Constitutive Splicing Research Articles

Expression of U1 small nuclear ribonucleoprotein 70K antisense transcript using APETALA3 promoter suppresses the development of sepals and petals.

U1 small nuclear ribonucleoprotein (snRNP)-70K (U1-70K), a U1 snRNP-specific protein, is involved in the early stages of spliceosome formation. In non-plant systems, it is involved in constitutive and alternative splicing. It has been shown that U1snRNP is dispensable for in vitro splicing of some animal pre-mRNAs, and inactivation of U1-70K in yeast (Saccharomyces cerevisiae) is not lethal. As in yeast and humans (Homo sapiens), plant U1-70K is coded by a single gene. In this study, we blocked the expression of Arabidopsis U1-70K in petals and stamens by expressing U1-70K antisense transcript using the AP3 (APETALA3) promoter specific to these floral organs. Flowers of transgenic Arabidopsis plants expressing U1-70K antisense transcript showed partially developed stamens and petals that are arrested at different stages of development. In some transgenic lines, flowers have rudimentary petals and stamens and are male sterile. The severity of the phenotype is correlated with the level of the antisense transcript. Molecular analysis of transgenic plants has confirmed that the observed phenotype is not due to disruption of whorl-specific homeotic genes, AP3 or PISTILLATA, responsible for petal and stamen development. The AP3 transcript was not detected in transgenic flowers with severe phenotype. Flowers of Arabidopsis plants transformed with a reporter gene driven by the same promoter showed no abnormalities. These results show that U1-70K is necessary for the development of sepals and petals and is an essential gene in plants.

Serine-Arginine-rich Protein p30 Directs Alternative Splicing of Glucocorticoid Receptor Pre-mRNA to Glucocorticoid Receptor β in Neutrophils

Glucocorticoid (GC) insensitivity is a major clinical challenge in the treatment of many inflammatory diseases. It has been shown previously that GC insensitivity, in several inflammatory cell types, is due to an overabundance of the beta isoform of the glucocorticoid receptor (GCRbeta) relative to the ligand binding isoform, GCRalpha. GCRbeta functions as a dominant inhibitor of GCRalpha action. A number of GCR isoforms are created from the same pre-mRNA transcript via alternative splicing, and the factor or factors that control alternative splicing of GCR pre-mRNA are of great importance. In the current study, we have identified the predominant alternative splicing factor present in human neutrophils, which is known to be exceptionally GC-insensitive. The predominant alternative splicing factor in neutrophils is SRp30c, which is one of several highly conserved serine-arginine-rich (SR) proteins that are involved in both constitutive and alternative splicing in eukaryotic cells. Inhibition of SRp30c expression with antisense oligonucleotide strongly inhibited expression of GCRbeta and stimulated expression of GCRalpha. Antisense molecules targeted to other SR proteins had no effect. Our data indicate that SRp30c is necessary for alternative splicing of the GCR pre-mRNA to create mRNA encoding GCRbeta.

Ectopic expression of atRSZ33 reveals its function in splicing and causes pleiotropic changes in development.

Splicing provides an additional level in the regulation of gene expression and contributes to proteome diversity. Herein, we report the functional characterization of a recently described plant-specific protein, atRSZ33, which has characteristic features of a serine/arginine-rich protein and the ability to interact with other splicing factors, implying that this protein might be involved in constitutive and/or alternative splicing. Overexpression of atRSZ33 leads to alteration of splicing patterns of atSRp30 and atSRp34/SR1, indicating that atRSZ33 is indeed a splicing factor. Moreover, atRSZ33 is a regulator of its own expression, as splicing of its pre-mRNA is changed in transgenic plants. Investigations by promoter-beta-glucuronidase (GUS) fusion and in situ hybridization revealed that atRSZ33 is expressed during embryogenesis and early stages of seedling formation, as well as in flower and root development. Ectopic expression of atRSZ33 caused pleiotropic changes in plant development resulting in increased cell expansion and changed polarization of cell elongation and division. In addition, changes in activity of an auxin-responsive promoter suggest that auxin signaling is disturbed in these transgenic plants.

