Initiation of Parental Genome Reprogramming in Fertilized Oocyte by Splicing Kinase SRPK1-Catalyzed Protamine Phosphorylation.

The paternal genome undergoes a massive exchange of histone with protamine for compaction into sperm during spermiogenesis. Upon fertilization, this process is potently reversed, which is essential for parental genome reprogramming and subsequent activation; however, it remains poorly understood how this fundamental process is initiated and regulated. Here, we report that the previously characterized splicing kinase SRPK1 initiates this life-beginning event by catalyzing site-specific phosphorylation of protamine, thereby triggering protamine-to-histone exchange in the fertilized oocyte. Interestingly, protamine undergoes a DNA-dependent phase transition to gel-like condensates and SRPK1-mediated phosphorylation likely helps open up such structures to enhance protamine dismissal by nucleoplasmin (NPM2) and enable the recruitment of HIRA for H3.3 deposition. Remarkably, genome-wide assay for transposase-accessible chromatin sequencing (ATAC-seq) analysis reveals that selective chromatin accessibility in both sperm and MII oocytes is largely erased in early pronuclei in a protamine phosphorylation-dependent manner, suggesting that SRPK1-catalyzed phosphorylation initiates a highly synchronized reorganization program in both parental genomes.

Release of SR Proteins from CLK1 by SRPK1: A Symbiotic Kinase System for Phosphorylation Control of Pre-mRNA Splicing.

Phosphorylation has been generally thought to activate the SR family of splicing factors for efficient splice-site recognition, but this idea is incompatible with an early observation that overexpression of an SR protein kinase, such as the CDC2-like kinase 1 (CLK1), weakens splice-site selection. Here, we report that CLK1 binds SR proteins but lacks the mechanism to release phosphorylated SR proteins, thus functionally inactivating the splicing factors. Interestingly, CLK1 overcomes this dilemma through a symbiotic relationship with the serine-arginine protein kinase 1 (SRPK1). We show that SRPK1 interacts with an RS-like domain in the N terminus of CLK1 to facilitate the release of phosphorylated SR proteins, which then promotes efficient splice-site recognition and subsequent spliceosome assembly. These findings reveal an unprecedented signaling mechanism by which two protein kinases fulfill separate catalytic features that are normally encoded in single kinases to institute phosphorylation control of pre-mRNA splicing in the nucleus.

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.

Disordered protein interactions for an ordered cellular transition: Cdc2-like kinase 1 is transported to the nucleus via its Ser-Arg protein substrate.

Serine-arginine (SR) proteins are essential splicing factors that promote numerous steps associated with mRNA processing and whose biological function is tightly regulated through multi-site phosphorylation. In the nucleus, the cdc2-like kinases (CLKs) phosphorylate SR proteins on their intrinsically disordered Arg-Ser (RS) domains, mobilizing them from storage speckles to the splicing machinery. The CLKs have disordered N termini that bind tightly to RS domains, enhancing SR protein phosphorylation. The N termini also promote nuclear localization of CLKs, but their transport mechanism is presently unknown. To explore cytoplasmic-nuclear transitions, several classical nuclear localization sequences in the N terminus of the CLK1 isoform were identified, but their mutation had no effect on subcellular localization. Rather, we found that CLK1 amplifies its presence in the nucleus by forming a stable complex with the SR protein substrate and appropriating its NLS for transport. These findings indicate that, along with their well-established roles in mRNA splicing, SR proteins use disordered protein-protein interactions to carry their kinase regulator from the cytoplasm to the nucleus.

Molecular interactions connecting the function of the serine-arginine-rich protein SRSF1 to protein phosphatase 1.

