A nonsense exon in the Tpm1 gene is silenced by hnRNP H and F

As well as generating protein isoform diversity, in some cases alternative splicing generates RNAs that harbor premature termination codons and that are subject to nonsense-mediated decay (NMD). We previously identified an apparent pseudo-exon in the rat α-tropomyosin (Tpm1) gene as a probable genuine alternatively spliced exon that causes NMD when spliced into Tpm1 RNA. Here, we report the analysis of cis-acting splicing regulatory elements within this “nonsense exon.” Guided by the data set of predicted splicing enhancer and silencer elements compiled by Zhang and Chasin, we made a series of mutations through the nonsense exon and found that like authentic exons it is densely packed with enhancer and silencer elements. Strikingly, 11 of 13 tested mutations behaved as predicted computationally. In particular, we found that a G-rich silencer at the 5′ end, which is crucial for skipping of the nonsense exon, functions by binding hnRNP-H and F.     

Role of an inhibitory pyrimidine element and polypyrimidine tract binding protein in repression of a regulated alpha-tropomyosin exon.

Splicing of exons 2 and 3 of a-tropomyosin (TM) involves mutually exclusive selection of either exon 3, which occurs in most cells, or of exon 2 in smooth muscle (SM) cells. The SM-specific selection of exon 2 results from the inhibition of exon 3. At least two essential cis-acting elements are required for exon 3 inhibition, the upstream and downstream regulatory elements (URE and DRE). These elements are essential for repression of TM exon 3 in SM cells, and also mediate a low level of repression of exon 3 in an in vitro 5' splice site competition assay in HeLa extracts. Here, we show that the DRE consists of at least two discrete components, a short region containing a number of UGC motifs, and an essential pyrimidine-rich tract (DY). We show that the specific sequence of the DY element is important and that DY is able to bind to factors in HeLa nuclear extracts that mediate a low background level of exon 3 skipping. Deletion of a sequence within DY identified as an optimal binding site for PTB impairs (1) regulation of splicing in vivo, (2) skipping of exon 3 in an in vitro 5' splice site competition, (3) the ability of DY competitors to affect the 5' splice site competition in vitro, and (4) binding of PTB to DY. Addition of recombinant PTB to in vitro splicing reactions is able to partially reverse the effects of the DY competitor RNA. The data are consistent with a model for regulation of TM splicing that involves the participation of both tissue-specific and general inhibitory factors and in which PTB plays a role in repressing both splice sites of exon 3.     

Exon and intron sequences, respectively, repress and activate splicing of a fibroblast growth factor receptor 2 alternative exon.

Two alternative exons, BEK and K-SAM, code for part of the ligand binding site of fibroblast growth factor receptor 2. Splicing of these exons is mutually exclusive, and the choice between them is made in a tissue-specific manner. We identify here pre-mRNA sequences involved in controlling splicing of the K-SAM exon. The short K-SAM exon sequence 5'-TAGGGCAGGC-3' inhibits splicing of the exon. This inhibition can be overcome by mutating either the exon's 5' or 3' splice site to make it correspond more closely to the relevant consensus sequence. Two separate sequence elements in the intron immediately downstream of the K-SAM exon, one of which is a sequence rich in pyrimidines, are both needed for efficient K-SAM exon splicing. This is no longer the case if either the exon's 5' or 3' splice site is reinforced. Furthermore, if the exon inhibitory sequence is removed, the intron sequences are not required for splicing of the K-SAM exon in a cell line which normally splices this exon. At least three elements are thus involved in controlling splicing of the K-SAM exon: suboptimal 5' and 3' splice sites, an exon inhibitory sequence, and intron activating sequences.     

