Activation of pre-mRNA splicing by human RNPS1 is regulated by CK2 phosphorylation

Human RNPS1 was originally characterized as a pre-mRNA splicing activator in vitro and was shown to regulate alternative splicing in vivo. RNPS1 was also identified as a protein component of the splicing-dependent mRNP complex, or exon-exon junction complex (EJC), and a role for RNPS1 in postsplicing processes has been proposed. Here we demonstrate that RNPS1 incorporates into active spliceosomes, enhances the formation of the ATP-dependent A complex, and promotes the generation of both intermediate and final spliced products. RNPS1 is phosphorylated in vivo and interacts with the CK2 (casein kinase II) protein kinase. Serine 53 (Ser-53) of RNPS1 was identified as the major phosphorylation site for CK2 in vitro, and the same site is also phosphorylated in vivo. The phosphorylation status of Ser-53 significantly affects splicing activation in vitro, but it does not perturb the nuclear localization of RNPS1. In vivo experiments indicated that the phosphorylation of RNPS1 at Ser-53 influences the efficiencies of both splicing and translation. We propose that RNPS1 is a splicing regulator whose activator function is controlled in part by CK2 phosphorylation.     

Human RNPS1 and its associated factors: a versatile alternative pre-mRNA splicing regulator in vivo

Human RNPS1 was originally purified and characterized as a pre-mRNA splicing activator, and its role in the postsplicing process has also been proposed recently. To search for factors that functionally interact with RNPS1, we performed a yeast two-hybrid screen with a human cDNA library. Four factors were identified: p54 (also called SRp54; a member of the SR protein family), human transformer 2β (hTra2β; an exonic splicing enhancer-binding protein), hLucA (a potential component of U1 snRNP), and pinin (also called DRS and MemA; a protein localized in nuclear speckles). The N-terminal region containing the serine-rich (S) domain, the central RNA recognition motif (RRM), and the C-terminal arginine/serine/proline-rich (RS/P) domain of RNPS1 interact with p54, pinin, and hTra2β, respectively. Protein-protein binding between RNPS1 and these factors was verified in vitro and in vivo. Overexpression of RNPS1 in HeLa cells induced exon skipping in a model β-globin pre-mRNA and a human tra-2β pre-mRNA. Coexpression of RNPS1 with p54 cooperatively stimulated exon inclusion in an ATP synthase γ-subunit pre-mRNA. The RS/P domain and RRM are necessary for the exon-skipping activity, whereas the S domain is important for the cooperative effect with p54. RNPS1 appears to be a versatile factor that regulates alternative splicing of a variety of pre-mRNAs.     

Human SAP18 mediates assembly of a splicing regulatory multiprotein complex via its ubiquitin-like fold

RNPS1, Acinus, and SAP18 form the apoptosis- and splicing-associated protein (ASAP) complex, which is also part of the exon junction complex. Whereas RNPS1 was originally identified as a general activator of mRNA processing, all three proteins have been found within functional spliceosomes. Both RNPS1 and Acinus contain typical motifs of splicing regulatory proteins including arginine/serine-rich domains. Due to the absence of such structural features, however, a function of SAP18 in splicing regulation is completely unknown. Here we have investigated splicing regulatory activities of the ASAP components. Whereas a full-length Acinus isoform displayed only limited splicing regulatory activity, both RNPS1 and, surprisingly, SAP18 strongly modulated splicing regulation. Detailed mutational analysis and three-dimensional modeling data revealed that the ubiquitin-like fold of SAP18 was required for efficient splicing regulatory activity. Coimmunoprecipitation and immunofluorescence experiments demonstrated that SAP18 assembles a nuclear speckle-localized splicing regulatory multiprotein complex including RNPS1 and Acinus via its ubiquitin-like fold. Our results therefore suggest a novel function of SAP18 in splicing regulation.     