Splicing generates many mRNA strands from a single precursor mRNA, expanding the proteome and enhancing intracellular diversity. Both initial assembly and activation of the spliceosome require an essential family of splicing factors called serine-arginine (SR) proteins. Protein phosphatase 1 (PP1) regulates the SR proteins by controlling phosphorylation of a C-terminal arginine-serine-rich (RS) domain. These modifications are vital for the subcellular localization and mRNA splicing function of the SR protein. Although PP1 has been shown to dephosphorylate the prototype SR protein splicing factor 1 (SRSF1), the molecular nature of this interaction is not understood. Here, using NMR spectroscopy, we identified two electrostatic residues in helix α2 and a hydrophobic residue in helix α1 in the RNA recognition motif 1 (RRM1) of SRSF1 that constitute a binding surface for PP1. Substitution of these residues dissociated SRSF1 from PP1 and enhanced phosphatase activity, reducing phosphorylation in the RS domain. These effects lead to shifts in alternative splicing patterns that parallel increases in SRSF1 diffusion from speckles to the nucleoplasm brought on by regiospecific decreases in RS domain phosphorylation. Overall, these findings establish a molecular and biological connection between PP1-targeted amino acids in an RRM with the phosphorylation state and mRNA-processing function of an SR protein.

Mobilization of a splicing factor through a nuclear kinase-kinase complex.

The splicing of mRNA is dependent on serine-arginine (SR) proteins that are mobilized from membrane-free, nuclear speckles to the nucleoplasm by the Cdc2-like kinases (CLKs). This movement is critical for SR protein-dependent assembly of the macromolecular spliceosome. Although CLK1 facilitates such trafficking through the phosphorylation of serine-proline dipeptides in the prototype SR protein SRSF1, an unrelated enzyme known as SR protein kinase 1 (SRPK1) performs the same function but does not efficiently modify these dipeptides in SRSF1. We now show that the ability of SRPK1 to mobilize SRSF1 from speckles to the nucleoplasm is dependent on active CLK1. Diffusion from speckles is promoted by the formation of an SRPK1-CLK1 complex that facilitates dissociation of SRSF1 from CLK1 and enhances the phosphorylation of several serine-proline dipeptides in this SR protein. Down-regulation of either kinase blocks EGF-stimulated mobilization of nuclear SRSF1. These findings establish a signaling pathway that connects SRPKs to SR protein activation through the associated CLK family of kinases.

Intra-domain Cross-talk Regulates Serine-arginine Protein Kinase 1-dependent Phosphorylation and Splicing Function of Transformer 2ß1.

Transformer 2ß1 (Tra2ß1) is a splicing effector protein composed of a core RNA recognition motif flanked by two arginine-serine-rich (RS) domains, RS1 and RS2. Although Tra2ß1-dependent splicing is regulated by phosphorylation, very little is known about how protein kinases phosphorylate these two RS domains. We now show that the serine-arginine protein kinase-1 (SRPK1) is a regulator of Tra2ß1 and promotes exon inclusion in the survival motor neuron gene 2 (SMN2). To understand how SRPK1 phosphorylates this splicing factor, we performed mass spectrometric and kinetic experiments. We found that SRPK1 specifically phosphorylates 21 serines in RS1, a process facilitated by a docking groove in the kinase domain. Although SRPK1 readily phosphorylates RS2 in a splice variant lacking the N-terminal RS domain (Tra2ß3), RS1 blocks phosphorylation of these serines in the full-length Tra2ß1. Thus, RS2 serves two new functions. First, RS2 positively regulates binding of the central RNA recognition motif to an exonic splicing enhancer sequence, a phenomenon reversed by SRPK1 phosphorylation on RS1. Second, RS2 enhances ligand exchange in the SRPK1 active site allowing highly efficient Tra2ß1 phosphorylation. These studies demonstrate that SRPK1 is a regulator of Tra2ß1 splicing function and that the individual RS domains engage in considerable cross-talk, assuming novel functions with regard to RNA binding, splicing, and SRPK1 catalysis.

Nuclear protein kinase CLK1 uses a non-traditional docking mechanism to select physiological substrates.

Phosphorylation-dependent cell communication requires enzymes that specifically recognize key proteins in a sea of similar, competing substrates. The protein kinases achieve this goal by utilizing docking grooves in the kinase domain or heterologous protein adaptors to reduce 'off pathway' targeting. We now provide evidence that the nuclear protein kinase CLK1 (cell division cycle2-like kinase 1) important for splicing regulation departs from these classic paradigms by using a novel self-association mechanism. The disordered N-terminus of CLK1 induces oligomerization, a necessary event for targeting its physiological substrates the SR protein (splicing factor containing a C-terminal RS domain) family of splicing factors. Increasing the CLK1 concentration enhances phosphorylation of the splicing regulator SRSF1 (SR protein splicing factor 1) compared with the general substrate myelin basic protein (MBP). In contrast, removal of the N-terminus or dilution of CLK1 induces monomer formation and reverses this specificity. CLK1 self-association also occurs in the nucleus, is induced by the N-terminus and is important for localization of the kinase in sub-nuclear compartments known as speckles. These findings present a new picture of substrate recognition for a protein kinase in which an intrinsically disordered domain is used to capture physiological targets with similar disordered domains in a large oligomeric complex while discriminating against non-physiological targets.