Global control of aberrant splice-site activation by auxiliary splicing sequences: evidence for a gradient in exon and intron definition

Auxiliary splicing signals play a major role in the regulation of constitutive and alternative pre-mRNA splicing, but their relative importance in selection of mutation-induced cryptic or de novo splice sites is poorly understood. Here, we show that exonic sequences between authentic and aberrant splice sites that were activated by splice-site mutations in human disease genes have lower frequencies of splicing enhancers and higher frequencies of splicing silencers than average exons. Conversely, sequences between authentic and intronic aberrant splice sites have more enhancers and less silencers than average introns. Exons that were skipped as a result of splice-site mutations were smaller, had lower SF2/ASF motif scores, a decreased availability of decoy splice sites and a higher density of silencers than exons in which splice-site mutation activated cryptic splice sites. These four variables were the strongest predictors of the two aberrant splicing events in a logistic regression model. Elimination or weakening of predicted silencers in two reporters consistently promoted use of intron-proximal splice sites if these elements were maintained at their original positions, with their modular combinations producing expected modification of splicing. Together, these results show the existence of a gradient in exon and intron definition at the level of pre-mRNA splicing and provide a basis for the development of computational tools that predict aberrant splicing outcomes.     

Multiple features contribute to efficient constitutive splicing of an unusually large exon

Vertebrate internal exons are usually between 50 and 400 nt long; exons outside this size range may require additional exonic and/or intronic sequences to be spliced into the mature mRNA. The mouse polymeric immunoglobulin receptor gene has a 654 nt exon that is efficiently spliced into the mRNA. We have examined this exon to identify features that contribute to its efficient splicing despite its large size; a large constitutive exon has not been studied previously. We found that a strong 5′ splice site is necessary for this exon to be spliced intact, but the splice sites alone were not sufficient to efficiently splice a large exon. At least two exonic sequences and one evolutionarily conserved intronic sequence also contribute to recognition of this exon. However, these elements have redundant activities as they could only be detected in conjunction with other mutations that reduced splicing efficiency. Several mutations activated cryptic 5′ splice sites that created smaller exons. Thus, the balance between use of these potential sites and the authentic 5′ splice site must be modulated by sequences that repress or enhance use of these sites, respectively. Also, sequences that enhance cryptic splice site use must be absent from this large exon.     

Exon definition may facilitate splice site selection in RNAs with multiple exons.

Interactions at the 3' end of the intron initiate spliceosome assembly and splice site selection in vertebrate pre-mRNAs. Multiple factors, including U1 small nuclear ribonucleoproteins (snRNPs), are involved in initial recognition at the 3' end of the intron. Experiments were designed to test the possibility that U1 snRNP interaction at the 3' end of the intron during early assembly functions to recognize and define the downstream exon and its resident 5' splice site. Splicing precursor RNAs constructed to have elongated second exons lacking 5' splice sites were deficient in spliceosome assembly and splicing activity in vitro. Similar substrates including a 5' splice site at the end of exon 2 assembled and spliced normally as long as the second exon was less than 300 nucleotides long. U2 snRNPs were required for protection of the 5' splice site terminating exon 2, suggesting direct communication during early assembly between factors binding the 3' and 5' splice sites bordering an exon. We suggest that exons are recognized and defined as units during early assembly by binding of factors to the 3' end of the intron, followed by a search for a downstream 5' splice site. In this view, only the presence of both a 3' and a 5' splice site in the correct orientation and within 300 nucleotides of one another will stable exon complexes be formed. Concerted recognition of exons may help explain the 300-nucleotide-length maximum of vertebrate internal exons, the mechanism whereby the splicing machinery ignores cryptic sites within introns, the mechanism whereby exon skipping is normally avoided, and the phenotypes of 5' splice site mutations that inhibit splicing of neighboring introns.     

A general role for splicing enhancers in exon definition

Exonic splicing enhancers (ESEs) facilitate exon definition by assisting in the recruitment of splicing factors to the adjacent intron. Here we demonstrate that suboptimal 5' and 3' splice sites are activated independently by ESEs when they are located on different exons. However, when they are situated within a single exon, the same weak 5' and 3' splice sites are activated simultaneously by a single ESE. These findings demonstrate that a single ESE promotes the recognition of both exon/intron junctions within the same step during exon definition. Our results suggest that ESEs recruit a multicomponent complex that minimally contains components of the splicing machinery required for 5' and 3' splice site selection.     

