A pyrimidine-rich exonic splicing suppressor binds multiple RNA splicing factors and inhibits spliceosome assembly

The bovine papillomavirus type 1 (BPV-1) exonic splicing suppressor (ESS) is juxtaposed immediately downstream of BPV-1 splicing enhancer 1 and negatively modulates selection of a suboptimal 3′ splice site at nucleotide 3225. The present study demonstrates that this pyrimidine-rich ESS inhibits utilization of upstream 3′ splice sites by blocking early steps in spliceosome assembly. Analysis of the proteins that bind to the ESS showed that the U-rich 5′ region binds U2AF⁶⁵ and polypyrimidine tract binding protein, the C-rich central part binds 35- and 54–55-kDa serine/arginine-rich (SR) proteins, and the AG-rich 3′ end binds alternative splicing factor/splicing factor 2. Mutational and functional studies indicated that the most critical region of the ESS maps to the central C-rich core (GGCUCCCCC). This core sequence, along with additional nonspecific downstream nucleotides, is sufficient for partial suppression of spliceosome assembly and splicing of BPV-1 pre-mRNAs. The inhibition of splicing by the ESS can be partially relieved by excess purified HeLa SR proteins, suggesting that the ESS suppresses pre-mRNA splicing by interfering with normal bridging and recruitment activities of SR proteins.     

Coevolutionary networks of splicing cis-regulatory elements

Accurate and efficient splicing of eukaryotic pre-mRNAs requires recognition by trans-acting factors of a complex array of cis-acting RNA elements. Here, we developed a generalized Bayesian network to model the coevolution of splicing cis elements in diverse eukaryotic taxa. Cross-exon but not cross-intron compensatory interactions between the 5' splice site (5'ss) and 3' splice site (3'ss) were observed in human/mouse, indicating that the exon is the primary evolutionary unit in mammals. Studied plants, fungi, and invertebrates exhibited exclusively cross-intron interactions, suggesting that intron definition drives evolution in these organisms. In mammals, 5'ss strength and the strength of several classes of exonic splicing silencers (ESSs) evolved in a correlated way, whereas specific exonic splicing enhancers (ESEs), including motifs associated with hTra2, SRp55, and SRp20, evolved in a compensatory manner relative to the 5'ss and 3'ss. Interactions between specific ESS or ESE motifs were not observed, suggesting that elements bound by different factors are not commonly interchangeable. Thus, the splicing elements defining exons coevolve in a way that preserves overall exon strength, allowing specific elements to substitute for loss or weakening of others.     

The SRm160/300 splicing coactivator is required for exon-enhancer function.

Exonic splicing enhancer (ESE) sequences are important for the recognition of splice sites in pre-mRNA. These sequences are bound by specific serine-arginine (SR) repeat proteins that promote the assembly of splicing complexes at adjacent splice sites. We have recently identified a splicing "coactivator," SRm160/300, which contains SRm160 (the SR nuclear matrix protein of 160 kDa) and a 300-kDa nuclear matrix antigen. In the present study, we show that SRm160/300 is required for a purine-rich ESE to promote the splicing of a pre-mRNA derived from the Drosophila doublesex gene. The association of SRm160/300 and U2 small nuclear ribonucleoprotein particle (snRNP) with this pre-mRNA requires both U1 snRNP and factors bound to the ESE. Independently of pre-mRNA, SRm160/300 specifically interacts with U2 snRNP and with a human homolog of the Drosophila alternative splicing regulator Transformer 2, which binds to purine-rich ESEs. The results suggest a model for ESE function in which the SRm160/300 splicing coactivator promotes critical interactions between ESE-bound "activators" and the snRNP machinery of the spliceosome.     

Identification of alternative splicing and negative splicing activity of a nonsegmented negative-strand RNA virus, Borna disease virus

Borna disease virus (BDV) is a nonsegmented negative-strand RNA virus that belongs to the Mononegavirales. Unlike other animal viruses of this order, BDV replicates and transcribes in the nucleus of infected cells. Previous studies have shown that BDV uses RNA splicing machinery for its mRNA expression. In the present study, we identified spliced RNAs that use an alternative 3' splice site, SA3, in BDV-infected cell lines as well as infected animal brain cells. Transient transfection analysis of cDNA clones of BDV RNA revealed that although SA3 is a favorable splice site in mammalian cells, utilization of SA3 is negatively regulated in infected cells. This negative splicing activity of the SA3 site is regulated by a putative cis-acting region, the exon splicing suppressor (ESS), within the polymerase exon of BDV. The BDV ESS contains similar motifs to other known ESSs present in viral and cellular genes. Furthermore, our results indicated that a functional polyadenylation signal just upstream of the BDV ESS is also involved in the regulation of alternative splicing of BDV. These observations represent the first documentation of complex RNA splicing in animal RNA viruses and also provide new insight into the mechanism of regulation of alternative splicing in animal viruses.     

