Alternative splicing of the Drosophila Dscam pre-mRNA is both temporally and spatially regulated

The Drosophila melanogaster Down syndrome cell adhesion molecule (Dscam) gene encodes an axon guidance receptor that can express 38,016 different mRNAs by virtue of alternative splicing. The Dscam gene contains 95 alternative exons that are organized into four clusters of 12, 48, 33, and 2 exons each. Although numerous Dscam mRNA isoforms can be synthesized, it remains to be determined whether different Dscam isoforms are synthesized at different times in development or in different tissues. We have investigated the alternative splicing of the Dscam exon 4 cluster, which contains 12 mutually exclusive alternative exons, and found that Dscam exon 4 alternative splicing is developmentally regulated. The most highly regulated exon, 4.2, is infrequently used in early embryos but is the predominant exon 4 variant used in adults. Moreover, the developmental regulation of exon 4.2 alternative splicing is conserved in D. yakuba. In addition, different adult tissues express distinct collections of Dscam mRNA isoforms. Given the role of Dscam in neural development, these results suggest that the regulation of alternative splicing plays an important role in determining the specificity of neuronal wiring. In addition, this work provides a framework to determine the mechanisms by which complex alternative splicing events are regulated.     

Regulation of neuron-specific alternative splicing of neurofibromatosis type 1 pre-mRNA

Neurofibromatosis type 1 (NF1) is one of the most common heritable autosomal dominant disorders. Alternative splicing modulates the function of neurofibromin, the NF1 gene product, by inserting the in-frame exon 23a into the region of NF1 mRNA that encodes the GTPase-activating protein-related domain. This insertion, which is predominantly skipped in neurons, reduces the ability of neurofibromin to regulate Ras by 10-fold. Here, we report that the neuron-specific Hu proteins control the production of the short protein isoform by suppressing inclusion of NF1 exon 23a, while TIA-1/TIAR proteins promote inclusion of this exon. We identify two binding sites for Hu proteins, located upstream and downstream of the regulated exon, and provide biochemical evidence that Hu proteins specifically block exon definition by preventing binding of essential splicing factors. In vitro analyses using nuclear extracts show that at the downstream site, Hu proteins prevent binding of U1 and U6 snRNPs to the 5′ splice site, while TIAR increases binding. Hu proteins also decrease U2AF binding at the 3′ splice site located upstream of exon 23a. In addition to providing the first mechanistic insight into tissue-specific control of NF1 splicing, these studies establish a novel strategy whereby Hu proteins regulate RNA processing.     

Activation of pre-mRNA splicing by human RNPS1 is regulated by CK2 phosphorylation

Human RNPS1 was originally characterized as a pre-mRNA splicing activator in vitro and was shown to regulate alternative splicing in vivo. RNPS1 was also identified as a protein component of the splicing-dependent mRNP complex, or exon-exon junction complex (EJC), and a role for RNPS1 in postsplicing processes has been proposed. Here we demonstrate that RNPS1 incorporates into active spliceosomes, enhances the formation of the ATP-dependent A complex, and promotes the generation of both intermediate and final spliced products. RNPS1 is phosphorylated in vivo and interacts with the CK2 (casein kinase II) protein kinase. Serine 53 (Ser-53) of RNPS1 was identified as the major phosphorylation site for CK2 in vitro, and the same site is also phosphorylated in vivo. The phosphorylation status of Ser-53 significantly affects splicing activation in vitro, but it does not perturb the nuclear localization of RNPS1. In vivo experiments indicated that the phosphorylation of RNPS1 at Ser-53 influences the efficiencies of both splicing and translation. We propose that RNPS1 is a splicing regulator whose activator function is controlled in part by CK2 phosphorylation.     

The neurofibromatosis type I pre-mRNA is a novel target of CELF protein-mediated splicing regulation

The CUG-BP and ETR-3 like factors (CELF) are a family of six highly conserved RNA-binding proteins that preferentially bind to UG-rich sequences. One of the key functions of these proteins is to mediate alternative splicing in a number of tissues, including brain, heart and muscle. To fully understand the function of CELF proteins, it is important to identify downstream targets of CELF proteins. In this communication, we report that neurofibromatosis type I (NF1) exon 23a is a novel target of CELF protein-mediated splicing regulation in neuron-like cells. NF1 regulates Ras signaling, and the isoform that excludes exon 23a shows 10 times greater ability to down-regulate Ras signaling than the isoform that includes exon 23a. Five of the six CELF proteins strongly suppress the inclusion of NF1 exon 23a. Over-expression or siRNA knockdown of these proteins in cell transfection experiments altered the levels of NF1 exon 23a inclusion. In vitro binding and splicing analyses demonstrate that CELF proteins block splicing through interfering with binding of U2AF⁶⁵. These studies, combined with our previous investigations demonstrating a role for Hu proteins and TIA-1/TIAR in controlling NF1 exon 23a inclusion, highlight the complex nature of regulation of this important alternative splicing event.     