Mutually exclusive splicing of alpha-tropomyosin exons enforced by an unusual lariat branch point location: implications for constitutive splicing

Alternative splicing of alpha-tropomyosin pre-mRNA involves mutually exclusive utilization of exons 2 and 3, exon 3 being preferentially selected in most cells. This mutually exclusive behavior is enforced by absolute incompatibility between the adjacent splice sites of the two exons, due to close proximity of the exon 3 branch point to exon 2. The branch point, with an associated polypyrimidine tract, is in an unusual location, 177 nt upstream of the acceptor, only 42 nt from the exon 2 splice donor site. Splicing of exon 2 to 3 is consequently blocked prior to formation of an active spliceosome complex. This block to splicing can be relieved by insertion of spacer elements that increase the donor site-branch point separation to 51-59 nt. The unconventional relative location of the constitutive cis splicing elements therefore provides a simple mechanistic basis for strict mutually exclusive splicing. These results not only demonstrate that the branch point is not specified by proximity to the splice acceptor site, but rather suggest that it is the acceptor site which is specified relative to the branch point.     

Efficient internal exon recognition depends on near equal contributions from the 3' and 5' splice sites

Pre-mRNA splicing is carried out by the spliceosome, which identifies exons and removes intervening introns. In vertebrates, most splice sites are initially recognized by the spliceosome across the exon, because most exons are small and surrounded by large introns. This gene architecture predicts that efficient exon recognition depends largely on the strength of the flanking 3' and 5' splice sites. However, it is unknown if the 3' or the 5' splice site dominates the exon recognition process. Here, we test the 3' and 5' splice site contributions towards efficient exon recognition by systematically replacing the splice sites of an internal exon with sequences of different splice site strengths. We show that the presence of an optimal splice site does not guarantee exon inclusion and that the best predictor for exon recognition is the sum of both splice site scores. Using a genome-wide approach, we demonstrate that the combined 3' and 5' splice site strengths of internal exons provide a much more significant separator between constitutive and alternative exons than either the 3' or the 5' splice site strength alone.     

The cardiac troponin T alternative exon contains a novel purine-rich positive splicing element.

We have characterized a novel positive-acting splicing element within the developmentally regulated alternative exon (exon 5) of the cardiac troponin T (cTNT) gene. The exon splicing element (ESE) is internal to the exon portions of the splice sites and is required for splicing to the 3' splice site but not the 5' splice site flanking the exon. Sequence comparisons between cTNT exon 5 and other exons that contain regions required for splicing reveal a common purine-rich motif. Sequence within cTNT exon 5 or a synthetic purine-rich motif facilitates splicing of heterologous alternative and constitutive splice sites in vivo. Interestingly, the ESE is not required for the preferential inclusion of cTNT exon 5 observed in primary skeletal muscle cultures. Our results strongly suggest that the purine-rich ESE serves as a general splicing element that is recognized by the constitutive splicing machinery.     

During in vivo maturation of eukaryotic nuclear mRNA, splicing yields excised exon circles.

Circular splicing has already been described on nuclear pre-mRNA for certain splice sites far apart in the multi exonic ETS-1 gene and in the single 1.2 kb exon of the Sry locus. To date, it is unclear how splice site juxtaposition occurs in normal and circular splicing. The splice site selection of an internal exon is likely to involve pairing between splice sites across that exon. Based on this, we predict that, albeit at low frequency, internal exons yield circular RNA by splicing as an error-prone mechanism of exon juxtaposition or, perhaps more interestingly, as a regulated mechanism on alternative exons. To address this question, the circular exon formation was analyzed at three ETS-1 internal exons (one alternative spliced exon and two constitutive), in human cell line and blood cell samples. Here, we show by RT-PCR and sequencing that exon circular splicing occurs at the three individual exons that we examined. RNase protection experiments suggest that there is no correlation between exon circle expression and exon skipping.     

Alternative splicing of a human alpha-tropomyosin muscle-specific exon: identification of determining sequences.