Evidence for the function of an exonic splicing enhancer after the first catalytic step of pre-mRNA splicing

Exonic splicing enhancers (ESEs) activate pre-mRNA splicing by promoting the use of the flanking splice sites. They are recognized by members of the serine/arginine-rich (SR) family of proteins, such as splicing factor 2/alternative splicing factor (SF2/ASF), which recruit basal splicing factors to form the initial complexes during spliceosome assembly. The in vitro splicing kinetics of an ESE-dependent IgM pre-mRNA suggested that an SF2/ASF-specific ESE has additional functions later in the splicing reaction, after the completion of the first catalytic step. A bimolecular exon ligation assay, which physically uncouples the first and second catalytic steps of splicing in a trans-splicing reaction, was adapted to test the function of the ESE after the first step. A 3' exon containing the SF2/ASF-specific ESE underwent bimolecular exon ligation, whereas 3' exons without the ESE or with control sequences did not. The ESE-dependent trans-splicing reaction occurred after inactivation of U1 or U2 small nuclear ribonucleoprotein particles, compatible with a functional assay for events after the first step of splicing. The ESE-dependent step appears to take place before the ATP-independent part of the second catalytic step. Bimolecular exon ligation also occurred in an S100 cytosolic extract, requiring both the SF2/ASF-dependent ESE and complementation with SF2/ASF. These data suggest that some ESEs can act late in the splicing reaction, together with appropriate SR proteins, to enhance the second catalytic step of splicing.     

Interaction between the RNA binding domains of Ser-Arg splicing factor 1 and U1-70K snRNP protein determines early spliceosome assembly

It has been widely accepted that the early spliceosome assembly begins with U1 small nuclear ribonucleoprotein (U1 snRNP) binding to the 5' splice site (5'SS), which is assisted by the Ser/Arg (SR)-rich proteins in mammalian cells. In this process, the RS domain of SR proteins is thought to directly interact with the RS motif of U1-70K, which is subject to regulation by RS domain phosphorylation. Here we report that the early spliceosome assembly event is mediated by the RNA recognition domains (RRM) of serine/arginine-rich splicing factor 1 (SRSF1), which bridges the RRM of U1-70K to pre-mRNA by using the surface opposite to the RNA binding site. Specific mutation in the RRM of SRSF1 that disrupted the RRM-RRM interaction also inhibits the formation of spliceosomal E complex and splicing. We further demonstrate that the hypo-phosphorylated RS domain of SRSF1 interacts with its own RRM, thus competing with U1-70K binding, whereas the hyper-phosphorylated RS domain permits the formation of a ternary complex containing ESE, an SR protein, and U1 snRNP. Therefore, phosphorylation of the RS domain in SRSF1 appears to induce a key molecular switch from intra- to intermolecular interactions, suggesting a plausible mechanism for the documented requirement for the phosphorylation/dephosphorylation cycle during pre-mRNA splicing.     

Pre-mRNA splicing in plants: characterization of Ser/Arg splicing factors.

The fact that animal introns are not spliced out in plants suggests that recognition of pre-mRNA splice sites differs between the two kingdoms. In plants, little is known about proteins required for splicing, as no plant in vitro splicing system is available. Several essential splicing factors from animals, such as SF2/ASF and SC-35, belong to a family of highly conserved proteins consisting of one or two RNA binding domain(s) (RRM) and a C-terminal Ser/Arg-rich (SR or RS) domain. These animal SR proteins are required for splice site recognition and spliceosome assembly. We have screened for similar proteins in plants by using monoclonal antibodies specific for a phosphoserine epitope of the SR proteins (mAb1O4) or for SF2/ASF. These experiments demonstrate that plants do possess SR proteins, including SF2/ASF-like proteins. Similar to the animal SR proteins, this group of proteins can be isolated by two salt precipitations. However, compared to the animal SR proteins, which are highly conserved in size and number, SR proteins from Arabidopsis, carrot, and tobacco exhibit a complex pattern of intra- and interspecific variants. These plant SR proteins are able to complement inactive HeLa cell cytoplasmic S1OO extracts that are deficient in SR proteins, yielding functional splicing extracts. In addition, plant SR proteins were active in a heterologous alternative splicing assay. Thus, these plant SR proteins are authentic plant splicing factors.     