Regulation and substrate specificity of the SR protein kinase Clk/Sty.

SR proteins constitute a family of splicing factors that play key roles in both constitutive and regulated splicing in metazoan organisms. The proteins are extensively phosphorylated, and kinases capable of phosphorylating them have been identified. However, little is known about how these kinases function, for example, whether they target specific SR proteins or whether the kinases themselves are regulated. Here we describe properties of one such kinase, Clk/Sty, the founding member of the Clk/Sty family of dual-specificity kinases. Clk/Sty is autophosphorylated on both Ser/Thr and Thr residues, and using both direct kinase assays and SR protein-dependent splicing assays, we have analyzed the effects of each type of modification. We find not only that the pattern of phosphorylation on a specific SR protein substrate, ASF/SF2, is modulated by autophosphorylation but also that the ability of Clk/Sty to recognize different SR proteins is influenced by the extent and nature of autophosphorylation. Strikingly, phosphorylation of ASF/SF2 is sensitive to changes in Tyr, but not Ser/Thr, autophosphorylation while that of SC35 displays the opposite pattern. In contrast, phosphorylation of a third SR protein, SRp40, is unaffected by autophosphorylation. We also present biochemical data indicating that as expected for a factor directly involved in splicing control (but in contrast to recent reports), Clk/Sty is found in the nucleus of several different cell types.

Differential effects of hyperphosphorylation on splicing factor SRp55.

Members of the serine/arginine-rich (SR) protein family play an important role in both constitutive and regulated splicing of precursor mRNAs. Phosphorylation of the arginine/serine dipeptide-rich domain (RS domain) can modulate the activity and the subcellular localization of SR proteins. However, whether the SR protein family members are individually regulated and how this is achieved remain unclear. In this report we show that 5,6-dichloro-1 beta-D-ribofuranosyl-benzimidazole (DRB), an inhibitor of RNA polymerase II-dependent transcription, specifically induced hyperphosphorylation of SRp55 but not that of any other SR proteins tested. Hyperphosphorylation of SRp55 occurs at the RS domain and appears to require the RNA-binding activity. Upon DRB treatment, hyperphosphorylated SRp55 relocates to enlarged nuclear speckles. Intriguingly, SRp55 is specifically targeted for degradation by the proteasome upon overexpression of the SR protein kinase Clk/Sty. Although a destabilization signal is mapped within the C-terminal 43-amino acid segment of SRp55, its adjacent lysine/serine-rich RS domain is nevertheless critical for the Clk/Sty-mediated degradation. We report for the first time that SRp55 can be hyperphosphorylated under different circumstances whereby its fate is differentially influenced.

CDNA cloning and spatiotemporal expression during avian embryogenesis of hnRNP A1, a regulatory factor in alternative splicing

Alternative splicing and selective transport of RNA transcripts from cell nuclei are important regulatory mechanisms of gene expression during embryonic development. Here we report the molecular characterization and developmental expression in several tissue and organ systems of chicken hnRNP A1, a nucleo-cytoplasmic ‘shuttle’ protein which in mammalian systems has been shown to function in the regulation of RNA alternative splicing by antagonizing constitutive splicing factors such as SF2/ASF. We show that hnRNP A1 is represented in the chicken by a single gene which is widely expressed at early embryonic stages, with particularly high levels of expression in the brain, skin, developing gut, and other ectodermal and endodermal derivatives. At later stages, expression of its mRNA and protein product become progressively confined to specific organ primordia and cell types, where both transient and persistent expression patterns are observed. HnRNP A1 protein is expressed at sites of active neurogenesis in the developing central and peripheral nervous systems, regions of known extensive alternative splicing.

Conserved sequence elements associated with exon skipping.

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

Interactions between two fission yeast serine/arginine-rich proteins and their modulation by phosphorylation.

