Physiological state co-regulates thousands of mammalian mRNA splicing events at tandem splice sites and alternative exons

Thousands of tandem alternative splice sites (TASS) give rise to mRNA insertion/deletion variants with small size differences. Recent work has concentrated on the question of biological relevance in general, and the physiological regulation of TASS in particular. We have quantitatively studied 11 representative TASS cases in comparison to one mutually exclusive exon case and two cassette exons (CEs) using a panel of human and mouse tissues, as well as cultured cell lines. Tissues show small but significant differences in TASS isoform ratios, with a variance 4- to 20-fold lower than seen for CEs. Remarkably, in cultured cells, all studied alternative splicing (AS) cases showed a cell-density-dependent shift of isoform ratios with similar time series profiles. A respective genome-wide co-regulation of TASS splicing was shown by next-generation mRNA sequencing data. Moreover, data from human and mouse organs indicate that this co-regulation of TASS occurs in vivo, with brain showing the strongest difference to other organs. Together, the results indicate a physiological AS regulation mechanism that functions almost independently from the splice site context and sequence.     

The essential pre-mRNA splicing factor SF2 influences 5' splice site selection by activating proximal sites

SF2 is a 33 kd protein factor required for 5' splice site cleavage and lariat formation during pre-mRNA splicing in HeLa cell extracts. In addition to its essential role in constitutive splicing, SF2 can strongly influence 5' splice site selection. When pre-mRNAs containing multiple cis-competing 5' splice sites are spliced in vitro, high concentrations of purified SF2 promote the use of the 5' splice site closest to the 3' splice site. However, SF2 discriminates properly between authentic and cryptic splice sites. These effects of SF2 on splice site selection may reflect the cellular mechanisms that prevent exon skipping and ensure the accuracy of splicing. In addition, alterations in the concentration or activity of SF2, and of other general splicing factors, may serve to regulate alternative splicing in vivo.     

Control of BEK and K-SAM splice sites in alternative splicing of the fibroblast growth factor receptor 2 pre-mRNA.

The fibroblast growth factor receptor 2 gene pre-mRNA can be spliced by using either the K-SAM exon or the BEK exon. The exon chosen has a profound influence on the ligand-binding specificity of the receptor obtained. Cells make a choice between the two alternative exons by controlling use of both exons. Using fibroblast growth factor receptor 2 minigenes, we have shown that in cells normally using the K-SAM exon, the BEK exon is not used efficiently even in the absence of the K-SAM exon. This is because these cells apparently express a titratable repressor of BEK exon use. In cells normally using the BEK exon, the K-SAM exon is not used efficiently even in the absence of a functional BEK exon. Three purines in the K-SAM polypyrimidine tract are at least in part responsible for this, as their mutation to pyrimidines leads to efficient use of the K-SAM exon, while mutating the BEK polypyrimidine tract to include these purines stops BEK exon use.     

Ubiquitin-like protein Hub1 is required for pre-mRNA splicing and localization of an essential splicing factor in fission yeast

Hub1/Ubl5 is a member of the family of ubiquitin-like proteins (UBLs). The tertiary structure of Hub1 is similar to that of ubiquitin; however, it differs from known modifiers in that there is no conserved glycine residue near the C terminus which, in ubiquitin and UBLs, is required for covalent modification of target proteins. Instead, there is a conserved dityrosine motif proximal to the terminal nonconserved amino acid. In S. cerevisiae, high molecular weight adducts can be formed in vivo from Hub1, but the structure of these adducts is not known, and they could be either covalent or noncovalent. The budding yeast HUB1 gene is not essential, but Delta hub1 mutants display defects in mating. Here, we report that fission yeast hub1 is an essential gene, whose loss results in cell cycle defects and inefficient pre-mRNA splicing. A screen for Hub1 interactors identified Snu66, a component of the U4/U6.U5 tri-snRNP splicing complex. Furthermore, overexpression of Snu66 suppresses the lethality of a hub1ts mutant. In cells lacking functional hub1, the nuclear localization of Snu66 is disrupted, suggesting that an important role for Hub1 is the correct subcellular targeting of Snu66, although our data suggest that Hub1 is likely to perform other roles in splicing as well.     