Primary structures of the heterogeneous nuclear ribonucleoprotein A2, B1, and C2 proteins: a diversity of RNA binding proteins is generated by small peptide inserts.

We have isolated cDNAs for the major heterogeneous nuclear ribonucleoprotein (hnRNP) A2, B1, and C2 proteins and determined their nucleotide and deduced amino acid sequences. The A2 and B1 cDNAs are identical except for a 36-nucleotide in-frame insert in B1. Similarly, the sequence of the C2 protein cDNA is related to that of C1 in that C2 contains an extra 39 in-frame nucleotides. Therefore, the B1 amino acid sequence is identical to A2 except for the insertion of 12 amino acids near its amino terminus, and C1 and C2 are also identical to each other except for an extra 13 amino acids near the middle of C2. All three proteins are members of a large family of RNA binding proteins that contain the consensus sequence-type RNA binding domain (CS-RBD). The A2 and B1 proteins have a modular structure similar to that of the hnRNP protein A1: they contain two CS-RBDs and a glycine-rich auxiliary domain at the carboxyl terminus. The CS-RBDs of A2 and B1 have approximately 80% amino acid identity with those of A1, whereas the glycine-rich auxiliary domain is considerably more divergent with less than 30% of the amino acids being identical. These findings indicate that the addition of small peptides, probably by alternative pre-mRNA splicing, generates some of the diversity apparent among hnRNP proteins.     

A complex of nuclear proteins mediates SR protein binding to a purine-rich splicing enhancer.

A purine-rich splicing enhancer from a constitutive exon has been shown to shift the alternative splicing of calcitonin/CGRP pre-mRNA in vivo. Here, we demonstrate that the native repetitive GAA sequence comprises the optimal enhancer element and specifically binds a saturable complex of proteins required for general splicing in vitro. This complex contains a 37-kDa protein that directly binds the repetitive GAA sequence and SRp40, a member of the SR family of non-snRNP splicing factors. While purified SR proteins do not stably bind the repetitive GAA element, exogenous SR proteins become associated with the GAA element in the presence of nuclear extracts and stimulate GAA-dependent splicing. These results suggest that repetitive GAA sequences enhance splicing by binding a protein complex containing a sequence-specific RNA binding protein and a general splicing activator that, in turn, recruit additional SR proteins. This type of mechanism resembles the tra/tra-2-dependent recruitment of SR proteins to the Drosophila doublesex alternative splicing regulatory element.     

Serine-arginine (SR)-rich splicing factors have an exon-independent function in pre-mRNA splicing

Two distinct functions have been proposed for the serine-arginine (SR)-rich family of splicing factors. First, SR proteins are essential splicing factors and are thought to function by mediating protein-protein interactions within the intron during spliceosome assembly. Second, SR proteins bind to exonic enhancer sequences and recruit spliceosome components to adjacent introns. The latter activity is required for splice-site recognition and alternative splicing. Until now it has not been possible to determine whether the requirement for SR proteins in the basic splicing reaction is a secondary consequence of their exon-dependent recruitment function. Here we show that RNA substrates containing only 1 nt of exon sequence can undergo the first step of the splicing reaction in vitro and that this activity requires SR proteins. Thus, we provide direct evidence that SR proteins have both exon-independent and exon-dependent functions in pre-mRNA splicing.     

