Processive phosphorylation of alternative splicing factor/splicing factor 2

SR proteins, named for their multiple arginine/serine (RS) dipeptide repeats, are critical components of the spliceosome, influencing both constitutive and alternative splicing of pre-mRNA. SR protein function is regulated through phosphorylation of their RS domains by multiple kinases, including a family of evolutionarily conserved SR protein-specific kinases (SRPKs). The SRPK family of kinases is unique in that they are capable of phosphorylating repetitive RS domains with remarkable specificity and efficiency. Here, we carried out kinetic experiments specially developed to investigate how SRPK1 phosphorylates the model human SR protein, ASF/SF2. By using the start-trap strategy, we monitored the progress curve for ASF/SF2 phosphorylation in the absence and presence of an inhibitor peptide directed at the active site of SRPK1. ASF/SF2 modification is not altered when the inhibitor peptide (trap) is added with ATP (start). However, when the trap is added first and allowed to incubate for a specific delay time, the decrease in phosphate content of the enzyme-substrate complex follows a simple exponential decline corresponding to the release rate of SRPK1. These data demonstrate that SRPK1 phosphorylates a specific region within the RS domain of ASF/SF2 by using a fully processive catalytic mechanism, in which the splicing factor remains "locked" onto SRPK1 during RS domain modification.     

Identification of a plant serine-arginine-rich protein similar to the mammalian splicing factor SF2/ASF.

We show that the higher plant Arabidopsis thaliana has a serine-arginine-rich (SR) protein family whose members contain a phosphoepitope shared by the animal SR family of splicing factors. In addition, we report the cloning and characterization of a cDNA encoding a higher-plant SR protein from Arabidopsis, SR1, which has striking sequence and structural homology to the human splicing factor SF2/ASF. Similar to SF2/ASF, the plant SR1 protein promotes splice site switching in mammalian nuclear extracts. A novel feature of the Arabidopsis SR protein is a C-terminal domain containing a high concentration of proline, serine, and lysine residues (PSK domain), a composition reminiscent of histones. This domain includes a putative phosphorylation site for the mitotic kinase cyclin/p34cdc2.     

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.     

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.     

RNA splicing specificity determined by the coordinated action of RNA recognition motifs in SR proteins

Pre-mRNA splicing requires a large number of RNA-binding proteins that have one or more RNA-recognition motifs (RRMs). Among these is the SR protein family, whose members are essential for splicing and are able to commit pre-mRNAs to the splicing pathway with overlapping but distinct substrate specificity. Some SR proteins, such as SC35, contain an N-terminal RRM and a C-terminal arginine/serine-rich (RS) domain, whereas others, such as SF2/ASF, also contain a second, atypical RRM. Although both the RRMs and the RS domain of SR proteins are required for constitutive splicing, it is unclear which domain(s) defines their substrate specificity, and whether two RRMs in a given SR protein function independently or act coordinately. Using domain swaps between SC35 and SF2/ASF and a functional commitment assay, we demonstrate that individual domains are functional modules, RS domains are interchangeable, and substrate specificity is defined by the RRMs. The atypical RRM of SF2/ASF does not appear to function alone in splicing, but can either activate or suppress the splicing specificity of an N-terminal RRM. Therefore, multiple RRMs in SR proteins act coordinately to achieve a unique spectrum of pre-mRNA substrate specificity.     

Conservation in budding yeast of a kinase specific for SR splicing factors

SR protein kinases (SRPKs) and their substrates, the SR family of serine/arginine-rich pre-mRNA splicing factors, appear to be key regulators of alternative splicing. Although SR proteins have been well characterized through biochemical experiments in metazoans, their functions in vivo are unclear. Because of the strict splice site consensus and near absence of alternative splicing in Saccharomyces cerevisiae, it had been thought that budding yeast would lack an SRPK and its substrates. Here, we present structural, biochemical, and cell-biological evidence that directly demonstrates an SR protein kinase, Sky1p, as well as a number of SRPK substrates in S. cerevisiae. One of these substrates is Npl3p, an SR-like protein involved in mRNA export. This finding raises the provocative possibility that Sky1p, and by extension metazoan SRPKs, regulates mRNA export or the nucleocytoplasmic shuttling of RS domain proteins. The unexpected discovery of an SR protein kinase in budding yeast provides a foundation for genetic dissection of the biological functions of SR proteins and their kinases.     

