to modulate Tau splicing. In addition, 2D electrophoresis coupled to western-blotting suggest a possible change inbrain CELF2 activity induced by its hyperphosphorylation in DM1. Altogether, we showed that MBNL1 and CELF2 are implicated in the mis-splicing of Tau-exons 2 and 10, respectively, but not CELF1. Moreover, our Tau splicing study also suggests that two different mechanisms may contribute to the mis-splicing of several targets in DM1.
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Abstract RNA binding motif (RBM) proteins, RBM10v1, RBM10v2 and RBM5 have quite similar molecular structures with a high degree of the conserved domains. Alternative splicing of RBM10 pre-mRNA produces the two mRNA variants, RBM10v1 (exon 4-included) and RBM10v2 (exon 4-skipped). RBM10v1 has a 77 amino acids-domain coded by its exon 4, but RBM10v2 lacks it. I explored the alternative splicing of the RBM10 pre-mRNA by the above three RBMs in COS-7, lung adenocarcinoma A549 and differentiated mouse cardiomyocytes H9c2 cells. Firstly, COS-7 and A549 cells express both RBM10v1 and RBM10v2 mRNA variants in contrast to H9c2 cells which express RBM10v2 variant alone. Transfection experiments of RBM10v1, RBM10v2 or RBM5 were performed to examine the alternative splicing of RBM10v1 pre-mRNA in COS-7, A549 and H9c2 cells. The result showed that RBM10v1 includes, by itself, its own exon 4 of the pre-mRNA in contrast to RBM10v2 and RBM5 which exclude the exon 4. The inclusion of the exon 4 seems to be repressed in differentiated H9c2 cells.
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