The unexpected low number of genes in the human genome has triggered increasing attention to alternative pre-mRNA splicing, and serine/arginine-rich (SR) proteins have been correlated with the complex alternative splicing that is a characteristic of metazoans. SR proteins interact with RNA and splicing protein factors, and they also undergo reversible phosphorylation, thereby regulating constitutive and alternative splicing in mammals and Drosophila. However, it is not clear whether the features of SR proteins and alternative splicing are present in simple and genetically tractable organisms, such as yeasts. In the present study, we show that the SR-like proteins Srp1 and Srp2, found in the fission yeast Schizosaccharomyces pombe, interact with each other and the interaction is modulated by protein phosphorylation. By using Srp1 as bait in a yeast two-hybrid analysis, we specifically isolated Srp2 from a random screen. This Srp interaction was confirmed by a glutathione-S-transferase pull-down assay. We also found that the Srp1-Srp2 complex was phosphorylated at a reduced efficiency by a fission yeast SR-specific kinase, Dis1-suppression kinase (Dsk1). Conversely, Dsk1-mediated phosphorylation inhibited the formation of the Srp complex. These findings offer the first example in fission yeast for interactions between SR-related proteins and the modulation of the interactions by specific protein phosphorylation, suggesting that a mammalian-like SR protein function may exist in fission yeast.

The SR Protein SRp38 Represses Splicing in M Phase Cells

SR proteins constitute a family of pre-mRNA splicing factors that play important roles in both constitutive and regulated splicing. Here, we describe one member of the family, which we call SRp38, with unexpected properties. Unlike other SR proteins, SRp38 cannot activate splicing and is essentially inactive in splicing assays. However, dephosphorylation converts SRp38 to a potent, general repressor that inhibits splicing at an early step. To investigate the cellular function of SRp38, we examined its possible role in cell cycle control. We show first that splicing, like other steps in gene expression, is inhibited in extracts of mitotic cells. Strikingly, SRp38 was found to be dephosphorylated specifically in mitotic cells, and we show that dephosphorylated SRp38 is required for the observed splicing repression.

Functional inactivation of the SR family of splicing factors during a vaccinia virus infection.

SR proteins are essential splicing factors required for constitutive splicing and function as key regulators of alternative RNA splicing. We have shown that SR proteins purified from late adenovirus-infected cells (SR-Ad) are functionally inactivated as splicing enhancer or splicing repressor proteins by a virus-induced partial de-phosphorylation. Here, we show that SR proteins purified from late vaccinia-virus-infected cells (SR-VV) are also hypo-phosphorylated and functionally inactivated as splicing regulatory proteins. We further show that incubating SR-Ad proteins under conditions that restore the phospho-epitopes to the SR proteins results in the restoration of their activity as splicing enhancer and splicing repressor proteins. Interestingly, re-phosphorylation of SR-VV proteins only partially restored the splicing enhancer or splicing repressor phenotype to the SR proteins. Collectively, our results suggest that viral control of SR protein activity may be a common strategy used by DNA viruses to take control of the host cell RNA splicing machinery.

Regulation of alternative splicing by the ATP-dependent DEAD-box RNA helicase p72.

Although a number of ATP-dependent RNA helicases are important for constitutive RNA splicing, no helicases have been implicated in alternative RNA splicing. Here, we show that the abundant DEAD-box RNA helicase p72, but not its close relative p68, affects the splicing of alternative exons containing AC-rich exon enhancer elements. The effect of p72 was tested by using mini-genes that undergo different types of alternative splicing. When the concentration of p72 was increased in transient transfections, the inclusion of enhancer-containing CD44 alternative exons v4 and v5 increased using a mini-gene that contained these exons and their flanking introns inserted into a beta-globin gene. Other types of alternative splicing were not impacted by altering p72 concentrations. Mutation of the p72 helicase ATP-binding site or deletion of the carboxy-terminal region of the protein reduced the ability of the transfected protein to affect CD44 variable exon splicing. Use of in vitro extracts overexpressing p72 indicated that p72 becomes associated with complexes containing precursor RNA. Helicases have been implicated both in altering RNA-RNA interactions and in remodeling RNA-protein complexes. CD44 exon v4 contains a potential internal secondary structure element that base pairs the 5' splice site with a region inside the exon located between enhancer elements. Mutations that destroyed this complementarity modestly increased inclusion in the absence of p72 but still responded to increasing p72 concentration like the wild-type exon, suggesting that p72 might have effects on protein-RNA interactions. In agreement with this hypothesis, p72 was not able to restore the inclusion of an exon mutated for its major enhancer element. Our results suggest that RNA helicases may be important alternative splicing regulatory factors.