Conserved proline-directed phosphorylation regulates SR protein conformation and splicing function.

The alternative splicing of human genes is dependent on SR proteins, a family of essential splicing factors whose name derives from a signature C-terminal domain rich in arginine-serine dipeptide repeats (RS domains). Although the SRPKs (SR-specific protein kinases) phosphorylate these repeats, RS domains also contain prolines with flanking serines that are phosphorylated by a second family of protein kinases known as the CLKs (Cdc2-like kinases). The role of specific serine-proline phosphorylation within the RS domain has been difficult to assign since CLKs also phosphorylate arginine-serine dipeptides and, thus, display overlapping residue specificities with the SRPKs. In the present study, we address the effects of discrete serine-proline phosphorylation on the conformation and cellular function of the SR protein SRSF1 (SR protein splicing factor 1). Using chemical tagging and dephosphorylation experiments, we show that modification of serine-proline dipeptides broadly amplifies the conformational ensemble of SRSF1. The induction of these new structural forms triggers SRSF1 mobilization in the nucleus and alters its binding mechanism to an exonic splicing enhancer in precursor mRNA. These physical events correlate with changes in the alternative splicing of over 100 human genes based on a global splicing assay. Overall, these studies draw a direct causal relationship between a specific type of chemical modification in an SR protein and the regulation of alternative gene splicing programmes.

N-terminus of the protein kinase CLK1 induces SR protein hyperphosphorylation.

SR proteins are essential splicing factors that are regulated through multisite phosphorylation of their RS (arginine/serine-rich) domains by two major families of protein kinases. The SRPKs (SR-specific protein kinases) efficiently phosphorylate the arginine/serine dipeptides in the RS domain using a conserved docking groove in the kinase domain. In contrast, CLKs (Cdc2-like kinases) lack a docking groove and phosphorylate both arginine/serine and serine-proline dipeptides, modifications that generate a hyperphosphorylated state important for unique SR protein-dependent splicing activities. All CLKs contain long flexible N-terminal extensions (140-300 residues) that resemble the RS domains present in their substrate SR proteins. We showed that the N-terminus in CLK1 contacts both the kinase domain and the RS domain of the SR protein SRSF1 (SR protein splicing factor 1). This interaction not only is essential for facilitating hyperphosphorylation, but also induces co-operative binding of SRSF1 to RNA. The N-terminus of CLK1 enhances the total phosphoryl contents of a panel of physiological substrates including SRSF1, SRSF2, SRSF5 and Tra2ß1 (transformer 2ß1) by 2-3-fold. These findings suggest that CLK1-dependent hyperphosphorylation is the result of a general mechanism in which the N-terminus acts as a bridge connecting the kinase domain and the RS domain of the SR protein.

Recruiting a silent partner for activation of the protein kinase SRPK1.

The SRPK family of protein kinases regulates mRNA splicing by phosphorylating an essential group of factors known as SR proteins, so named for a C-terminal domain enriched in arginine-serine dipeptide repeats (RS domains). SRPKs phosphorylate RS domains at numerous sites altering SR protein subcellular localization and splicing function. The RS domains in these splicing factors differ considerably in overall length and dipeptide layout. Despite their importance, little is known about how these diverse RS domains interact with SRPKs and regulate SR protein phosphorylation. We now show that sequences distal to the SRPK1 consensus region in the RS domain of the prototype SR protein SRSF1 are not passive as originally thought but rather play active roles in accelerating phosphorylation rates. Located in the C-terminal end of the RS domain, this nonconsensus region up-regulates rate-limiting ADP release through the nucleotide release factor, a structural module in SRPK1 composed of two noncontiguous sequence elements outside the kinase core domain. The data show that the RS domain in SRSF1 is multifunctional and that sequences once thought to be catalytically silent can be recruited to enhance the efficiency of SR protein phosphorylation.

