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

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.     

U1 snRNP-dependent function of TIAR in the regulation of alternative RNA processing of the human calcitonin/CGRP pre-mRNA

Alternative RNA processing of human calcitonin/CGRP pre-mRNA is regulated by an intronic enhancer element. Previous studies have demonstrated that multiple sequence motifs within the enhancer and a number of trans-acting factors play critical roles in the regulation. Here, we report the identification of TIAR as a novel player in the regulation of human calcitonin/CGRP alternative RNA processing. TIAR binds to the U tract sequence motif downstream of a pseudo 5' splice site within the previously characterized intron enhancer element. Binding of TIAR promotes inclusion of the alternative 3'-terminal exon located more than 200 nucleotides upstream from the U tract. In cells that preferentially include this exon, overexpression of a mutant TIAR that lacks the RNA binding domains suppressed inclusion of this exon. In this report, we also demonstrate an unusual novel interaction between U6 snRNA and the pseudo 5' splice site, which was shown previously to bind U1 snRNA. Interestingly, TIAR binding to the U tract sequence depends on the interaction of not only U1 but also U6 snRNA with the pseudo 5' splice site. Conversely, TIAR binding promotes U6 snRNA binding to its target. The synergistic relationship between TIAR and U6 snRNA strongly suggests a novel role of U6 snRNP in regulated alternative RNA processing.     

TIA proteins are necessary but not sufficient for the tissue-specific splicing of the myosin phosphatase targeting subunit 1

We are using the tissue-specific splicing of myosin phosphatase targeting subunit (MYPT1) as a model to investigate smooth muscle phenotypic diversity. We previously identified a U-rich intronic enhancer flanking the 5' splice site (IE1), and a bipartite exonic enhancer/suppressor, that regulate splicing of the MYPT1 central alternative exon. Here we show that T-cell inhibitor of apoptosis (TIA-1) and T-cell inhibitor of apoptosis-related (TIAR) proteins bind to the IE1. Co-transfection of TIA expression vectors with a MYPT1 mini-gene construct increase splicing of the central alternative exon. TIA proteins do not enhance splicing when the palindromic exonic splicing enhancer (ESE) is mutated, indicating that TIAs are necessary but not sufficient for splicing. The ESE specifically binds SRp55 and SRp20 proteins, supporting a model in which both SR and TIA proteins binding to their cis-elements are required for the recruitment of the splicing complex to a weak 5' splice site. Inactivation of TIA proteins in the DT40 cell line (TIA-1(-/-)TIAR(+/-)) reduced the splicing of the central alternative exon of the endogenous MYPT1 as well as stably transfected MYPT1 minigene constructs. Splicing of the MYPT1 3' alternative exon and the MLC(17) alternative exon were unaffected, suggesting that TIA proteins regulate a subset of smooth muscle/nonmuscle alternative splicing reactions. Finally, reduced RNA binding and reduced expression of the TIA and SR proteins in phasic (gizzard) smooth muscle around hatching coincided with the switch from exon inclusion to exon skipping, suggesting that loss of TIA and SR enhancer activity may play a role in the developmental switch in MYPT1 splicing.     

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.     

hnRNP A1 contacts exon 5 to promote exon 6 inclusion of apoptotic Fas gene

Fas is a transmembrane cell surface protein recognized by Fas ligand (FasL). When FasL binds to Fas, the target cells undergo apoptosis. A soluble Fas molecule that lacks the transmembrane domain is produced from skipping of exon 6 encoding this region in alternative splicing procedure. The soluble Fas molecule has the opposite function of intact Fas molecule, protecting cells from apoptosis. Here we show that knockdown of hnRNP A1 promotes exon 6 skipping of Fas pre-mRNA, whereas overexpression of hnRNP A1 reduces exon 6 skipping. Based on the bioinformatics approach, we have hypothesized that hnRNP A1 functions through interrupting 5′ splice site selection of exon 5 by interacting with its potential binding site close to 5′ splice site of exon 5. Consistent with our hypothesis, we demonstrate that mutations of the hnRNP A1 binding site on exon 5 disrupted the effects of hnRNP A1 on exon 6 inclusion. RNA pull-down assay and then western blot analysis with hnRNP A1 antibody prove that hnRNP A1 contacts the potential binding site RNA sequence on exon 5 but not the mutant sequence. In addition, we show that the mutation of 5′ splice site on exon 5 to a less conserved sequence destructed the effects of hnRNP A1 on exon 6 inclusion. Therefore we conclude that hnRNP A1 interacts with exon 5 to promote distal exon 6 inclusion of Fas pre-mRNA. Our study reveals a novel alternative splicing mechanism of Fas pre-mRNA.     

