Exon identity established through differential antagonism between exonic splicing silencer-bound hnRNP A1 and enhancer-bound SR proteins.

SR proteins recognize exonic splicing enhancer (ESE) elements and promote exon use, whereas certain hnRNP proteins bind to exonic splicing silencer (ESS) elements and block exon recognition. We investigated how ESS3 in HIV-1 tat exon 3 blocks splicing promoted by one SR protein (SC35) but not another (SF2/ASF). hnRNP A1 mediates silencing by binding initially to a required high-affinity site in ESS3, which then promotes further hnRNP A1 association with the upstream region of the exon. Both SC35 and SF2/ASF recognize upstream ESE motifs, but only SF2/ASF prevents secondary hnRNP A1 binding, presumably by blocking its cooperative propagation along the exon. The differential antagonism between a negative and two positive regulators exemplifies how inclusion of an alternative exon can be modulated.     

hnRNP H and hnRNP F complex with Fox2 to silence fibroblast growth factor receptor 2 exon IIIc

The heterogeneous nuclear ribonucleoprotein H (hnRNP) family of proteins has been shown to activate exon inclusion by binding intronic G triplets. Much less is known, however, about how hnRNP H and hnRNP F silence exons. In this study, we identify hnRNP H and hnRNP F proteins as being novel silencers of fibroblast growth factor receptor 2 exon IIIc. In cells that normally include this exon, we show that the overexpression of either hnRNP H1 or hnRNP F resulted in the dramatic silencing of exon IIIc. In cells that normally skip exon IIIc, skipping was disrupted when RNA interference was used to knock down both hnRNP H and hnRNP F. We show that an exonic GGG motif overlapped a critical exonic splicing enhancer, which was predicted to bind the SR protein ASF/SF2. Furthermore, the expression of ASF/SF2 reversed the silencing of exon IIIc caused by the expression of hnRNP H1. We show that hnRNP H and hnRNP F proteins are present in a complex with Fox2 and that the presence of Fox allows hnRNP H1 to better compete with ASF/SF2 for binding to exon IIIc. These results establish hnRNP H and hnRNP F as being repressors of exon inclusion and suggest that Fox proteins enhance their ability to antagonize ASF/SF2.     

A disease-associated polymorphism alters splicing of the human CD45 phosphatase gene by disrupting combinatorial repression by heterogeneous nuclear ribonucleoproteins (hnRNPs)

Alternative splicing is typically controlled by complexes of regulatory proteins that bind to sequences within or flanking variable exons. The identification of regulatory sequence motifs and the characterization of sequence motifs bound by splicing regulatory proteins have been essential to predicting splicing regulation. The activation-responsive sequence (ARS) motif has previously been identified in several exons that undergo changes in splicing upon T cell activation. hnRNP L binds to this ARS motif and regulates ARS-containing exons; however, hnRNP L does not function alone. Interestingly, the proteins that bind together with hnRNP L differ for different exons that contain the ARS core motif. Here we undertake a systematic mutational analysis of the best characterized context of the ARS motif, namely the ESS1 sequence from CD45 exon 4, to understand the determinants of binding specificity among the components of the ESS1 regulatory complex and the relationship between protein binding and function. We demonstrate that different mutations within the ARS motif affect specific aspects of regulatory function and disrupt the binding of distinct proteins. Most notably, we demonstrate that the C77G polymorphism, which correlates with autoimmune disease susceptibility in humans, disrupts exon silencing by preventing the redundant activity of hnRNPs K and E2 to compensate for the weakened function of hnRNP L. Therefore, these studies provide an important example of the functional relevance of combinatorial function in splicing regulation and suggest that additional polymorphisms may similarly disrupt function of the ESS1 silencer.     

Silencer elements as possible inhibitors of pseudoexon splicing

Human pre-mRNAs contain a definite number of exons and several pseudoexons which are located within intronic regions. We applied a computational approach to address the question of how pseudoexons are neglected in favor of exons and to possibly identify sequence elements preventing pseudoexon splicing. A search for possible splicing silencers was carried out on a pseudoexon selection that resembled exons in terms of splice site strength and exon splicing enhancer (ESE) representation; three motifs were retrieved through hexamer composition comparisons. One of these functions as a powerful silencer in transfection-based splicing assays and matches a previously identified silencer sequence with hnRNP H binding ability. The other two motifs are novel and failed to induce skipping of a constitutive exon, indicating that they might act as weak repressors or in synergy with other unidentified elements. All three motifs are enriched in pseudoexons compared with intronic regions and display higher frequencies in intronless gene-coding sequences compared with exons. We consider that a subpopulation of pseudoexons might rely on negative regulators for splicing repression; this hypothesis, if experimentally verified, might improve our understanding of exonic splicing regulatory sequences and provide the identification of a novel mutation target for human genetic diseases.     