C6 pyridinium ceramide influences alternative pre-mRNA splicing by inhibiting protein phosphatase-1.

Alternative pre-mRNA processing is a central element of eukaryotic gene regulation. The cell frequently alters the use of alternative exons in response to physiological stimuli. Ceramides are lipid-signaling molecules composed of sphingosine and a fatty acid. Previously, water-insoluble ceramides were shown to change alternative splicing and decrease SR-protein phosphorylation by activating protein phosphatase-1 (PP1). To gain further mechanistical insight into ceramide-mediated alternative splicing, we analyzed the effect of C6 pyridinium ceramide (PyrCer) on alternative splice site selection. PyrCer is a water-soluble ceramide analog that is under investigation as a cancer drug. We found that PyrCer binds to the PP1 catalytic subunit and inhibits the dephosphorylation of several splicing regulatory proteins containing the evolutionarily conserved RVxF PP1-binding motif (including PSF/SFPQ, Tra2-beta1 and SF2/ASF). In contrast to natural ceramides, PyrCer promotes phosphorylation of splicing factors. Exons that are regulated by PyrCer have in common suboptimal splice sites, are unusually short and share two 4-nt motifs, GAAR and CAAG. They are dependent on PSF/SFPQ, whose phosphorylation is regulated by PyrCer. Our results indicate that lipids can influence pre-mRNA processing by regulating the phosphorylation status of specific regulatory factors, which is mediated by protein phosphatase activity.

Splicing kinase SRPK1 conforms to the landscape of its SR protein substrate.

The splicing function of SR proteins is regulated by multisite phosphorylation of their C-terminal RS (arginine-serine rich) domains. SRPK1 has been shown to phosphorylate the prototype SR protein SRSF1 using a directional mechanism in which 11 serines flanked by arginines are sequentially fed from a docking groove in the large lobe of the kinase domain to the active site. Although this process is expected to operate on lengthy arginine-serine repeats (≥8), many SR proteins contain smaller repeats of only 1-4 dipeptides, raising the question of how alternate RS domain configurations are phosphorylated. To address this, we studied a splice variant of Tra2ß that contains a C-terminal RS domain with short arginine-serine repeats [Tra2ß(ΔN)]. We showed that SRPK1 selectively phosphorylates several serines near the C-terminus of the RS domain. SRPK1 uses a distributive mechanism for Tra2ß(ΔN) where the rate-limiting step is the dissociation of the protein substrate rather than nucleotide exchange as in the case of SRSF1. Although a functioning docking groove is required for efficient SRSF1 phosphorylation, this conserved structural element is dispensable for Tra2ß(ΔN) phosphorylation. These large shifts in mechanism are likely to account for the slower net turnover rate of Tra2ß(ΔN) compared to SRSF1 and may signal fundamental differences in phosphorylation among SR proteins with distinctive arginine-serine profiles. Overall, these data indicate that SRPK1 conforms to changes in RS domain architecture using a flexible kinetic mechanism and selective usage of a conserved docking groove.

Kinetic mechanism of human histidine triad nucleotide binding protein 1.