The carboxy terminal WD domain of the pre-mRNA splicing factor Prp17p is critical for function

In Saccharomyces cerevisiae, Prp17p is required for the efficient completion of the second step of pre-mRNA splicing. The function and interacting factors for this protein have not been elucidated. We have performed a mutational analysis of yPrp17p to identify protein domains critical for function. A series of deletions were made throughout the region spanning the N-terminal 158 amino acids of the protein, which do not contain any identified structural motifs. The C-terminal portion (amino acids 160-455) contains a WD domain containing seven WD repeats. We determined that a minimal functional Prp17p consists of the WD domain and 40 amino acids N-terminal to it. We generated a three-dimensional model of the WD repeats in Prp17p based on the crystal structure of the beta-transducin WD domain. This model was used to identify potentially important amino acids for in vivo functional characterization. Through analysis of mutations in four different loops of Prp17p that lie between beta strands in the WD repeats, we have identified four amino acids, 235TETG238, that are critical for function. These amino acids are predicted to be surface exposed and may be involved in interactions that are important for splicing. Temperature-sensitive prp17 alleles with mutations of these four amino acids are defective for the second step of splicing and are synthetically lethal with a U5 snRNA loop I mutation, which is also required for the second step of splicing. These data reinforce the functional significance of this region within the WD domain of Prp17p in the second step of splicing.     

A conserved Drosophila transportin-serine/arginine-rich (SR) protein permits nuclear import of Drosophila SR protein splicing factors and their antagonist repressor splicing factor 1

Members of the highly conserved serine/arginine-rich (SR) protein family are nuclear factors involved in splicing of metazoan mRNA precursors. In mammals, two nuclear import receptors, transportin (TRN)-SR1 and TRN-SR2, are responsible for targeting SR proteins to the nucleus. Distinctive features in the nuclear localization signal between Drosophila and mammalian SR proteins prompted us to examine the mechanism by which Drosophila SR proteins and their antagonist repressor splicing factor 1 (RSF1) are imported into nucleus. Herein, we report the identification and characterization of a Drosophila importin beta-family protein (dTRN-SR), homologous to TRN-SR2, that specifically interacts with both SR proteins and RSF1. dTRN-SR has a broad localization in the cytoplasm and the nucleus, whereas an N-terminal deletion mutant colocalizes with SR proteins in nuclear speckles. Far Western experiments established that the RS domain of SR proteins and the GRS domain of RSF1 are required for the direct interaction with dTRN-SR, an interaction that can be modulated by phosphorylation. Using the yeast model system in which nuclear import of Drosophila SR proteins and RSF1 is impaired, we demonstrate that complementation with dTRN-SR is sufficient to target these proteins to the nucleus. Together, the results imply that the mechanism by which SR proteins are imported to the nucleus is conserved between Drosophila and humans.     

FUBP1 is a general splicing factor facilitating 3′ splice site recognition and splicing of long introns

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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.     

The role of alternative pre-mRNA splicing in regulating the structure and function of skeletal protein 4.1

Alternative pre-mRNA splicing plays a major role in regulating cell type-specific expression of the protein 4.1 family of skeletal proteins. The biological importance of alternative splicing as a mechanism for 4.1 gene regulation is underscored by studies of the prototypical 4.1R gene in erythroid cells: activation of exon 16 inclusion in mRna at the erythroblast stage greatly enhances the ability of newly synthesized 4.1R protein to bind spectrin and actin, and thus assemble into a stable membrane skeleton. This gain-of- function has profound effects on the biophysical properties of deformability and membrane strength that are critical to red cell survival in the circulation. Another example of developmentally regulated splicing occurs in differentiating mammary epithelial cells in culture, where cell morphogenesis is accompanied by a splicing switch that reversibly activates inclusion of alternative exon muscle. Few other genes are known to be so richly endowed with regulated switches in pre-mRna splicing making the 4.1R gene an interesting paradigm for the role of alternative splicing as a mediator of cell function. Recent evidence that other members of the 4.1 gene family are also regulated by alternative splicing suggests, moreover, that this phenomenon is of general importance in regulating the structure of this class of skeletal proteins.     

A novel protein factor is required for use of distal alternative 5'' splice sites in vitro.

Adenovirus E1A pre-mRNA was used as a model to examine alternative 5' splice site selection during in vitro splicing reactions. Strong preference for the downstream 13S 5' splice site over the upstream 12S or 9S 5' splice sites was observed. However, the 12S 5' splice site was used efficiently when a mutant pre-mRNA lacking the 13S 5' splice site was processed, and 12S splicing from this substrate was not reduced by 13S splicing from a separate pre-mRNA, demonstrating that 13S splicing reduced 12S 5' splice site selection through a bona fide cis-competition. DEAE-cellulose chromatography of nuclear extract yielded two fractions with different splicing activities. The bound fraction contained all components required for efficient splicing of simple substrates but was unable to utilize alternative 5' splice sites. In contrast, the flow-through fraction, which by itself was inactive, contained an activity required for alternative splicing and was shown to stimulate 12S and 9S splicing, while reducing 13S splicing, when added to reactions carried out by the bound fraction. Furthermore, the activity, which we have called distal splicing factor (DSF), enhanced utilization of an upstream 5' splice site on a simian virus 40 early pre-mRNA, suggesting that the factor acts in a position-dependent, substrate-independent fashion. Several lines of evidence are presented suggesting that DSF is a non-small nuclear ribonucleoprotein protein. Finally, we describe a functional interaction between DSF and ASF, a protein that enhances use of downstream 5' splice sites.     