The apoptosis-promoting factor TIA-1 is a regulator of alternative pre-mRNA splicing

We report here that the apoptosis-promoting protein TIA-1 regulates alternative pre-mRNA splicing of the Drosophila melanogaster gene male-specific-lethal 2 and of the human apoptotic gene Fas. TIA-1 associates selectively with pre-mRNAs that contain 5' splice sites followed by U-rich sequences. TIA-1 binding to the U-rich stretches facilitates 5' splice site recognition by U1 snRNP. This activity is critical for activation of the weak 5' splice site of msl-2 and for modulating the choice of splice site partner in Fas. Structural and functional similarities with the Saccharomyces cerevisiae splicing factor Nam8 suggest striking evolutionary conservation of a mechanism of pre-mRNA splicing regulation that controls biological processes as diverse as meiosis in yeast, dosage compensation in fruit flies, or programmed cell death in humans.     

Two isoforms of the T-cell intracellular antigen 1 (TIA-1) splicing factor display distinct splicing regulation activities. Control of TIA-1 isoform ratio by TIA-1-related protein

TIA-1 (T-cell Intracellular Antigen 1) and TIAR (TIA-1-related protein) are RNA-binding proteins involved in the regulation of alternative pre-mRNA splicing and other aspects of RNA metabolism. Various isoforms of these proteins exist in mammals. For example, TIA-1 presents two major isoforms (TIA-1a and TIA-1b) generated by alternative splicing of exon 5 that differ by eleven amino acids exclusive of the TIA-1a isoform. Here we show that the relative expression of TIA-1 and TIAR isoforms varies in different human tissues and cell lines, suggesting distinct functional properties and regulated isoform expression. We report that whereas TIA-1 isoforms show similar subcellular distribution and RNA binding, TIA-1b displays enhanced splicing stimulatory activity compared with TIA-1a, both in vitro and in vivo. Interestingly, TIAR depletion from HeLa and mouse embryonic fibroblasts results in an increased ratio of TIA-1b/a expression, suggesting that TIAR regulates the relative expression of TIA-1 isoforms. Taken together, the results reveal distinct functional properties of TIA-1 isoforms and the existence of a regulatory network that controls isoform expression.     

A DNA damage signal activates and derepresses exon inclusion in Drosophila TAF1 alternative splicing

Signal-dependent alternative splicing is important for regulating gene expression in eukaryotes, yet our understanding of how signals impact splicing mechanisms is limited. A model to address this issue is alternative splicing of Drosophila TAF1 pre-mRNA in response to camptothecin (CPT)-induced DNA damage signals. CPT treatment of Drosophila S2 cells causes increased inclusion of TAF1 alternative cassette exons 12a and 13a through an ATR signaling pathway. To evaluate the role of TAF1 pre-mRNA sequences in the alternative splicing mechanism, we developed a TAF1 minigene (miniTAF1) and an S2 cell splicing assay that recapitulated key aspects of CPT-induced alternative splicing of endogenous TAF1. Analysis of miniTAF1 indicated that splice site strength underlies independent and distinct mechanisms that control exon 12a and 13a inclusion. Mutation of the exon 13a weak 5' splice site or weak 3' splice site to a consensus sequence was sufficient for constitutive exon 13a inclusion. In contrast, mutation of the exon 12a strong 5' splice site or moderate 3' splice site to a consensus sequence was only sufficient for constitutive exon 12a inclusion in the presence of CPT-induced signals. Analogous studies of the exon 13 3' splice site suggest that exon 12a inclusion involves signal-dependent pairing between constitutive and alternative splice sites. Finally, intronic elements identified by evolutionary conservation were necessary for full repression of exon 12a inclusion or full activation of exon 13a inclusion and may be targets of CPT-induced signals. In summary, this work defines the role of sequence elements in the regulation of TAF1 alternative splicing in response to a DNA damage signal.     