Identification of alternative splicing regulators by RNA interference in Drosophila

Alternative splicing is thought to be regulated by nonspliceosomal RNA binding proteins that modulate the association of core components of the spliceosome with the pre-mRNA. Although the majority of metazoan genes encode pre-mRNAs that are alternatively spliced, remarkably few splicing regulators are currently known. Here, we used RNA interference to examine the role of >70% of the Drosophila RNA-binding proteins in regulating alternative splicing. We identified 47 proteins as splicing regulators, 26 of which have not previously been implicated in alternative splicing. Many of the regulators we identified are nonspliceosomal RNA-binding proteins. However, our screen unexpectedly revealed that altering the concentration of certain core components of the spliceosome specifically modulates alternative splicing. These results significantly expand the number of known splicing regulators and reveal an extraordinary richness in the mechanisms that regulate alternative splicing.     

hnRNP U protein is required for normal pre-mRNA splicing and postnatal heart development and function

We report that mice lacking the heterogeneous nuclear ribonucleoprotein U (hnRNP U) in the heart develop lethal dilated cardiomyopathy and display numerous defects in cardiac pre-mRNA splicing. Mutant hearts have disorganized cardiomyocytes, impaired contractility, and abnormal excitation–contraction coupling activities. RNA-seq analyses of Hnrnpu mutant hearts revealed extensive defects in alternative splicing of pre-mRNAs encoding proteins known to be critical for normal heart development and function, including Titin and calcium/calmodulin-dependent protein kinase II delta (Camk2d). Loss of hnRNP U expression in cardiomyocytes also leads to aberrant splicing of the pre-mRNA encoding the excitation–contraction coupling component Junctin. We found that the protein product of an alternatively spliced Junctin isoform is N-glycosylated at a specific asparagine site that is required for interactions with specific protein partners. Our findings provide conclusive evidence for the essential role of hnRNP U in heart development and function and in the regulation of alternative splicing.The expression of more than 95% of human genes is affected by alternative pre-mRNA splicing (AS) (1, 2). Differentially spliced isoforms play distinct roles in a temporally and spatially specific manner (3), and mutations that lead to aberrant splicing are the cause of many human genetic diseases (4). RNA-binding proteins (RBPs) play a central role in the regulation of alternative splicing during development and disease. They function primarily by positively or negatively regulating splice-site recognition by the spliceosome (1). Many RBPs are expressed in specific tissues, and AS is regulated by the combinatorial activities of these factors on specific pre-mRNAs through their interactions with distinct regulatory sequences in pre-mRNA that function as splicing enhancers or silencers (5).The developing heart is one of the best studied systems where splicing changes occur during normal development, and mutations affecting specific splicing outcomes contribute to cardiomyopathy (6, 7). Although these mutations can either disrupt splicing elements or affect the expression of specific splicing factors, the latter mechanism is clearly responsible for the distinct splicing profiles at different developmental stages. For example, the dynamics of alternative splicing during postnatal heart development correlate with expression changes of many RBPs, including CUG-BP, Elav-like family member 1 (CELF1), Muscleblind-like 1 (MBNL1), and FOX proteins (8). Detailed biochemical studies have elucidated the mechanisms by which these splicing factors regulate splicing in a position- and context-dependent manner (9, 10). The function of other RBPs during heart development has also been studied. For example, two of the muscle-specific splicing factors, RBM20 and RBM24, play distinct roles in splicing regulation. RBM20 mainly acts as a splicing repressor, as its absence leads to multiple exon inclusion events in the heart. For example, the Titin gene is one of the major targets of RBM20 (11, 12). On the other hand, loss of RBM24 expression gives rise to many exon skipping events (13), implicating RBM24 as a splicing activator. Strikingly, there is little overlap between RBM20 and RBM24 splicing targets, suggesting that RBM20 and RBM24 are involved in regulating splicing of distinct groups of pre-mRNAs and there is little cross-talk between these two splicing factors.Distinct splicing activities have also been ascribed to general splicing factors (1). There are two major types of ubiquitously expressed RBPs: the heterogeneous nuclear ribonucleoproteins (hnRNPs) and serine/arginine (SR)-rich proteins. hnRNPs and SR proteins are generally believed to play opposite roles in splicing: SR proteins promote the inclusion of exons during splicing, whereas hnRNP proteins repress inclusion (1). The function of certain SR proteins has been studied in the mouse heart through the conditional knockout approach. Srsf1 deletion in the heart leads to lethal dilated cardiomyopathy (DCM) (death occurs 6–8 wk after birth) (14). SRSF1 is required for the cardiac-specific splicing of Cypher (also called Ldb3) pre-mRNA, and the regulation of alternative splicing of calcium/calmodulin-dependent protein kinase II delta (Camk2d) and cardiac Troponin T (cTnT) during heart development. In particular, the persistent splicing of a neuronal isoform of Camk2d and its ability to enhance excitation and contraction coupling (ECC) activity in Srsf1 mutant cardiomyocytes have been proposed as a possible cause of the phenotype in mutant mice (14). Ablation of another SR protein, SRSF10 (SRp38), from the mouse also leads to heart defects (15). SRSF10 has been shown to regulate the splicing of Triadin, an important component of ECC machinery (15). Interestingly, conditional deletion of Srsf2 from the heart leads to DCM with little splicing misregulation but instead affects the expression of the calcium channel Ryr2 (16). It is striking that these SR proteins affect ECC activity in the heart by directly regulating the expression/splicing of distinct players in this machinery. Because these studies were conducted before the advent of next-generation RNA sequencing, only a few splicing targets specifically regulated by these SR proteins were identified. A more comprehensive study of the effects of deleting the genes encoding these proteins from the heart on the splicing program has not been reported.In contrast to SR proteins, specific functions of hnRNP proteins in cardiac pre-mRNA splicing have not been determined. In this report, we selectively inactivated the expression of one of the most abundant hnRNP proteins—hnRNP U—in the heart. We found that Hnrnpu mutant mice develop a lethal DCM phenotype, with death occurring around 2 wk after birth. There are multiple cardiac defects in mutant hearts accompanied by many splicing alterations. Some of these splicing targets are commonly regulated by hnRNP U and other SR and RBM proteins. We also identified many hnRNP U-specific splicing targets in the heart, including an ECC component Junctin. The protein product of the alternatively spliced Junctin isoform is N-glycosylated at a specific asparagine site in Hnrnpu mutant cells and could contribute to abnormal cardiac function. Our study also enables comparisons of the roles of different splicing factors in heart development and function.     