Nuclear Pnn/DRS protein binds to spliced mRNPs and participates in mRNA processing and export via interaction with RNPS1

Pnn/DRS protein is associated with desmosomes and colocalizes with splicing factors in nuclear speckled domains. The potential interaction of Pnn with RNPS1, a pre-mRNA splicing factor and a component of the exon-exon junction complex, prompted us to examine whether Pnn is involved in nuclear mRNA processing. By immunoprecipitation, we found that Pnn associates preferentially with mRNAs produced by splicing in vitro. Oligonucleotide-directed RNase H digestion revealed that Pnn binds to the spliced mRNAs at a position immediately upstream of the splice junction and that 5' splice site utilization determines the location of Pnn in alternatively spliced mRNAs. Immunoprecipitation further showed that Pnn binds to mRNAs produced from a transiently expressed reporter in vivo. Although associated with mRNPs, Pnn is a nuclear-restricted protein as revealed by the heterokaryon assay. Overexpression of an amino-terminal fragment of Pnn that directly interacts with RNPS1 leads to blockage of pre-mRNA splicing. However, although suppression of Pnn expression shows no significant effect on splicing, it leads to some extent to nuclear accumulation of bulk poly(A)(+) RNA. Therefore, Pnn may participate, via its interaction with RNPS1, in mRNA metabolism in the nucleus, including mRNA splicing and export.     

Proteins associated with the exon junction complex also control the alternative splicing of apoptotic regulators

Several apoptotic regulators, including Bcl-x, are alternatively spliced to produce isoforms with opposite functions. We have used an RNA interference strategy to map the regulatory landscape controlling the expression of the Bcl-x splice variants in human cells. Depleting proteins known as core (Y14 and eIF4A3) or auxiliary (RNPS1, Acinus, and SAP18) components of the exon junction complex (EJC) improved the production of the proapoptotic Bcl-x(S) splice variant. This effect was not seen when we depleted EJC proteins that typically participate in mRNA export (UAP56, Aly/Ref, and TAP) or that associate with the EJC to enforce nonsense-mediated RNA decay (MNL51, Upf1, Upf2, and Upf3b). Core and auxiliary EJC components modulated Bcl-x splicing through different cis-acting elements, further suggesting that this activity is distinct from the established EJC function. In support of a direct role in splicing control, recombinant eIF4A3, Y14, and Magoh proteins associated preferentially with the endogenous Bcl-x pre-mRNA, interacted with a model Bcl-x pre-mRNA in early splicing complexes, and specifically shifted Bcl-x alternative splicing in nuclear extracts. Finally, the depletion of Y14, eIF4A3, RNPS1, SAP18, and Acinus also encouraged the production of other proapoptotic splice variants, suggesting that EJC-associated components are important regulators of apoptosis acting at the alternative splicing level.     

Function of conserved domains of hnRNP A1 and other hnRNP A/B proteins.

hnRNP A1 is a pre-mRNA binding protein that antagonizes the alternative splicing activity of splicing factors SF2/ASF or SC35, causing activation of distal 5' splice sites. The structural requirements for hnRNP A1 function were determined by mutagenesis of recombinant human hnRNP A1. Two conserved Phe residues in the RNP-1 submotif of each of two RNA recognition motifs appear to be involved in specific RNA-protein interactions and are essential for modulating alternative splicing. These residues are not required for general pre-mRNA binding or RNA annealing activity. The C-terminal Gly-rich domain is necessary for alternative splicing activity, for stable RNA binding and for optimal RNA annealing activity. hnRNP A1B, which is an alternatively spliced isoform of hnRNP A1 with a longer Gly-rich domain, binds more strongly to pre-mRNA but has only limited alternative splicing activity. In contrast, hnRNP A2 and B1, which have 68% amino acid identity with hnRNP A1, bind more weakly to pre-mRNA and have stronger splice site switching activities than hnRNP A1. We propose that specific combinations of antagonistic hnRNP A/B and SR proteins are involved in regulating alternative splicing of distinct subsets of cellular premRNAs.     