Conserved sequence elements associated with exon skipping

One of the major forms of alternative splicing, which generates multiple mRNA isoforms differing in the precise combinations of their exon sequences, is exon skipping. While in constitutive splicing all exons are included, in the skipped pattern(s) one or more exons are skipped. The regulation of this process is still not well understood; so far, cis- regulatory elements (such as exonic splicing enhancers) were identified in individual cases. We therefore set to investigate the possibility that exon skipping is controlled by sequences in the adjacent introns. We employed a computer analysis on 54 sequences documented as undergoing exon skipping, and identified two motifs both in the upstream and downstream introns of the skipped exons. One motif is highly enriched in pyrimidines (mostly C residues), and the other motif is highly enriched in purines (mostly G residues). The two motifs differ from the known cis-elements present at the 5′ and 3′ splice site. Interestingly, the two motifs are complementary, and their relative positional order is conserved in the flanking introns. These suggest that base pairing interactions can underlie a mechanism that involves secondary structure to regulate exon skipping. Remarkably, the two motifs are conserved in mouse orthologous genes that undergo exon skipping.     

hnRNP A1 controls HIV-1 mRNA splicing through cooperative binding to intron and exon splicing silencers in the context of a conserved secondary structure

The removal of the second intron in the HIV-1 rev/tat pre-mRNAs, which involves the joining of splice site SD4 to SA7, is inhibited by hnRNP A1 by a mechanism that requires the intronic splicing silencer (ISS) and the exon splicing silencer (ESS3). In this study, we have determined the RNA secondary structure and the hnRNP A1 binding sites within the 3' splice site region by phylogenetic comparison and chemical/enzymatic probing. A biochemical characterization of the RNA/protein complexes demonstrates that hnRNP A1 binds specifically to primarily three sites, the ISS, a novel UAG motif in the exon splicing enhancer (ESE) and the ESS3 element, which are all situated in experimentally supported stem loop structures. A mutational analysis of the ISS region revealed that the core hnRNP A1 binding site directly overlaps with a major branchpoint used in splicing to SA7, thereby providing a direct explanation for the inhibition of U2 snRNP association with the pre-mRNA by hnRNP A1. Binding of hnRNP A1 to the ISS core site is inhibited by RNA structure but strongly stimulated by the exonic silencer, ESS3. Moreover, the ISS also stimulate binding of hnRNP A1 to the exonic splicing regulators ESS3 and the ESE. Our results suggest a model where a network is formed between hnRNP A1 molecules situated at discrete sites in the intron and exon and that these interactions preclude the recognition of essential splicing signals including the branch point.     

Heterogeneous ribonucleoprotein C displays a repressor activity mediated by T-cell intracellular antigen-1-related/like protein to modulate Fas exon 6 splicing through a mechanism involving Hu antigen R

T-cell intracellular antigen (TIA)-proteins are known regulators of alternative pre-mRNA splicing. In this study, pull-down experiments and mass spectrometry indicate that TIAR/TIAL1 and hnRNP C1/C2 are associated in HeLa nuclear extracts. Co-immunoprecipitation and GST-pull-down assays confirmed this interaction. Interestingly, binding requires the glutamine-rich (Q-rich) C-terminal domain of TIAR and the leucine-rich plus acidic residues-rich C-terminal domains of hnRNP C1/C2. This interaction also occurs in an RNA-dependent manner. Recombinant GFP-TIAR and RFP-hnRNP C1 proteins display partial nuclear co-localization when overexpressed in HeLa cells, and this requires the Q-rich domain of TIAR. hnRNP C1 overexpression in the presence of rate-limiting amounts of TIAR in HeLa and HEK293 cells affects alternative splicing of Fas and FGFR2 minigenes, promoting Fas exon 6 and FGFR2 exon K-SAM skipping, respectively. The repressor activity of hnRNP C1 on Fas exon 6 splicing is mediated by Hu antigen R (HuR). Experiments involving tethering approaches showed that the repressor capacity of hnRNP C1 is associated with an exonic splicing silencer in Fas exon 6. This effect was reversed by splice-site strengthening and is linked to its basic leucine zipper-like motif. These results suggest that hnRNP C1/C2 acts as a bridge between HuR and TIAR to modulate alternative Fas splicing.     