Predictive identification of exonic splicing enhancers in human genes.

Specific short oligonucleotide sequences that enhance pre-mRNA splicing when present in exons, termed exonic splicing enhancers (ESEs), play important roles in constitutive and alternative splicing. A computational method, RESCUE-ESE, was developed that predicts which sequences have ESE activity by statistical analysis of exon-intron and splice site composition. When large data sets of human gene sequences were used, this method identified 10 predicted ESE motifs. Representatives of all 10 motifs were found to display enhancer activity in vivo, whereas point mutants of these sequences exhibited sharply reduced activity. The motifs identified enable prediction of the splicing phenotypes of exonic mutations in human genes.

Splicing signals and factors in plant intron removal

Constitutive splicing of the potato invertase miniexon 2 (9 nt long) requires a branchpoint sequence positioned around 50 nt upstream of the 5′ splice site of the adjacent intron and a U11 element found just downstream of the branchpoint in the upstream intron [Simpson, Hedley, Watters, Clark, McQuade, Machray and Brown (2000) RNA 6, 422–433]. The sensitivity of this in vivo plant splicing system has been used to demonstrate exon scanning in plants, and to characterize plant intronic elements, such as branchpoint and poly-pyrimidine tract sequences. Plant introns differ from their vertebrate and yeast couterparts in being UA- or U-rich (up to 85% UA). One of the key differences in splicing between plants and other eukaryotes lies in early intron recognition, which is thought to be mediated by UA-binding proteins. We are adopting three approaches to studying the RNA-protein interactions in plant splicing. First, overexpression of plant splicing factors and, in particular, UA-binding proteins, in conjunction with a range of mini-exon mutants. Secondly, the sequences of around 65% of vertebrate and yeast splicing factors have high-quality matches to Arabidopsis proteins, opening the door to identification and analysis of gene knockouts. Finally, to discover plant-specific proteins involved in splicing and in, for example, rRNA or small nuclear RNA processing, green fluorescent protein-cDNA fusion libraries in viral vectors are being screened.

Half pint Regulates Alternative Splice Site Selection in Drosophila

Alternative splicing is used by metazoans to increase protein diversity and to alter gene expression during development. However, few factors that control splice site choice in vivo have been identified. Here we describe a factor, Half pint (Hfp), that regulates RNA splicing in Drosophila. Females harboring hypomorphic mutations in hfp lay short eggs and show defects in germline mitosis, nuclear morphology, and RNA localization during oogenesis. We find that hfp encodes the Drosophila ortholog of human PUF60 and functions in both constitutive and alternative splicing in vivo. In particular, hfp mutants display striking defects in the developmentally regulated splicing of ovarian tumor ( otu). Furthermore, transgenic expression of the missing otu splice form can rescue the ovarian phenotypes of hfp.

Stability of a PKCI-1-related mRNA is controlled by the splicing factor ASF/SF2: a novel function for SR proteins.

Pre-mRNA splicing is a widely used regulatory mechanism for controlling gene expression, and a family of conserved proteins, SR proteins, participate in both constitutive and alternative splicing. Here we describe a novel function for the SR protein ASF/SF2. We used an embryonic chicken cDNA library to screen for differential mRNA expression in the chicken B-cell line DT40-ASF, expressing or not expressing ASF/SF2. Remarkably, out of 3 x 10(6) clones screened, only one, isolated several times independently, showed ASF/SF2-related differential expression. The isolated cDNA, referred to here as PKCI-r (for PKCI-related), is closely related to the protein kinase C interacting protein (PKCI-1) gene. Transcript levels were increased approximately sixfold in ASF/SF2-depleted cells compared with cells expressing ASF/SF2, indicating a negative role for the SR protein. Strikingly, inhibition of ASF/SF2 expression had no significant effect on PKCI-r splicing, or transcription, but markedly increased the half-life of PKCI-r mRNA (6.6-fold). Similarly, increased mRNA stability was also observed upon expression of exogenous PKCI-r mRNA in cells depleted of ASF/SF2. ASF/SF2 bound to a discrete region containing a purine-rich sequence in the 3' UTR of the PKCI-r transcript, and deletion of this region eliminated ASF/SF2-mediated regulation of transcript stability. Together these data indicate a novel, direct effect of ASF/SF2 on PKCI-r mRNA stability. Therefore, ASF/SF2, and perhaps other SR proteins, affects gene expression in vertebrate cells through regulation of mRNA stability as well as splicing.