Chemical correction of pre-mRNA splicing defects associated with sequestration of muscleblind-like 1 protein by expanded r(CAG)-containing transcripts

Recently, it was reported that expanded r(CAG) triplet repeats (r(CAG)(exp)) associated with untreatable neurological diseases cause pre-mRNA mis-splicing likely due to sequestration of muscleblind-like 1 (MBNL1) splicing factor. Bioactive small molecules that bind the 5'CAG/3'GAC motif found in r(CAG)(exp) hairpin structure were identified by using RNA binding studies and virtual screening/chemical similarity searching. Specifically, a benzylguanidine-containing small molecule was found to improve pre-mRNA alternative splicing of MBNL1-sensitive exons in cells expressing the toxic r(CAG)(exp). The compound was identified by first studying the binding of RNA 1 × 1 nucleotide internal loops to small molecules known to have affinity for nucleic acids. Those studies identified 4',6-diamidino-2-phenylindole (DAPI) as a specific binder to RNAs with the 5'CAG/3'GAC motif. DAPI was then used as a query molecule in a shape- and chemistry alignment-based virtual screen to identify compounds with improved properties, which identified 4-guanidinophenyl 4-guanidinobenzoate, a small molecule that improves pre-mRNA splicing defects associated with the r(CAG)(exp)-MBNL1 complex. This compound may facilitate the development of therapeutics to treat diseases caused by r(CAG)(exp) and could serve as a useful chemical tool to dissect the mechanisms of r(CAG)(exp) toxicity. The approach used in these studies, defining the small RNA motifs that bind small molecules with known affinity for nucleic acids and then using virtual screening to optimize them for bioactivity, may be generally applicable for designing small molecules that target other RNAs in the human genomic sequence.     

Consequences of regulated pre-mRNA splicing in the immune system

Alternative splicing is widely recognized to be a ubiquitous and crucial mechanism for generating protein diversity and regulating protein expression. Numerous immunologically relevant genes have been found to undergo alternative splicing; however, there has been little effort to develop a coherent picture of how alternative splicing might be used as a general mechanism to regulate the function of the immune system. In this review, I summarize the mechanisms by which splicing is controlled in T cells, and discuss the role of alternative splicing and alternative isoform expression in the regulation of T-cell activation and function.     

Alternative pre-mRNA splicing: the logic of combinatorial control

Alternative splicing of mRNA precursors is a versatile mechanism of gene expression regulation that accounts for a considerable proportion of proteomic complexity in higher eukaryotes. Its modulation is achieved through the combinatorial interplay of positive and negative regulatory signals present in the RNA, which are recognized by complexes composed of members of the hnRNP and SR protein families.     

Alternative splicing of CD200 is regulated by an exonic splicing enhancer and SF2/ASF

CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200_tr) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200.     

Interaction of muscleblind, CUG-BP1 and hnRNP H proteins in DM1-associated aberrant IR splicing

In myotonic dystrophy (DM1), both inactivation of muscleblind proteins and increased levels of CUG-BP1 are reported. These events have been shown to contribute independently to aberrant splicing of a subset RNAs. We demonstrate that steady-state levels of the splice regulator, hnRNP H, are elevated in DM1 myoblasts and that increased hnRNP H levels in normal myoblasts results in the inhibition of insulin receptor (IR) exon 11 splicing in a manner similar to that observed in DM1. In normal myoblasts, overexpression of either hnRNP H or CUG-BP1 results in the formation of an RNA-dependent suppressor complex consisting of both hnRNP H and CUG-BP1, which is required to maximally inhibit IR exon 11 inclusion. Elevated levels of MBNL1 show RNA-independent interaction with hnRNP H and dampen the inhibitory activity of increased hnRNP H levels on IR splicing in normal myoblasts. In DM1 myoblasts, overexpression of MBNL1 in conjunction with si-RNA mediated depletion of hnRNP H contributes to partial rescue of the IR splicing defect. These data demonstrate that coordinated physical and functional interactions between hnRNP H, CUG-BP1 and MBNL1 dictate IR splicing in normal and DM1 myoblasts.     

Alternative splicing of RNA triplets is often regulated and accelerates proteome evolution

Thousands of human genes contain introns ending in NAGNAG (N any nucleotide), where both NAGs can function as 3' splice sites, yielding isoforms that differ by inclusion/exclusion of three bases. However, few models exist for how such splicing might be regulated, and some studies have concluded that NAGNAG splicing is purely stochastic and nonfunctional. Here, we used deep RNA-Seq data from 16 human and eight mouse tissues to analyze the regulation and evolution of NAGNAG splicing. Using both biological and technical replicates to estimate false discovery rates, we estimate that at least 25% of alternatively spliced NAGNAGs undergo tissue-specific regulation in mammals, and alternative splicing of strongly tissue-specific NAGNAGs was 10 times as likely to be conserved between species as was splicing of non-tissue-specific events, implying selective maintenance. Preferential use of the distal NAG was associated with distinct sequence features, including a more distal location of the branch point and presence of a pyrimidine immediately before the first NAG, and alteration of these features in a splicing reporter shifted splicing away from the distal site. Strikingly, alignments of orthologous exons revealed a ～15-fold increase in the frequency of three base pair gaps at 3' splice sites relative to nearby exon positions in both mammals and in Drosophila. Alternative splicing of NAGNAGs in human was associated with dramatically increased frequency of exon length changes at orthologous exon boundaries in rodents, and a model involving point mutations that create, destroy, or alter NAGNAGs can explain both the increased frequency and biased codon composition of gained/lost sequence observed at the beginnings of exons. This study shows that NAGNAG alternative splicing generates widespread differences between the proteomes of mammalian tissues, and suggests that the evolutionary trajectories of mammalian proteins are strongly biased by the locations and phases of the introns that interrupt coding sequences.     

Neuronal cell type–specific alternative splicing is regulated by the KH domain protein SLM1

The unique functional properties and molecular identity of neuronal cell populations rely on cell type–specific gene expression programs. Alternative splicing represents a powerful mechanism for expanding the capacity of genomes to generate molecular diversity. Neuronal cells exhibit particularly extensive alternative splicing regulation. We report a highly selective expression of the KH domain–containing splicing regulators SLM1 and SLM2 in the mouse brain. Conditional ablation of SLM1 resulted in a severe defect in the neuronal isoform content of the polymorphic synaptic receptors neurexin-1, -2, and -3. Thus, cell type–specific expression of SLM1 provides a mechanism for shaping the molecular repertoires of synaptic adhesion molecules in neuronal populations in vivo.