The human alpha-tropomyosin gene hTMnm has two mutually exclusive versions of exon 5 (NM and SK), one of which is expressed specifically in skeletal muscle (exon SK). A minigene construct expresses only the nonmuscle (NM) isoform when transfected into COS-1 cells and both forms when transfected into myoblasts. Twenty-four mutants were produced to determine why the SK exon is not expressed in COS cells. The results showed that exons NM and SK are not in competition for splicing to the flanking exons and that there is no intrinsic barrier to splicing between the exons. Instead, exon SK is skipped whenever there are flanking introns. Splicing of exon SK was induced when the branch site sequence 70 nucleotides upstream of the exon was mutated to resemble the consensus and when the extremities of the exon itself were changed to the corresponding NM sequence. Precise swaps of the NM and SK exon sequences showed that the exon sequence effect was dominant to that of intron sequences. The mechanism of regulation appears to be unlike that of other tropomyosin genes. We propose that exclusion of exon SK arises because its 3' splicing signals are weak and are prevented by an exon-specific repressor from competing for splice site recognition.     

hnRNP F directs formation of an exon 4 minus variant of tumor-associated NADH oxidase (ENOX2)

HUVEC or mouse 3T3 cells infected with SV-40 generate within 3 to 5 days post-infection an ENOX2 species corresponding to the exon-4 minus splice variant of a tumor-associated NADH oxidase (ENOX2 or tNOX) expressed at the cancer cell surface. This study was to seek evidence for splicing factors that might direct formation of the exon 4 minus ENOX2 splice variant. To determine if silencing of ENOX2 exon 4 occurs because of motifs located in exon 4, transfections were performed on MCF-10A (mammary non-cancer), BT-20 (mammary cancer), and HeLa (cervical cancer) cells using a GFP minigene construct containing either a constitutively spliced exon (albumin exon 2) or the alternatively spliced ENOX2 exon 4 between the two GFP halves. Removal of exon 4 from the processed RNA of the GFP minigene construct occurred with HeLa and to a lesser extent with BT-20 but not in non-cancer MCF-10A cells. The Splicing Rainbow Program was used to identify all of the possible hnRNPs binding sites of exon 4 of ENOX2. There are 8 Exonic Splicing Silencers (ESSs) for hnRNP binding in the exon 4 sequences. Each of these sites were mutated by site-directed mutagenesis to test if any were responsible for the splicing skip. Results showed MutG75 ESS mutation changed the GFP expression which is a sign of splicing silence, while other mutations did not. As MutG75 changed the ESS binding site for hnRNP F, this result suggests that hnRNP F directs formation of the exon 4 minus variant of ENOX2.     

Branch point selection in alternative splicing of tropomyosin pre-mRNAs.

The rat tropomyosin 1 gene gives rise to two mRNAs encoding rat fibroblast TM-1 and skeletal muscle beta-tropomyosin via an alternative splicing mechanism. The gene is comprised of 11 exons. Exons 1 through 5 and exons 8 and 9 are common to all mRNAs expressed from this gene. Exons 6 and 11 are used in fibroblasts as well as smooth muscle whereas exons 7 and 10 are used exclusively in skeletal muscle. In the present studies we have focused on the mutually exclusive internal alternative splice choice involving exon 6 (fibroblast-type splice) and exon 7 (skeletal muscle-type splice). To study the mechanism and regulation of alternative splice site selection we have characterized the branch points used in processing of the tropomyosin pre-mRNAs in vitro using nuclear extracts obtained from HeLa cells. Splicing of exon 5 to exon 6 (fibroblast-type splice) involves the use of three branch points located 25, 29, and 36 nucleotides upstream of the 3' splice site of exon 6. Splicing of exon 6 (fibroblast-type splice) or exon 7 (skeletal muscle type-splice) to exon 8 involves the use of the same branch point located 24 nucleotides upstream of this shared 3' splice site. In contrast, the splicing of exon 5 to exon 7 (skeletal muscle-type splice) involves the use of three branch sites located 144, 147 and 153 nucleotides, upstream of the 3' splice site of exon 7. In addition, the pyrimidine content of the region between these unusual branch points and the 3' splice site of exon 7 was found to be greater than 80%. These studies raise the possibility that the use of branch points located a long distance from a 3' splice site may be an essential feature of some alternatively spliced exons. The possible significance of these unusual branch points as well as a role for the polypyrimidine stretch in intron 6 in splice site selection are discussed.     

Regulation of alternative 3' splice site selection by constitutive splicing factors.