The RNA-binding protein Staufen1 is increased in DM1 skeletal muscle and promotes alternative pre-mRNA splicing

In myotonic dystrophy type 1 (DM1), dystrophia myotonica protein kinase messenger ribonucleic acids (RNAs; mRNAs) with expanded CUG repeats (CUG(exp)) aggregate in the nucleus and become toxic to cells by sequestering and/or misregulating RNA-binding proteins, resulting in aberrant alternative splicing. In this paper, we find that the RNA-binding protein Staufen1 is markedly and specifically increased in skeletal muscle from DM1 mouse models and patients. We show that Staufen1 interacts with mutant CUG(exp) mRNAs and promotes their nuclear export and translation. This effect is critically dependent on the third double-stranded RNA-binding domain of Staufen1 and shuttling of Staufen1 into the nucleus via its nuclear localization signal. Moreover, we uncover a new role of Staufen1 in splicing regulation. Overexpression of Staufen1 rescues alternative splicing of two key pre-mRNAs known to be aberrantly spliced in DM1, suggesting its increased expression represents an adaptive response to the pathology. Altogether, our results unravel a novel function for Staufen1 in splicing regulation and indicate that it may positively modulate the complex DM1 phenotype, thereby revealing its potential as a therapeutic target.     

Requirement for SLU7 in yeast pre-mRNA splicing is dictated by the distance between the branchpoint and the 3' splice site.

Yeast pre-mRNA splicing factors SLU7 and PRP16 are required for cleavage of the 3' splice site and exon ligation in vitro. Using natural and model precursor RNAs, we found that SLU7 is dispensable for splicing of RNAs in which the 3' splice site is in close proximity to the branchpoint. SLU7 is only required when the interval between the branchpoint and the 3' splice site is greater than 7 nt. In contrast, PRP16 is essential for splicing of all pre-mRNAs tested. Immunoprecipitation of the products of step 1 by anti-SLU7 antibodies demonstrates that SLU7 is a component of the spliceosome. Recruitment of SLU7 to the spliceosome is greatly enhanced by prior addition of PRP16. PRP16 is liberated from the spliceosome after completion of step 2, whereas SLU7 remains bound to the excised intron and spliced mature RNA until the spliceosome disassembles, in a reaction that requires ATP.     

p68 RNA helicase is an essential human splicing factor that acts at the U1 snRNA-5' splice site duplex

Modulation of the interaction between U1 snRNP and the 5' splice site (5'ss) is a key event that governs 5'ss recognition and spliceosome assembly. Using the methylene blue-mediated cross-linking method (Z. R. Liu, A. M. Wilkie, M. J. Clemens, and C. W. Smith, RNA 2:611-621, 1996), a 65-kDa protein (p65) was shown to interact with the U1-5'ss duplex during spliceosome assembly (Z. R. Liu, B. Sargueil, and C. W. Smith, Mol. Cell. Biol. 18:6910-6920, 1998). In this report, p65 was identified as p68 RNA helicase and shown to be essential for in vitro pre-mRNA splicing. Depletion of endogenous p68 RNA helicase does not affect the loading of the U1 snRNP to the 5'ss during early stage of splicing. However, dissociation of the U1 from the 5'ss is largely inhibited. The data suggest that p68 RNA helicase functions in destabilizing the U1-5'ss interactions. Furthermore, depletion of p68 RNA helicase arrested spliceosome assembly at the prespliceosome stage, suggesting that p68 may play a role in the transition from prespliceosome to spliceosome.     

A novel function for human factor C1 (HCF-1), a host protein required for herpes simplex virus infection,in pre-mRNA splicing

Human factor C1 (HCF-1) is needed for the expression of herpes simplex virus 1 (HSV-1) immediate-early genes in infected mammalian cells. Here, we provide evidence that HCF-1 is required for spliceosome assembly and splicing in mammalian nuclear extracts. HCF-1 interacts with complexes containing splicing snRNPs in uninfected mammalian cells and is a stable component of the spliceosome complex. We show that a missense mutation in HCF-1 in the BHK21 hamster cell line tsBN67, at the non-permissive temperature, inhibits the protein's interaction with U1 and U5 splicing snRNPs, causes inefficient spliceosome assembly and inhibits splicing. Transient expression of wild-type HCF-1 in tsBN67 cells restores splicing at the non-permissive temperature. The inhibition of splicing in tsBN67 cells correlates with the temperature-sensitive cell cycle arrest phenotype, suggesting that HCF-1-dependent splicing events may be required for cell cycle progression.     