Position-dependent effects of hnRNP A1/A2 in SMN1/2 exon7 splicing

Heterogeneous nuclear ribonucleoprotein A1 and A2 (hnRNP A1/2) is a ubiquitously expressed RNA binding protein known to bind intronic or exonic splicing silencer. Binding of hnRNP A1/2 to survival of motor neuron gene (SMN1/2) exon 7 and flanking sequences strongly inhibits the inclusion of exon 7, which causes spinal muscular atrophy, a common genetic disorder. However, the role of hnRNP A1/2 on the side away from exon 7 is unclear. Here using antisense oligonucleotides, we fished an intronic splicing enhancer (ISE) near the 3′-splice site (SS) of intron 7 of SMN1/2. Mutagenesis identified the efficient motif of the ISE as “UAGUAGG”, coupled with RNA pull down and protein overexpression, we proved that hnRNP A1/2 binding to the ISE promotes the inclusion of SMN1/2 exon 7. Using MS2-tethering array and “UAGGGU” motif walking, we further uncovered that effects of hnRNP A1/2 on SMN1/2 exon 7 splicing are position-dependent: exon 7 inclusion is inhibited when hnRNP A1/2 binds proximal to the 5′SS of intron 7, promoted when its binds proximal to the 3′SS. These data provide new insights into the splicing regulatory mechanism of SMN1/2.     

An extended inhibitory context causes skipping of exon 7 of SMN2 in spinal muscular atrophy

SMN1 and SMN2 represent the two nearly identical copies of the survival of motor neuron gene in humans. The most frequent cause of spinal muscular atrophy (SMA) is loss of SMN1 accompanied by the inability of SMN2 to compensate due to an inhibitory mutation at position 6 in exon 7 (C6U) that causes exon 7 exclusion. How this single exonic nucleotide regulates exon 7 recognition has been of major interest. Based on score matrices and in vitro assays, abrogation of an exonic splicing enhancer (ESE) associated with SF2/ASF has been considered as the cause of exon 7 exclusion. However, a recent report supports the creation of an exonic splicing silencer (ESS) associated with hnRNP A1 as the determining factor for exon 7 exclusion. Here we show that C6U strengthens an inhibitory context that covers a larger sequence than the hnRNP A1 binding site. The inhibitory context can also be strengthened by the addition of a G residue at the first position of exon 7 in SMN1, promoting exon 7 skipping despite the presence of SF2/ASF binding site. Through in vivo selection and a series of mutations we demonstrate that the strengthening of the extended inhibitory context at the 5' end of exon 7 is exercised through overlapping sequence motifs that collaborate to regulate exon usage.     

Roles for SR proteins and hnRNP A1 in the regulation of c-src exon N1

The splicing of the c-src exon N1 is controlled by an intricate combination of positive and negative RNA elements. Most previous work on these sequences focused on intronic elements found upstream and downstream of exon N1. However, it was demonstrated that the 5' half of the N1 exon itself acts as a splicing enhancer in vivo. Here we examine the function of this regulatory element in vitro. We show that a mutation in this sequence decreases splicing of the N1 exon in vitro. Proteins binding to this element were identified as hnRNP A1, hnRNP H, hnRNP F, and SF2/ASF by site-specific cross-linking and immunoprecipitation. The binding of these proteins to the RNA was eliminated by a mutation in the exonic element. The activities of hnRNP A1 and SF2/ASF on N1 splicing were examined by adding purified protein to in vitro splicing reactions. SF2/ASF and another SR protein, SC35, are both able to stimulate splicing of c-src pre-mRNA. However, splicing activation by SF2/ASF is dependent on the N1 exon enhancer element whereas activation by SC35 is not. In contrast to SF2/ASF and in agreement with other systems, hnRNP A1 repressed c-src splicing in vitro. The negative activity of hnRNP A1 on splicing was compared with that of PTB, a protein previously demonstrated to repress splicing in this system. Both proteins repress exon N1 splicing, and both counteract the enhancing activity of the SR proteins. Removal of the PTB binding sites upstream of N1 prevents PTB-mediated repression but does not affect A1-mediated repression. Thus, hnRNP A1 and PTB use different mechanisms to repress c-src splicing. Our results link the activity of these well-known exonic splicing regulators, SF2/ASF and hnRNP A1, to the splicing of an exon primarily controlled by intronic factors.     