A proposed signaling motif for nuclear import in mRNA processing via the formation of arginine claw

Phosphorylation of proteins by kinases is the most commonly studied class of posttranslational modification, yet its structural consequences are not well understood. The human SR (serine-arginine) protein ASF/SF2 relies on the processive phosphorylation of the serine residues of eight consecutive arginine-serine (RS) dipeptide repeats at the C terminus by SRPK1 before it can be transported into the nucleus. This SR protein plays critical roles in spliceosome assembly, pre-mRNA splicing, and mRNA export, and the phosphorylation process of the RS repeats has been extensively studied experimentally. However, knowledge of the conformational changes associated with the phosphorylation of this simple sequence and how it triggers the importation of the SR protein is lacking. Here, we have carried out extensive molecular dynamics simulations to show that phosphorylation of the eight RS repeats significantly alters the peptide's conformation and leads to the formation of very stable structures that are likely to be involved in the recognition, binding, and transport of the SR protein. Specifically, we found an unusual symmetry-broken phase of conformations of the repetitive and quasi-symmetric phosphorylated peptide sequence. One of the main characteristics of these conformations is the exposed phosphate groups on the periphery, which possibly could serve as the recognition platform for the transport protein transportin-SR2.     

Utilization of host SR protein kinases and RNA-splicing machinery during viral replication

Although the viral genome is often quite small, it encodes a broad series of proteins. The virus takes advantage of the host-RNA-processing machinery to provide the alternative splicing capability necessary for the expression of this proteomic diversity. Serine-arginine-rich (SR) proteins and the kinases that activate them are central to this alternative splicing machinery. In studies reported here, we use the HIV genome as a model. We show that HIV expression decreases overall SR protein/activity. However, we also show that HIV expression is significantly increased (20-fold) when one of the SR proteins, SRp75 is phosphorylated by SR protein kinase (SRPK)2. Thus, inhibitors of SRPK2 and perhaps of functionally related kinases, such as SRPK1, could be useful antiviral agents. Here, we develop this hypothesis and show that HIV expression down-regulates SR proteins in Flp-In293 cells, resulting in only low-level HIV expression in these cells. However, increasing SRPK2 function up-regulates HIV expression. In addition, we introduce SR protein phosphorylation inhibitor 340 (SRPIN340), which preferentially inhibits SRPK1 and SRPK2 and down-regulates SRp75. Although an isonicotinamide compound, SPRIN340 (or its derivatives) remain to be optimized for better specificity and lower cytotoxicity, we show here that SRPIN340 suppresses propagation of Sindbis virus in plaque assay and variably suppresses HIV production. Thus, we show that SRPK, a well known kinase in the cellular RNA-processing machinery, is used by at least some viruses for propagation and hence suggest that SRPIN340 or its derivatives may be useful for curbing viral diseases.     

A molecular link between SR protein dephosphorylation and mRNA export

In metazoans, multiple RNA-binding proteins, including the shuttling serine/arginine-rich (SR)-splicing factors, function as adapters for mRNA nuclear export by interacting with the export receptor TAP/nuclear export factor 1 (NXF1). Yet, it is unclear how interactions between adapters and TAP are regulated. Here, we demonstrate that the SR proteins 9G8 and ASF/SF2 exhibit higher affinity for TAP/NXF1 when hypophosphorylated. 9G8 is recruited to the pre-mRNA in a hyperphosphorylated form but becomes hypophosphorylated during splicing both in vivo and in vitro. TAP preferentially binds spliced mRNA-protein complexes compared with pre-mRNA-protein complexes. Thus, the phosphorylation state of the SR protein adapters may underlie the selectivity of TAP-mediated export of spliced mRNA.     

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.     

Transportin-SR2 mediates nuclear import of phosphorylated SR proteins

Serine/arginine-rich proteins (SR proteins) are a family of nuclear factors that play important roles in both constitutive and regulated precursor mRNA splicing. The domain rich in arginine/serine (RS) repeats (RS domain) serves as both a nuclear and subnuclear localization signal. We previously identified an importin beta family protein, transportin-SR2 (TRN-SR2), that specifically interacts with phosphorylated RS domains. A TRN-SR2 mutant deficient in Ran binding colocalizes with SR proteins in nuclear speckles, suggesting a role of TRN-SR2 in nuclear targeting of SR proteins. Using in vitro import assays, we here show that nuclear import of SR protein fusions requires cytosolic factors, and that the RS domain becomes phosphorylated in the import reaction. Reconstitution of SR protein import by using recombinant transport factors clearly demonstrates that TRN-SR2 is capable of targeting phosphorylated, but not unphosphorylated, SR proteins to the nucleus. Therefore, RS domain phosphorylation is critical for TRN-SR2-mediated nuclear import. Interestingly, we found that the RNA-binding activity of SR proteins confers temperature sensitivity to their nuclear import. Finally, we show that TRN-SR2 interacts with a nucleoporin and is targeted not only to the nuclear envelope but also to nuclear speckles in vitro. Thus, TRN-SR2 may perhaps escort SR protein cargoes to nuclear subdomains.     