Antagonistic effects of the SRp30c protein and cryptic 5' splice sites on the alternative splicing of the apoptotic regulator Bcl-x

Alternative 5' splice site selection allows Bcl-x to produce two isoforms with opposite effects on apoptosis. The pro-apoptotic Bcl-x(S) variant is up-regulated by ceramide and down-regulated by protein kinase C through specific cis-acting exonic elements, one of which is bound by SAP155. Splicing to the Bcl-x(S) 5' splice site is also enforced by heterogeneous nuclear ribonucleoprotein (hnRNP) F/H proteins and by Sam68 in cooperation with hnRNP A1. Here, we have characterized exon elements that influence splicing to the 5' splice site of the anti-apoptotic Bcl-x(L) isoform. Within a 86-nucleotide region (B3) located immediately upstream of the Bcl-x(L) donor site we have identified two elements (ML2 and AM2) that stimulate splicing to the Bcl-x(L) 5' splice site. SRp30c binds to these elements and can shift splicing to the 5' splice site of Bcl-x(L) in an ML2/AM2-dependent manner in vitro and in vivo. The B3 region also contains an element that represses the use of Bcl-x(L). This element is bound by U1 small nuclear ribonucleoprotein and contains two 5' splice sites that can be used when the Bcl-x(L) 5' splice site is mutated or the ML2/AM2 elements are deleted. Conversely, mutating the cryptic 5' splice sites stimulates splicing to the Bcl-x(L) site. Thus, SRp30c stimulates splicing to the downstream 5' splice site of Bcl-x(L), thereby attenuating the repressive effect of upstream U1 snRNP binding sites.     

A novel splicing factor, Yju2, is associated with NTC and acts after Prp2 in promoting the first catalytic reaction of pre-mRNA splicing

The Prp19-associated complex (NTC) is essential for pre-mRNA splicing and is associated with the spliceosome during spliceosome activation. NTC is required for specifying interactions of U5 and U6 with pre-mRNA to stabilize their association with the spliceosome after dissociation of U4. Here, we show that a novel splicing factor, Yju2, is associated with components of NTC, and that it is required for pre-mRNA splicing both in vivo and in vitro. During spliceosome assembly, Yju2 is associated with the spliceosome at nearly the same time as NTC but is destabilized after the first catalytic reaction, whereas other NTC components remain associated until the reaction is complete. Extracts depleted of Yju2 could be complemented by recombinant Yju2, suggesting that Yju2 and NTC are not entirely in association with each other. Yju2 is not required for the binding of NTC to the spliceosome or for NTC-mediated spliceosome activation. Complementation analysis of the affinity-isolated spliceosome formed in Yju2-depleted extracts demonstrated that Yju2 acts in concert with an unidentified heat-resistant factor(s) in an ATP-independent manner to promote the first catalytic reaction of pre-mRNA splicing after Prp2-mediated structural rearrangement of the spliceosome.     

A 38 nt region and its flanking sequences within gag of Friend murine leukemia virus are crucial for splicing at the correct 5′ and 3′ splice sites

The genome of the Friend murine leukemia virus (Fr‐MLV) contains a 5′ splice site (5′ss) located at 205 nt and a 3′ss located at 5489 nt. In our previous studies, it was shown that if the HindIII–BglII (879–1904 bp) fragment within gag is deleted from the proA8m1 vector, which carries the entire Fr‐MLV sequence, then cryptic splicing of env‐mRNA occurs. Here, attempts were made to identify the genomic segment(s) in this region that is/are essential to correct splicing. First, vectors with a serially truncated HindIII–BglII fragment were constructed. The vector, in which a 38 bp fragment (1612–1649 bp) is deleted or reversed in proA8m1, only produced splice variants. It was found that a 38 nt region within gag contains important elements that positively regulate splicing at the correct splice sites. Further analyses of a series of vectors carrying the 38 bp fragment and its flanking sequences showed that a region (1183–1611 nt) upstream of the 38 nt fragment also contains sequences that positively or negatively influence splicing at the correct splice sites. The SphI–NdeI (5140–5400 bp) fragment just upstream of the 3′ss was deleted from vectors that carried the 38 bp fragment and its flanking sequences, which yielded correctly spliced mRNA; interestingly, these deleted vectors showed cryptic splicing. These findings suggest that the 5140–5400 nt region located just upstream of the 3′ss is required for the splicing function of the 38 nt fragment and its flanking sequences.