Characterization of U2AF(6), a splicing factor related to U2AF(35).

The essential splicing factor U2AF (U2 auxiliary factor) is a heterodimer composed of 65-kDa (U2AF(65)) and 35-kDa (U2AF(35)) subunits. U2AF(35) has multiple functions in pre-mRNA splicing. First, U2AF(35) has been shown to function by directly interacting with the AG at the 3' splice site. Second, U2AF(35) is thought to play a role in the recruitment of U2AF(65) by serine-arginine-rich (SR) proteins in enhancer-dependent splicing. It has been proposed that the physical interaction between the arginine-serine-rich (RS) domain of U2AF(35) and SR proteins is important for this activity. However, other data suggest that this may not be the case. Here, we report the identification of a mammalian gene that encodes a 26-kDa protein bearing strong sequence similarity to U2AF(35), designated U2AF(26). The N-terminal 187 amino acids of U2AF(35) and U2AF(26) are nearly identical. However, the C-terminal domain of U2AF(26) lacks many characteristics of the U2AF(35) RS domain and, therefore, might be incapable of interacting with SR proteins. We show that U2AF(26) can associate with U2AF(65) and can functionally substitute for U2AF(35) in both constitutive and enhancer-dependent splicing, demonstrating that the RS domain of the small U2AF subunit is not required for splicing enhancer function. Finally, we show that U2AF(26) functions by enhancing the binding of U2AF(65) to weak 3' splice sites. These studies identify U2AF(26) as a mammalian splicing factor and demonstrate that distinct U2AF complexes can participate in pre-mRNA splicing. Based on its sequence and functional similarity to U2AF(35), U2AF(26) may play a role in regulating alternative splicing.

SRm160 splicing coactivator promotes transcript 3'-end cleavage.

Individual steps in the processing of pre-mRNA, including 5'-end cap formation, splicing, and 3'-end processing (cleavage and polyadenylation) are highly integrated and can influence one another. In addition, prior splicing can influence downstream steps in gene expression, including export of mRNA from the nucleus. However, the factors and mechanisms coordinating these steps in the maturation of pre-mRNA transcripts are not well understood. In the present study we demonstrate that SRm160 (for serine/arginine repeat-related nuclear matrix protein of 160 kDa), a coactivator of constitutive and exon enhancer-dependent splicing, participates in 3'-end formation. Increased levels of SRm160 promoted the 3'-end cleavage of transcripts both in vivo and in vitro. Remarkably, at high levels in vivo SRm160 activated the 3'-end cleavage and cytoplasmic accumulation of unspliced pre-mRNAs, thereby uncoupling the requirement for splicing to promote the 3'-end formation and nuclear release of these transcripts. Consistent with a role in 3'-end formation coupled to splicing, SRm160 was found to associate specifically with the cleavage polyadenylation specificity factor and to stimulate the 3'-end cleavage of splicing-active pre-mRNAs more efficiently than that of splicing-inactive pre-mRNAs in vitro. The results provide evidence for a role for SRm160 in mRNA 3'-end formation and suggest that the level of this splicing coactivator is important for the proper coordination of pre-mRNA processing events.

Mutational analysis of a plant branchpoint and polypyrimidine tract required for constitutive splicing of a mini-exon.