Human histidine triad nucleotide binding protein 1 (hHint1) is a member of a ubiquitous and ancient branch of the histidine triad protein superfamily. hHint1 is a homodimeric protein that catalyzes the hydrolysis of model substrates, phosphoramidate and acyl adenylate, with a high efficiency. Recently, catalytically inactive hHint1 has been identified as the cause of inherited peripheral neuropathy [Zimon, M., et al. (2012) Nat. Genet. 44, 1080-1083]. We have conducted the first detailed kinetic mechanistic studies of hHint1 and have found that the reaction mechanism is consistent with a double-displacement mechanism, in which the active site nucleophile His112 is first adenylylated by the substrate, followed by hydrolysis of the AMP-enzyme intermediate. A transient burst phase followed by a linear phase from the stopped-flow fluorescence assay indicated that enzyme adenylylation was faster than the subsequent intermediate hydrolysis and product release. Solvent viscosity experiments suggested that both chemical transformation and diffusion-sensitive events (product release or protein conformational change) limit the overall turnover. The catalytic trapping experiments and data simulation indicated that the true koff rate of the final product AMP is unlikely to control the overall kcat. Therefore, a protein conformational change associated with product release is likely rate-limiting. In addition, the rate of Hint1 adenylylation was found to be dependent on two residues with pKa values of 6.5 and 8, with the former pKa agreeing well with the nuclear magnetic resonance titration results for the pKa of the active site nucleophile His112. In comparison to the uncatalyzed rates, hHint1 was shown to enhance acyl-AMP and AMP phosphoramidate hydrolysis by 10(6)-10(8)-fold. Taken together, our analysis indicates that hHint1 catalyzes the hydrolysis of phosphoramidate and acyl adenylate with high efficiency, through a mechanism that relies on rapid adenylylation of the active residue, His112, while being partially rate-limited by intermediate hydrolysis and product release associated with a conformational change. Given the high degree of sequence homology of Hint proteins across all kingdoms of life, it is likely that their kinetic and catalytic mechanisms will be similar to those elucidated for hHint1.

Nucleotide release sequences in the protein kinase SRPK1 accelerate substrate phosphorylation.

Protein kinases are essential signaling enzymes that transfer phosphates from bound ATP to select amino acids in protein targets. For most kinases, the phosphoryl transfer step is highly efficient, while the rate-limiting step for substrate processing involves slow release of the product ADP. It is generally thought that structural factors intrinsic to the kinase domain and the nucleotide-binding pocket control this step and consequently the efficiency of protein phosphorylation for these cases. However, the kinase domains of protein kinases are commonly flanked by sequences that regulate catalytic function. To address whether such sequences could alter nucleotide exchange and, thus, regulate protein phosphorylation, the presence of activating residues external to the kinase domain was probed in the serine protein kinase SRPK1. Deletion analyses indicate that a small segment of a large spacer insert domain and a portion of an N-terminal extension function cooperatively to increase nucleotide exchange. The data point to a new mode of protein kinase regulation in which select sequences outside the kinase domain constitute a nucleotide release factor that likely interacts with the small lobe of the kinase domain and enhances protein substrate phosphorylation through increases in ADP dissociation rate.

SRPK1 regulates RNA binding in a pre-spliceosomal complex using a catalytic bypass mechanism.

Serine-arginine protein kinase 1 (SRPK1) phosphorylates serine-arginine (SR) proteins in the cytoplasm, directing them to the nucleus for splicing function. SRPK1 has also been detected in the nucleus but its function here is still not fully understood. We now demonstrate that nuclear SRPK1 can regulate U1-70K, a protein component of the uridine-rich 1 small nuclear ribonucleoprotein (U1 snRNP) that binds SR proteins and facilitates 5' splice-site selection in precursor mRNA. We found that SRPK1 uses a large, disordered domain to bind U1-70K, regulating the interaction of an exonic splicing enhancer (ESE) to the associated SR protein. Surprisingly, the catalytic activity of SRPK1 is not required for this phenomenon. Instead, SRPK1 associates directly with the N-terminus of U1-70K and alters the regulatory function of the distal C-terminus, modifying interactions between the U1-70K:SR protein complex and the ESE. Disruption of SRPK1 binding to this complex affects the alternative splicing of genes modulated by the C-terminus of U1-70K. Such findings suggest that, in addition to operating as a traditional serine-modifying catalyst, SRPK1 can also bypass this intrinsic activity to regulate RNA contacts in an early pre-spliceosomal complex.

Applying the brakes to multisite SR protein phosphorylation: substrate-induced effects on the splicing kinase SRPK1.