Role for Fox-1/Fox-2 in mediating the neuronal pathway of calcitonin/calcitonin gene-related peptide alternative RNA processing

Although multiple regulatory elements and protein factors are known to regulate the non-neuronal pathway of alternative processing of the calcitonin/calcitonin gene-related peptide (CGRP) pre-mRNA, the mechanisms controlling the neuron-specific pathway have remained elusive. Here we report the identification of Fox-1 and Fox-2 proteins as novel regulators that mediate the neuron-specific splicing pattern. Fox-1 and Fox-2 proteins function to repress exon 4 inclusion, and this effect depends on two UGCAUG elements surrounding the 3' splice site of the calcitonin-specific exon 4. In neuron-like cells, mutation of a subset of UGCAUG elements promotes the non-neuronal pattern in which exon 4 is included. In HeLa cells, overexpression of Fox-1 or Fox-2 protein decreases exon 4 inclusion. Fox-1 and Fox-2 proteins interact with the UGCAUG elements specifically and regulate splicing by blocking U2AF(65) binding to the 3' splice site upstream of exon 4. We further investigated the inter-relationship between the UGCAUG silencer elements and the previously identified intronic and exonic splicing regulatory elements and found that exon 4 is regulated by an intricate balance of positive and negative regulation. These results define a critical role for Fox-1 and Fox-2 proteins in exon 4 inclusion of calcitonin/CGRP pre-mRNA and establish a regulatory network that controls the fate of exon 4.     

Regulation of Fas alternative splicing by antagonistic effects of TIA-1 and PTB on exon definition

Fas exon 6 can be included or skipped to generate mRNAs encoding, respectively, a membrane bound form of the receptor that promotes apoptosis or a soluble isoform that prevents programmed cell death. We report that the apoptosis-inducing protein TIA-1 promotes U1 snRNP binding to the 5' splice site of intron 6, which in turn facilitates exon definition by enhancing U2AF binding to the 3' splice site of intron 5. The polypyrimidine tract binding protein (PTB) promotes exon skipping by binding to an exonic splicing silencer and inhibiting the association of U2AF and U2 snRNP with the upstream 3' splice site, without affecting recognition of the downstream 5' splice site by U1. Remarkably, U1 snRNP-mediated recognition of the 5' splice site is required both for efficient U2AF binding and for U2AF inhibition by PTB. We propose that TIA-1 and PTB regulate Fas splicing and possibly Fas-mediated apoptosis by targeting molecular events that lead to exon definition.     

TIA1 prevents skipping of a critical exon associated with spinal muscular atrophy

Prevention of skipping of exon 7 during pre-mRNA splicing of Survival Motor Neuron 2 (SMN2) holds the promise for cure of spinal muscular atrophy (SMA), a leading genetic cause of infant mortality. Here, we report T-cell-restricted intracellular antigen 1 (TIA1) and TIA1-related (TIAR) proteins as intron-associated positive regulators of SMN2 exon 7 splicing. We show that TIA1/TIAR stimulate exon recognition in an entirely novel context in which intronic U-rich motifs are separated from the 5' splice site by overlapping inhibitory elements. TIA1 and TIAR are modular proteins with three N-terminal RNA recognition motifs (RRMs) and a C-terminal glutamine-rich (Q-rich) domain. Our results reveal that any one RRM in combination with a Q domain is necessary and sufficient for TIA1-associated regulation of SMN2 exon 7 splicing in vivo. We also show that increased expression of TIA1 counteracts the inhibitory effect of polypyrimidine tract binding protein, a ubiquitously expressed factor recently implicated in regulation of SMN exon 7 splicing. Our findings expand the scope of TIA1/TIAR in genome-wide regulation of alternative splicing under normal and pathological conditions.     

Alternative splicing of the neurofibromatosis type I pre-mRNA

NF1 (neurofibromatosis type?I) is a common genetic disease that affects one in 3500 individuals. The disease is completely penetrant but shows variable phenotypic expression in patients. NF1 is a large gene, and its pre-mRNA undergoes alternative splicing. The NF1 protein, neurofibromin, is involved in diverse signalling cascades. One of the best characterized functions of NF1 is its function as a Ras-GAP (GTPase-activating protein). NF1 exon 23a is an alternative exon that lies within the GAP-related domain of neurofibromin. This exon is predominantly included in most tissues, and it is skipped in CNS (central nervous system) neurons. The isoform in which exon 23a is skipped has 10?times higher Ras-GAP activity than the isoform in which exon 23a is included. Exon 23a inclusion is tightly regulated by at least three different families of RNA-binding proteins: CELF {CUG-BP (cytosine-uridine-guanine-binding protein) and ETR-3 [ELAV (embryonic lethal abnormal vision)-type RNA-binding protein]-like factor}, Hu and TIA-1 (T-cell intracellular antigen 1)/TIAR (T-cell intracellular antigen 1-related protein). The CELF and Hu proteins promote exon 23a skipping, while the TIA-1/TIAR proteins promote its inclusion. The widespread clinical variability that is observed among NF1 patients cannot be explained by NF1 mutations alone and it is believed that modifier genes may have a role in the variability. We suggest that the regulation of alternative splicing may act as a modifier to contribute to the variable expression in NF1 patients.     