Modulation of alternative pre-mRNA splicing in vivo by pinin

Pre-mRNA splicing occurs in a large macromolecular RNA-protein complex called the spliceosome. The major components of the spliceosome include snRNP and SR proteins. We have previously identified an SR-like protein, pinin (pnn), which is localized not only in nuclear speckles but also at desmosomes. The nuclear localization of pnn is a dynamic process because pnn can be found not only with SR proteins in nuclear speckles but also in enlarged speckles following treatment of cells with RNA polymerase II inhibitors, DRB, and alpha-amanitin. Using adenovirus E1A and chimeric calcitonin/dhfr construct as a splicing reporter minigene in combination with cellular cotransfection, we found that pnn regulates alternative 5(') and 3(') splicing by decreasing the use of distal splice sites. Regulation of 5(') splice site choice was also observed for RNPS1, a general splicing activator that interacts with pnn in nuclear speckles. The regulatory ability of pnn in alternative 5(') splicing, however, was not dependent on RNPS1 and a pnn mutant, lacking the N-terminal 167 amino acids, behaved like a dominant negative species, inhibiting E1A splicing when applied in splicing assays. These results provide direct evidence that pnn functions as a splicing regulator which participates itself directly in splicing reaction or indirectly via other components of splicing machinery.     

ASAP,a novel protein complex involved in RNA processing and apoptosis

Different isoforms of a protein complex termed the apoptosis- and splicing-associated protein (ASAP) were isolated from HeLa cell extract. ASAP complexes are composed of the polypeptides SAP18 and RNPS1 and different isoforms of the Acinus protein. While Acinus had previously been implicated in apoptosis and was recently identified as a component of the spliceosome, RNPS1 has been described as a general activator of RNA processing. Addition of ASAP isoforms to in vitro splicing reactions inhibits RNA processing mediated by ASF/SF2, by SC35, or by RNPS1. Additionally, microinjection of ASAP complexes into mammalian cells resulted in acceleration of cell death. Importantly, after induction of apoptosis the ASAP complex disassembles. Taken together, our results suggest an important role for the ASAP complexes in linking RNA processing and apoptosis.     

A simple whole cell lysate system for in vitro splicing reveals a stepwise assembly of the exon-exon junction complex

Pre-mRNA splicing removes introns and leaves in its wake a multiprotein complex near the exon-exon junctions of mRNAs. This complex, termed the exon-exon junction complex (EJC), contains at least seven proteins and provides a link between pre-mRNA splicing and downstream events, including transport, localization, and nonsense-mediated mRNA decay. Using a simple whole cell lysate system we developed for in vitro splicing, we prepared lysates from cells transfected with tagged EJC proteins and studied the association of these proteins with pre-mRNA, splicing intermediates, and mRNA, as well as formation of the EJC during splicing. Three of the EJC components, Aly/REF, RNPS1, and SRm160, are found on pre-mRNA by the time the spliceosome is formed, whereas Upf3b associates with splicing intermediates during or immediately after the first catalytic step of the splicing reaction (cleavage of exon 1 and intron-lariat formation). In contrast, Y14 and magoh, which remain stably associated with mRNA after export to the cytoplasm, join the EJC during or after completion of exon-exon ligation. These findings indicate that EJC formation is an ordered pathway that involves stepwise association of components and is coupled to specific intermediates of the splicing reaction.     

Regulation of mammalian pre-mRNA splicing

In eukaryotes, most protein-coding genes contain introns which are removed by precursor messenger RNA (pre-mRNA) splicing. Alternative splicing is a process by which multiple messenger RNAs (mRNAs) are generated from a single pre-mRNA, resulting in functionally distinct proteins. Recent genome-wide analyses of alternative splicing indicated that in higher eukaryotes alternative splicing is an important mechanism that generates proteomic complexity and regulates gene expression. Mis-regulation of splicing causes a wide range of human diseases. This review describes the current understanding of pre-mRNA splicing and the mechanisms that regulate mammalian pre-mRNA splicing. It also discusses emerging directions in the field of alternative splicing. Supported by the Program of “one Hundred Talented people” of the Chinese Academy of Sciences.     

Functional analysis of pre-mRNA splicing factor SF2/ASF structural domains.