Regulation and differential role of the tissue factor isoforms in cardiovascular biology

Alternative pre-mRNA splicing is an essential mechanism regulating protein diversity and functional plasticity of the proteome in response to environmental changes. Several factors are involved in this regulatory mechanism, such as serine/arginine-rich (SR) proteins, the Cdc2-like kinase (Clk) family, the dual-specificity tyrosine phosphorylation regulated kinases, the SR protein kinases (SRPK) 1 and 2, the protein kinase B (PKB,Akt), and the DNA topoisomerase I (DNA topo I). Dynamic changes of the phosphorylation state of SR proteins, mediated by the kinases mentioned previously, play an important role in alternative splicing regulation. Through alternative splicing of the tissue factor (TF) pre-mRNA, two naturally occurring forms of TF, the primary initiator of blood coagulation, are expressed in humans-soluble alternatively spliced (as)TF and membrane-bound "full length" (fl)TF. Both isoforms are known to circulate in blood. flTF, rather than asTF, appears to be the major contributor to the thrombogenicity of vascular wall and blood. asTF has been linked more closely to increased cell survival and angiogenesis. We found the expression of asTF and flTF to be reduced in the myocardium of patients with dilated cardiomyopathy, indicating a role of TF in maintaining myocardial structure. Moreover, we demonstrated proinflammatory cytokines to immediately upregulate the expression of both TF isoforms, which was differentially regulated by SR proteins as well as Clks and DNA topo I. We and others have shown that asTF induces cell proliferation, survival, and angiogenesis via signaling pathways different from flTF-induced effects. These data indicate that both TF isoforms influence diverse processes in cardiovascular (patho)biology and are potential targets for antithrombotic, pro-survival, and proangiogenic therapeutic strategies.     

CLK1 reorganizes the splicing factor U1-70K for early spliceosomal protein assembly

Early spliceosome assembly requires phosphorylation of U1-70K, a constituent of the U1 small nuclear ribonucleoprotein (snRNP), but it is unclear which sites are phosphorylated, and by what enzyme, and how such modification regulates function. By profiling the proteome, we found that the Cdc2-like kinase 1 (CLK1) phosphorylates Ser-226 in the C terminus of U1-70K. This releases U1-70K from subnuclear granules facilitating interaction with U1 snRNP and the serine-arginine (SR) protein SRSF1, critical steps in establishing the 5′ splice site. CLK1 breaks contacts between the C terminus and the RNA recognition motif (RRM) in U1-70K releasing the RRM to bind SRSF1. This reorganization also permits stable interactions between U1-70K and several proteins associated with U1 snRNP. Nuclear induction of the SR protein kinase 1 (SRPK1) facilitates CLK1 dissociation from U1-70K, recycling the kinase for catalysis. These studies demonstrate that CLK1 plays a vital, signal-dependent role in early spliceosomal protein assembly by contouring U1-70K for protein–protein multitasking.