Modulation of 5'' splice site choice in pre-messenger RNA by two distinct steps.

Ser/Arg-rich proteins (SR proteins) are essential splicing factors that commit pre-messenger RNAs to splicing and also modulate 5' splice site choice in the presence or absence of functional U1 small nuclear ribonucleoproteins (snRNPs). Here, we perturbed the U1 snRNP in HeLa cell nuclear extract by detaching the U1-specific A protein using a 2'-O-methyl oligonucleotide (L2) complementary to its binding site in U1 RNA. In this extract, the standard adenovirus substrate is spliced normally, but excess amounts of SR proteins do not exclusively switch splicing from the normal 5' splice site to a proximal site (site 125 within the adenovirus intron), suggesting that modulation of 5' splice site choice exerted by SR proteins requires integrity of the U1 snRNP. The observation that splicing does not necessarily follow U1 binding indicates that interactions between the U1 snRNP and components assembled on the 3' splice site via SR proteins may also be critical for 5' splice site selection. Accordingly, we found that SR proteins promote the binding of the U2 snRNP to the branch site and stabilize the complex formed on a 3'-half substrate in the presence or absence of functional U1 snRNPs. A novel U2/U6/3'-half substrate crosslink was also detected and promoted by SR proteins. Our results suggest that SR proteins in collaboration with the U1 snRNP function in two distinct steps to modulate 5' splice site selection.     

Reversible phosphorylation differentially affects nuclear and cytoplasmic functions of splicing factor 2/alternative splicing factor

The Ser/Arg-rich (SR) proteins constitute a family of highly conserved nuclear phosphoproteins that are involved in many steps of mRNA metabolism. Previously, we demonstrated that shuttling SR proteins can associate with translating ribosomes and enhance translation of reporter mRNAs both in vivo and in vitro. Here, we show that endogenous, cytoplasmic splicing factor 2/alternative splicing factor (SF2/ASF) associated with the translation machinery is hypophosphorylated, suggesting that the phosphorylation state of the Arg-Ser-rich (RS) domain may influence the role of SF2/ASF in cytoplasmic RNA processing. In agreement, we show that mutations mimicking a hypophosphorylated RS domain strongly increased SF2/ASF binding to cytoplasmic mRNA and its activity in translation. We also demonstrate that, whereas the RS domain is not required for the function of SF2/ASF in mRNA translation in vivo or in vitro, its second RNA recognition motif (RRM)2 plays a critical role in this process. Taken together, these data suggest that RS-domain phosphorylation may influence the association of SF2/ASF with mRNA, whereas RRM2 may play an important role in mediating protein-protein interactions during translation. These data are consistent with a model whereby reversible protein phosphorylation differentially regulates the subcellular localization and activity of shuttling SR proteins.