To investigate how a protein kinase interacts with its protein substrate during extended, multisite phosphorylation, the kinetic mechanism of a protein kinase involved in mRNA splicing control was investigated using rapid quench flow techniques. The protein kinase SRPK1 phosphorylates ~10 serines in the arginine--serine-rich domain (RS domain) of the SR protein SRSF1 in a C- to N-terminal direction, a modification that directs this essential splicing factor from the cytoplasm to the nucleus. Transient-state kinetic experiments illustrate that the first phosphate is added rapidly onto the RS domain of SRSF1 (t(1/2) = 0.1 s) followed by slower, multisite phosphorylation at the remaining serines (t(1/2) = 15 s). Mutagenesis experiments suggest that efficient phosphorylation rates are maintained by an extensive hydrogen bonding and electrostatic network between the RS domain of the SR protein and the active site and docking groove of the kinase. Catalytic trapping and viscosometric experiments demonstrate that while the phosphoryl transfer step is fast, ADP release limits multisite phosphorylation. By studying phosphate incorporation into selectively pre-phosphorylated forms of the enzyme-substrate complex, the kinetic mechanism for site-specific phosphorylation along the reaction coordinate was assessed. The binding affinity of the SR protein, the phosphoryl transfer rate, and ADP exchange rate were found to decline significantly as a function of progressive phosphorylation in the RS domain. These findings indicate that the protein substrate actively modulates initiation, extension, and termination events associated with prolonged, multisite phosphorylation.

A conserved sequence motif bridges two protein kinases for enhanced phosphorylation and nuclear function of a splicing factor.

The assembly and activation of the spliceosome rely upon the phosphorylation of an essential family of splicing factors known as the serine-arginine (SR) proteins. Although it has been demonstrated recently that two enzyme families, the SR protein kinases (SRPKs) and the Cdc2-like kinases (CLKs), can function as a complex to efficiently phosphorylate these SR proteins in the nucleus, the molecular features involved in such a connection are unknown. In this study, we identified a group of conserved residues in the large lobe of SRPK1 that interact with the N terminus of CLK1 stabilizing the SRPK1-CLK1 complex. Mutations in this motif not only disrupt formation of the kinase-kinase complex but also impair SRPK1-dependent release of the phospho-SR protein from CLK1. The binding motif potently up-regulates CLK1-specific phosphorylation sites, enhances SR protein diffusion from nuclear speckles, and impacts the alternative splicing of several target genes. These results indicate that CLK1 binds a conserved, electronegative surface on SRPK1, thereby controlling SR protein phosphorylation levels for enhanced subnuclear trafficking and alternative splicing regulation.

Matrix Rigidity Controls Epithelial-Mesenchymal Plasticity and Tumor Metastasis via a Mechanoresponsive EPHA2/LYN Complex.

Mechanical cues from the extracellular matrix (ECM) regulate various cellular processes via distinct mechanotransduction pathways. In breast cancer, increased ECM stiffness promotes epithelial-to-mesenchymal transition (EMT), cell invasion, and metastasis. Here, we identify a mechanosensitive EPHA2/LYN protein complex regulating EMT and metastasis in response to increasing ECM stiffness during tumor progression. High ECM stiffness leads to ligand-independent phosphorylation of ephrin receptor EPHA2, which recruits and activates the LYN kinase. LYN phosphorylates the EMT transcription factor TWIST1 to release TWIST1 from its cytoplasmic anchor G3BP2 to enter the nucleus, thus triggering EMT and invasion. Genetic and pharmacological inhibition of this pathway prevents breast tumor invasion and metastasis in vivo. In human breast cancer samples, activation of this pathway correlates with collagen fiber alignment, a marker of increasing ECM stiffness. Our findings reveal an EPHA2/LYN/TWIST1 mechanotransduction pathway that responds to mechanical signals from the tumor microenvironment to drive EMT, invasion, and metastasis.

Redirecting SR Protein Nuclear Trafficking through an Allosteric Platform.

Although phosphorylation directs serine-arginine (SR) proteins from nuclear storage speckles to the nucleoplasm for splicing function, dephosphorylation paradoxically induces similar movement, raising the question of how such chemical modifications are balanced in these essential splicing factors. In this new study, we investigated the interaction of protein phosphatase 1 (PP1) with the SR protein splicing factor (SRSF1) to understand the foundation of these opposing effects in the nucleus. We found that RNA recognition motif 1 (RRM1) in SRSF1 binds PP1 and represses its catalytic function through an allosteric mechanism. Disruption of RRM1-PP1 interactions reduces the phosphorylation status of the RS domain in vitro and in cells, redirecting SRSF1 in the nucleus. The data imply that an allosteric SR protein-phosphatase platform balances phosphorylation levels in a "goldilocks" region for the proper subnuclear storage of an SR protein splicing factor.