The splicing of precursor messenger RNA (pre-mRNA) occurs at the spliceosome, a multimegadalton complex composed of 5 small nuclear ribonucleoproteins (U1-6 snRNP) and over 100 proteins (1, 2). The assembly of this catalytic machine is a multistep process involving the exchange of numerous RNA-protein components leading to the active spliceosome. The first assembly stage involves U1 snRNP association at the 5′ splice site in pre-mRNA, an event guided by essential splicing factors known as the serine-arginine (SR) proteins, named for a C-terminal domain rich in arginine-serine (RS) dipeptides (3, 4). SR proteins contain one or two N-terminal RNA recognition motifs (RRMs) that bind exonic splicing enhancer (ESE) sequences near pre-mRNA splice sites (5). The RRMs are also important for U1 snRNP association at splice sites. An RRM (RRM1) from the SR protein SRSF1 (also known as ASF/SF2) binds the RRM from U1-70K, an essential protein substituent of U1 snRNP (6). Since U1 snRNP recognizes an intronic sequence from −2 to +6 along the 5′ splice site that includes only two conserved bases (GU), physical coupling of U1-70K to the SR protein that binds ESEs (five to seven nucleotides) provides additional specificity for proper 5′ splice-site assignment (7, 8). Importantly, these protein–protein assembly events are tightly regulated through SR protein phosphorylation. RS domain phosphorylation severs internal RS–RRM contacts within SRSF1, freeing its RRMs for both pre-mRNA binding and association with phosphorylated U1-70K in the U1 snRNP (6, 9, 10). While these posttranslational modifications are important for protein assembly, SR protein dephosphorylation is also required for attaining the fully active, mature spliceosome (11–13).Much is known about how phosphorylation regulates the structure and function of SR proteins. SR protein kinase 1 (SRPK1) phosphorylates Arg-Ser dipeptides in the RS domain of SRSF1 generating a “hypo-phosphorylated” state that allows SR-specific transportin TRN-SR2 binding and nuclear localization (14–16). This modified state of SRSF1 includes mostly phosphorylation of Arg-Ser dipeptides toward the N terminus of the RS domain, a region referred to as RS1 (residues 204 to 224). In the nucleus, SRSF1 largely resides in nonmembrane compartments known as speckles (17). The Cdc2-like kinase 1 (CLK1) phosphorylates three additional sites (Ser-Pro dipeptides) in the SRSF1 RS domain generating a “hyper-phosphorylated” state that enhances SRSF1 diffusion from speckles where it then binds pre-mRNA initiating early spliceosome assembly (18, 19). Nuclear CLK1 releases autoinhibitory contacts within SRSF1 allowing association of an RRM (RRM1) with the RRM in U1-70K (6). NMR studies showed that CLK1 phosphorylation breaks contacts between the RS domain and several residues in RRM1 liberating RRM1 (9). Unlike SRPK1 that contains a conserved docking groove in its kinase domain, CLK1 relies upon a lengthy, disordered N terminus that recognizes the disordered RS domains in SR proteins (20, 21). This generates high-affinity substrate recognition but does not efficiently release the phospho-SR protein. Epidermal growth factor (EGF) stimulation solves this dilemma by increasing nuclear SRPK1 where it binds the N terminus of CLK1, releasing phospho-SR proteins from CLK1 (22). SRPK1–CLK1 complex formation also promotes efficient CLK1-specific phosphorylation of Ser-Pro dipeptides and diffusion of SR proteins from speckles for splicing (22, 23). Thus, CLK1 recruits SRPK1 for enhanced phosphorylation and kinase recycling.Although U1-70K binding requires a phosphorylated SR protein (24, 25), U1-70K must also undergo phosphorylation to accept SR proteins. Previous complementation studies using U1 snRNP-depleted nuclear extracts and purified U1 snRNP incubated with either ATP or ATP-γS have shown that U1-70K phosphorylation supports spliceosome assembly and splicing activity whereas phosphatase-resistant thiophosphorylation allows assembly but not splicing (26). Such findings suggest that, similar to SR proteins, U1-70K must be phosphorylated early for spliceosome assembly and then dephosphorylated later for catalytic function. However, it is not clear which sites are modified, and by what protein kinase, and how such phosphorylation regulates U1-70K structure. SRPK1 has been shown to copurify with U1 snRNP and thus could serve this function, but it also binds CLK1 in the nucleus raising the question of whether it acts alone or in a kinase–kinase complex (22, 23, 27). To evaluate a possible role for CLK1, we used a combination of affinity-purification and phosphoproteomic approaches to identify CLK1 interactors and substrates. This dual-proteomic strategy pinpointed a CLK1-dependent phosphorylation site on U1-70K (Ser-226). We found that Ser-226 phosphorylation releases an internal contact between the C terminus and RRM in U1-70K. This structural change enhances U1-70K diffusion from subnuclear storage compartments, allows physical attachment of the U1-70K RRM to the RRMs in SRSF1, and promotes U1-70K binding to U1 snRNP-associated proteins. EGF stimulation induces nuclear SRPK1 translocation where it promotes CLK1 release from U1-70K, recycling the kinase for catalysis. These studies identify a prominent role for nuclear CLK1 in the assembly of vital protein complexes involved in early spliceosomal development.     

The splicing-factor oncoprotein SF2/ASF activates mTORC1

The splicing factor SF2/ASF is an oncoprotein that is up-regulated in many cancers and can transform immortal rodent fibroblasts when slightly overexpressed. The mTOR signaling pathway is activated in many cancers, and pharmacological blockers of this pathway are in clinical trials as anticancer drugs. We examined the activity of the mTOR pathway in cells transformed by SF2/ASF and found that this splicing factor activates the mTORC1 branch of the pathway, as measured by S6K and eIF4EBP1 phosphorylation. This activation is specific to mTORC1 because no activation of Akt, an mTORC2 substrate, was detected. mTORC1 activation by SF2/ASF bypasses upstream PI3K/Akt signaling and is essential for SF2/ASF-mediated transformation, as inhibition of mTOR by rapamycin blocked transformation by SF2/ASF in vitro and in vivo. Moreover, shRNA-mediated knockdown of mTOR, or of the specific mTORC1 and mTORC2 components Raptor and Rictor, abolished the tumorigenic potential of cells overexpressing SF2/ASF. These results suggest that clinical tumors with SF2/ASF up-regulation could be especially sensitive to mTOR inhibitors.     

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.