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.     

Accurate selection of a 5' splice site requires sequences within fibronectin alternative exon B.

Inclusion of fibronectin alternative exon B in mRNA is developmentally regulated. Here we demonstrate that exon B contains two unique purine-rich sequence tracts, PRE1 and PRE2, that are important for proper 5' splice site selection both in vivo and in vitro. Targeted mutations of both PREs decreased the inclusion of exon B in the mRNA by 50% in vivo. Deletion or mutation of the PREs reduced removal of the downstream intron, but not the upstream intron, and induced the activation of cryptic 5' splice sites in vitro. PRE-mediated 5' splice selection activity appears sensitive to position and sequence context. A well characterized exon sequence enhancer that normally acts on the upstream 3' splice site can partially rescue proper exon B 5' splice site selection. In addition, we found that PRE 5' splice selection activity was preserved when exon B was inserted into a heterologous pre-mRNA substrate. Possible roles of these unique activities in modulating exon B splicing are considered.     

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.     

A highly stable duplex structure sequesters the 5' splice site region of hnRNP A1 alternative exon 7B.

Exon 7B in the hnRNP A1 pre-mRNA is alternatively spliced to yield A1 and A1(B), two proteins that differ in their ability to modulate 5' splice site selection. Sequencing the murine intron downstream of exon 7B revealed the existence of several regions of similarity to the corresponding human intron. In vitro splicing assays indicate that an 84-nt region (CE6IO) decreases splicing to the proximal 5' splice site in a pre-mRNA carrying the 5' splice sites of exon 7 and 7B. In vivo, the CE6IO element promotes exon 7B skipping in pre-mRNAs expressed from a mini-gene containing the hnRNP A1 alternative splicing unit. Using oligonucleotide-targeted RNase H cleavage assays, we provide support for the existence of highly stable base pairing interactions between CE6IO and the 5' splice site region of exon 7B. Duplex formation occurs in naked pre-mRNA, resists incubation in splicing extracts, and is associated with a reduction in the assembly of U1 snRNP-dependent complexes to the 5' splice site of exon 7B. Our results demonstrate that pre-mRNA secondary structure plays an important role in promoting exon 7B skipping in the A1 pre-mRNA.     

Mechanistic control of carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) splice isoforms by the heterogeneous nuclear ribonuclear proteins hnRNP L, hnRNP A1, and hnRNP M

Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed in a variety of cell types and is implicated in carcinogenesis. Alternative splicing of CEACAM1 pre-mRNA generates two cytoplasmic domain splice variants characterized by the inclusion (L-isoform) or exclusion (S-isoform) of exon 7. Here we show that the alternative splicing of CEACAM1 pre-mRNA is regulated by novel cis elements residing in exon 7. We report the presence of three exon regulatory elements that lead to the inclusion or exclusion of exon 7 CEACAM1 mRNA in ZR75 breast cancer cells. Heterologous splicing reporter assays demonstrated that the maintenance of authentic alternative splicing mechanisms were independent of the CEACAM1 intron sequence context. We show that forced expression of these exon regulatory elements could alter CEACAM1 splicing in HEK-293 cells. Using RNA affinity chromatography, three members of the heterogeneous nuclear ribonucleoprotein family (hnRNP L, hnRNP A1, and hnRNP M) were identified. RNA immunoprecipitation of hnRNP L and hnRNP A1 revealed a binding motif located central and 3' to exon 7, respectively. Depletion of hnRNP A1 or L by RNAi in HEK-293 cells promoted exon 7 inclusion, whereas overexpression led to exclusion of the variable exon. By contrast, overexpression of hnRNP M showed exon 7 inclusion and production of CEACAM1-L mRNA. Finally, stress-induced cytoplasmic accumulation of hnRNP A1 in MDA-MB-468 cells dynamically alters the CEACAM1-S:CEACAM1:L ratio in favor of the l-isoform. Thus, we have elucidated the molecular factors that control the mechanism of splice-site recognition in the alternative splicing regulation of CEACAM1.