The branchpoint sequence and associated polypyrimidine tract are firmly established splicing signals in vertebrates. In plants, however, these signals have not been characterized in detail. The potato invertase mini-exon 2 (9 nt) requires a branchpoint sequence positioned around 50 nt upstream of the 5' splice site of the neighboring intron and a U11 element found adjacent to the branchpoint in the upstream intron (Simpson et al., RNA, 2000, 6:422-433). Utilizing the sensitivity of this plant splicing system, these elements have been characterized by systematic mutation and analysis of the effect on inclusion of the mini-exon. Mutation of the branchpoint sequence in all possible positions demonstrated that branchpoints matching the consensus, CURAY, were most efficient at supporting splicing. Branchpoint sequences that differed from this consensus were still able to permit mini-exon inclusion but at greatly reduced levels. Mutation of the downstream U11 element suggested that it functioned as a polypyrimidine tract rather than a UA-rich element, common to plant introns. The minimum sequence requirement of the polypyrimidine tract for efficient splicing was two closely positioned groups of uridines 3-4 nt long (<6 nt apart) that, within the context of the mini-exon system, required being close (<14 nt) to the branchpoint sequence. The functional characterization of the branchpoint sequence and polypyrimidine tract defines these sequences in plants for the first time, and firmly establishes polypyrimidine tracts as important signals in splicing of at least some plant introns.

Multiple cis elements regulate an alternative splicing event at 4.1R pre-mRNA during erythroid differentiation

AbstractThe inclusion of exon 16 in the mature protein 4.1R messenger RNA (mRNA) is a critical event in red blood cell membrane biogenesis. It occurs during late erythroid development and results in inclusion of the 10-kd domain needed for stabilization of the spectrin/actin lattice. In this study, an experimental model was established in murine erythroleukemia cells that reproduces the endogenous exon 16 splicing patterns from a transfected minigene. Exon 16 was excluded in predifferentiated and predominantly included after induction. This suggests that the minigene contained exon and abutting intronic sequences sufficient for splicing regulation. A systematic analysis of the cis-acting regulatory sequences that reside within the exon and flanking introns was performed. Results showed that (1) the upstream intron of 4.1R pre-mRNA is required for exon recognition and it displays 2 enhancer elements, a distal element acting in differentiating cells and a proximal constitutive enhancer that resides within the 25 nucleotides preceding the acceptor site; (2) the exon itself contains a strong constitutive splicing silencer; (3) the exon has a weak 5′ splice site; and (4) the downstream intron contains at least 2 splicing enhancer elements acting in differentiating cells, a proximal element at the vicinity of the 5′ splice site, and a distal element containing 3 copies of the UGCAUG motif. These results suggest that the interplay between negative and positive elements may determine the inclusion or exclusion of exon 16. The activation of the enhancer elements in late erythroid differentiation may play an important role in the retention of exon 16.

Multiple interactions between SRm160 and SR family proteins in enhancer-dependent splicing and development of C. elegans

Background: SR family and SR-related proteins assemble on exonic splicing enhancer (ESE) sequences to promote both constitutive and regulated splicing. The SRm160 splicing coactivator, an SR-related nuclear matrix protein of 160 kDa, is important for the splicing of specific constitutive and ESE-dependent pre-mRNAs.Results: In the present study, we show that SRm160 is required to promote pre-mRNA splicing mediated by a large population of functional ESE sequences within a randomized 18 nucleotide sequence. This suggests that it functions as a general coactivator by interacting with different SR family/SR-related proteins bound to different ESE sequences. Consistent with this, several SR family and SR-related proteins coimmunoprecipitated specifically with SRm160 in the presence of low salt. We used RNA interference (RNAi) in Caenorhabditis elegans to determine whether interactions between CeSRm160 and different CeSR family proteins are important in a whole-organism context. Previously we showed that RNAi of CeSRm160 and individual CeSR family genes other than CeSF2/ASF results in no obvious phenotype, which is indicative of gene redundancy. In the present study, we demonstrate that RNAi of CeSRm160 in combination with any CeSR family gene results in the production of unfertilized oocytes by the injected mother.Conclusions: The observation that simultaneous suppression of CeSRm160 and individual CeSR family proteins results in a distinct phenotype is indicative of critical functional interactions between these factors. Our results provide biochemical and genetic evidence indicating that interactions between SRm160 and multiple SR family proteins are important for both optimal splicing activity and for proper development.