We have devised an in vitro splicing assay in which the mutually exclusive exons 2 and 3 of alpha-tropomyosin act as competing 3' splice sites for joining to exon 1. Splicing in normal HeLa cell nuclear extracts results in almost exclusive joining of exons 1 and 3. Splicing in decreased nuclear extract concentrations and decreased ionic strength results in increased 1-2 splicing. We have used this assay to determine the role of three constitutive pre-mRNA splicing factors on alternative 3' splice site selection. Polypyrimidine tract binding protein (PTB) was found to inhibit the splicing of introns containing a strong binding site for this factor. However, the inhibitory effect of PTB could be partially reversed if pre-mRNAs were preincubated with U2 auxiliary factor (U2AF) prior to splicing in PTB-supplemented extracts. For alpha-tropomyosin, regulation of splicing by PTB and U2AF primarily affected the joining of exons 1-3 with no dramatic increases in 1-2 splicing being detected. Preincubation of pre-mRNAs with SR proteins led to small increases in 1-2 splicing. However, if pre-mRNAs were preincubated with SR proteins followed by splicing in PTB-supplemented extracts, there was a nearly complete reversal of the normal 1-2 to 1-3 splicing ratios. Thus, multiple pairwise, and sometimes antagonizing, interactions between constitutive pre-mRNA splicing factors and the pre-mRNA can regulate 3' splice site selection.     

Multisite and bidirectional exonic splicing enhancer in CD44 alternative exon v3

The human CD44 gene encodes multiple isoforms of a transmembrane protein that differ in their extracellular domains as a result of alternative splicing of its variable exons. Expression of CD44 is tightly regulated according to the type and physiological status of a cell, with expression of high molecular weight isoforms by inclusion of variable exons and low molecular weight isoforms containing few or no variable exons. Human CD44 variable exon 3 (v3) can follow a specific alternative splicing route different from that affecting other variable exons. Here we map and functionally describe the splicing enhancer element within CD44 exon v3 which regulates its inclusion in the final mRNA. The v3 splicing enhancer is a multisite bipartite element consisting of a tandem nonamer, the XX motif, and an heptamer, the Y motif, located centrally in the exon. Each of the three sites of this multisite enhancer partially retains its splicing enhancing capacity independently from each other in CD44 and shows full enhancing function in gene contexts different from CD44. We further demonstrate that these motifs act cooperatively as at least two motifs are needed to maintain exon inclusion. Their action is differential with respect to the splice-site target abutting v3. The first X motif acts on the 3' splice site, the second X motif acts on both splice sites (as a bidirectional exonic splicing enhancer), and the Y motif acts on the 5' splice site. We also show that the multisite v3 splicing enhancer is functional irrespective of flanking intron length and spatial organization within v3.     

A combinatorial role for exon, intron and splice site sequences in splicing in maize

Plant introns are typically AU-rich or U-rich, and this feature has been shown to be important for splicing. In maize, however, about 20% of the introns exceed 50% GC, and most of them are efficiently spliced. A series of constructs has been designed to analyze the cis requirements for splicing of the GC-rich Bz2 maize intron and two other GC-rich intron derivatives. By manipulating exon, intron and splice site sequences it is shown that exons can play an important role in intron definition: changes in exon sequences can increase splicing efficiency of a GC-rich intron from 17% to 86%. The relative difference, or base compositional contrast, in GC and U content between exon and intron sequences in the vicinity of splice sites, rather than the absolute base-content of the intron or exons, correlates with splicing efficiency. It is also shown that GC-rich intron constructs that are poorly spliced can be partially rescued by an improved 3' splice site.     

Switch in 3' splice site recognition between exon definition and splicing catalysis is important for sex-lethal autoregulation

Maintenance of female sexual identity in Drosophila melanogaster involves an autoregulatory loop in which the protein Sex-lethal (SXL) promotes skipping of exon 3 from its own pre-mRNA. We have used transient transfection of Drosophila Schneider cells to analyze the role of exon 3 splice sites in regulation. Our results indicate that exon 3 repression requires competition between the 5' splice sites of exons 2 and 3 but is independent of their relative strength. Two 3' splice site AG's precede exon 3. We report here that, while the distal site plays a critical role in defining the exon, the proximal site is preferentially used for the actual splicing reaction, arguing for a switch in 3' splice site recognition between exon definition and splicing catalysis. Remarkably, the presence of the two 3' splice sites is important for the efficient regulation by SXL, suggesting that SXL interferes with molecular events occurring between initial splice site communication across the exon and the splice site pairing that leads to intron removal.