Depolarization and CaM kinase IV modulate NMDA receptor splicing through two essential RNA elements

Alternative splicing controls the activity of many proteins important for neuronal excitation, but the signal-transduction pathways that affect spliced isoform expression are not well understood. One particularly interesting system of alternative splicing is exon 21 (E21) of the NMDA receptor 1 (NMDAR1 E21), which controls the trafficking of NMDA receptors to the plasma membrane and is repressed by Ca⁺⁺/calmodulin-dependent protein kinase (CaMK) IV signaling. Here, we characterize the splicing of NMDAR1 E21. We find that E21 splicing is reversibly repressed by neuronal depolarization, and we identify two RNA elements within the exon that function together to mediate the inducible repression. One of these exonic elements is similar to an intronic CaMK IV–responsive RNA element (CaRRE) originally identified in the 3′ splice site of the BK channel STREX exon, but not previously observed within an exon. The other element is a new RNA motif. Introduction of either of these two motifs, called CaRRE type 1 and CaRRE type 2, into a heterologous constitutive exon can confer CaMK IV–dependent repression on the new exon. Thus, either exonic CaRRE can be sufficient for CaMK IV–induced repression. Single nucleotide scanning mutagenesis defined consensus sequences for these two CaRRE motifs. A genome-wide motif search and subsequent RT-PCR validation identified a group of depolarization-regulated alternative exons carrying CaRRE consensus sequences. Many of these exons are likely to alter neuronal function. Thus, these two RNA elements define a group of co-regulated splicing events that respond to a common stimulus in neurons to alter their activity.     

Decrease in hnRNP A/B expression during erythropoiesis mediates a pre-mRNA splicing switch

A physiologically important alternative pre-mRNA splicing switch, involving activation of protein 4.1R exon 16 (E16) splicing, is required for the establishment of proper mechanical integrity of the erythrocyte membrane during erythropoiesis. Here we identify a conserved exonic splicing silencer element (CE(16)) in E16 that interacts with hnRNP A/B proteins and plays a role in repression of E16 splicing during early erythropoiesis. Experiments with model pre-mRNAs showed that CE(16) can repress splicing of upstream introns, and that mutagenesis or replacement of CE(16) can relieve this inhibition. An affinity selection assay with biotinylated CE(16) RNA demonstrated specific binding of hnRNP A/B proteins. Depletion of hnRNP A/B proteins from nuclear extract significantly increased E16 inclusion, while repletion with recombinant hnRNP A/B restored E16 silencing. Most importantly, differentiating mouse erythroblasts exhibited a stage-specific activation of the E16 splicing switch in concert with a dramatic and specific down-regulation of hnRNP A/B protein expression. These findings demonstrate that natural developmental changes in hnRNP A/B proteins can effect physiologically important switches in pre-mRNA splicing.     

G runs in Cystathionine β-Synthase c.833C/c.844_845ins68 mRNA are splicing silencers of pathogenic 3′ splice sites

The c.844_845ins68 is an evolutionary conserved polymorphism of the Cystathionine β-Synthase gene that segregates with the pathogenic c.833C mutation and consists of a 68nt insertion duplicating the 3′ splice site between intron 7 and exon 8. The gene rearrangement brought two GGGG runs close to each other and generated a splicing control element that allows the constitutive selection of the more distal 3′ splice site in the c.844_854ins68 carriers. In this study, we have characterized functionally the two G4 runs within the duplication and have found that they work as silencers of the upstream potentially pathogenic 3′ splice sites has been functionally characterized. This selection allows skipping of both the 68nt-insertion and the c.833C mutation, and is essential to preserve the wild-type ORF. Knocking down hnRNP H and F expression modulated the rescue of the proximal 3′ splice site more than hnRNP H alone. These observations suggest that hnRNP H/F contribute jointly to prevention of CBS deficiency in c.844_854ins68 carriers by silencing the potentially pathogenic upstream acceptor site.     

Modulation of exon skipping and inclusion by heterogeneous nuclear ribonucleoprotein A1 and pre-mRNA splicing factor SF2/ASF.

The essential splicing factor SF2/ASF and the heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) modulate alternative splicing in vitro of pre-mRNAs that contain 5' splice sites of comparable strengths competing for a common 3' splice site. Using natural and model pre-mRNAs, we have examined whether the ratio of SF2/ASF to hnRNP A1 also regulates other modes of alternative splicing in vitro. We found that an excess of SF2/ASF effectively prevents inappropriate exon skipping and also influences the selection of mutually exclusive tissue-specific exons in natural beta-tropomyosin pre-mRNA. In contrast, an excess of hnRNP A1 does not cause inappropriate exon skipping in natural constitutively or alternatively spliced pre-mRNAs. Although hnRNP A1 can promote alternative exon skipping, this effect is not universal and is dependent, e.g., on the size of the internal alternative exon and on the strength of the polypyrimidine tract in the preceding intron. With appropriate alternative exons, an excess of SF2/ASF promotes exon inclusion, whereas an excess of hnRNP A1 causes exon skipping. We propose that in some cases the ratio of SF2/ASF to hnRNP A1 may play a role in regulating alternative splicing by exon inclusion or skipping through the antagonistic effects of these proteins on alternative splice site selection.