Human pre-mRNA splicing factor SF2/ASF has an activity required for general splicing in vitro and promotes utilization of proximal alternative 5' splice sites in a concentration-dependent manner by opposing hnRNP A1. We introduced selected mutations in the N-terminal RNA recognition motif (RRM) and the C-terminal Arg/Ser (RS) domain of SF2/ASF, and assayed the resulting recombinant proteins for constitutive and alternative splicing in vitro and for binding to pre-mRNA and mRNA. Mutants inactive in constitutive splicing can affect alternative splice site selection, demonstrating that these activities involve distinct molecular interactions. Specific protein-RNA contact mediated by Phe56 and Phe58 in the RNP-1 submotif of the SF2/ASF RRM are essential for constitutive splicing, although they are not required for RRM-mediated binding to pre-mRNA. The RS domain is also required for constitutive splicing activity and both Arg and Ser residues are important. Analysis of domain deletion mutants demonstrated strong synergy between the RRM and a central degenerate RRM repeat in binding to RNA. These two domains are sufficient for alternative splicing activity in the absence of an RS domain.     

The serine/arginine-rich protein family in rice plays important roles in constitutive and alternative splicing of pre-mRNA

Ser/Arg-rich (SR) proteins play important roles in the constitutive and alternative splicing of pre-mRNA. We isolated 20 rice (Oryza sativa) genes encoding SR proteins, of which six contain plant-specific characteristics. To determine whether SR proteins modulate splicing efficiency and alternative splicing of pre-mRNA in rice, we used transient assays in rice protoplasts by cotransformation of SR protein genes with the rice Waxy(b) (Wx(b))-beta-glucuronidase fusion gene. The results showed that plant-specific RSp29 and RSZp23, an SR protein homologous to human 9G8, enhanced splicing and altered the alternative 5' splice sites of Wx(b) intron 1. The resulting splicing pattern was unique to each SR protein; RSp29 stimulated splicing at the distal site, and RSZp23 enhanced splicing at the proximal site. Results of domain-swapping experiments between plant-specific RSp29 and SCL26, which is a homolog of human SC35, showed the importance of RNA recognition motif 1 and the Arg/Ser-rich (RS) domain for the enhancement of splicing efficiencies. Overexpression of plant-specific RSZ36 and SRp33b, a homolog of human ASF/SF2, in transgenic rice changed the alternative splicing patterns of their own pre-mRNAs and those of other SR proteins. These results show that SR proteins play important roles in constitutive and alternative splicing of rice pre-mRNA.     

The haplo-spliceo-transcriptome: common variations in alternative splicing in the human population

Numerous inherited human genetic disorders are caused by defects in pre-mRNA splicing. Two recent studies have added a new twist to the link between genetic variation and pre-mRNA splicing by identifying SNPs that correlate with heritable changes in alternative splicing but do not cause disease. This suggests that allele-specific alternative splicing is a mechanism that accounts for individual variation in the human population.     

Modulation of RNA splicing as a potential treatment for cancer

Close to 90% of human genes are transcribed into pre-mRNA that undergoes alternative splicing, producing multiple mRNAs and proteins from single genes. This process is largely responsible for human proteome diversity, and about half of genetic disease-causing mutations affect splicing. Splice-switching oligonucleotides (SSOs) comprise an emerging class of antisense therapeutics that modify gene expression by directing pre-mRNA splice site usage. Bauman et al. investigated an SSO that up-regulated the expression of an anti-cancer splice variant while simultaneously eliminating an over-expressed cancer-causing splice variant. This was accomplished by targeting pre-mRNA of the apoptotic regulator Bcl-x, which is alternatively spliced to express anti- and pro-apoptotic splice variants Bcl-xL and Bcl-xS, respectively. High expression of Bcl-xL is a hallmark of many cancers and is considered a general mechanism used by cancer cells to evade apoptosis. Redirection of Bcl-x pre-mRNA splicing from Bcl-xL to -xS by SSO induced apoptotic and chemosensitizing effects in various cancer cell lines. Importantly, the paper shows that delivery of Bcl-x SSO using a lipid nanoparticle redirected Bcl-x splicing and reduced tumor burden in melanoma lung metastases. This was the first demonstration of SSO efficacy in tumors in vivo. SSOs are not limited to be solely potential anti-cancer drugs.?SSOs were first applied to repair aberrant splicing in thalassemia, a genetic disease, they have been used to create novel proteins (e.g., ?7TNFR1), and they have recently progressed to clinical trials for patients with Duchenne muscular dystrophy.