Directional Phosphorylation and Nuclear Transport of the Splicing Factor SRSF1 Is Regulated by an RNA Recognition Motif

Abstract

Multisite phosphorylation is required for the biological function of serine-arginine (SR) proteins, a family of essential regulators of mRNA splicing. These modifications are catalyzed by serine-arginine protein kinases (SRPKs) that phosphorylate numerous serines in arginine-serine-rich (RS) domains of SR proteins using a directional, C-to-N-terminal mechanism. The present studies explore how SRPKs govern this highly biased phosphorylation reaction and investigate biological roles of the observed directional phosphorylation mechanism. Using NMR spectroscopy with two separately expressed domains of SRSF1, we showed that several residues in the RNA-binding motif 2 interact with the N-terminal region of the RS domain (RS1). These contacts provide a structural framework that balances the activities of SRPK1 and the protein phosphatase PP1, thereby regulating the phosphoryl content of the RS domain. Disruption of the implicated intramolecular RNA-binding motif 2-RS domain interaction impairs both the directional phosphorylation mechanism and the nuclear translocation of SRSF1 demonstrating that the intrinsic phosphorylation bias is obligatory for SR protein biological function.

Keywords: NMR; RS domain; SR protein; kinetics; phosphorylation.

Figure 1. RRM2 Induces Directional Phosphorylation of RS1 in SRSF1

A) Model of the SRPK1-mediated directional phosphorylation of SRSF1. SRPK1 contains N- and C-terminal lobes that recognize Arg-Ser repeats. SRSF1 is composed of two RRMs and a C-terminal RS domain divided into RS1 and RS2 segments. For clarity, only the Arg-Ser repeats from RS1 of the RS domain are shown. B–D) LysC cleavage of SRPK1-phosphorylated cl-SRSF1 (B), cl-SR(ΔRRM1) (C), and cl-nRS (D) at low and high ATP concentrations monitored by autoradiography. *RRM2 represents an RRM2 variant in which five lysines are mutated to arginines (K138R, K165R, K174R, K179R & K193R). A cartoon of each construct is shown with indication of the sizes of the two fragments resulting from LysC treatment. E) Bar graph showing N/C ratios for the cleavage substrates at low and high ATP concentrations. Ratios for cl-nRS at high ATP are not shown due to the lack of reactivity with LysC (see text).

Figure 2. NMR observation of intermolecular complex formation between SRSF1-RRM2 and the solubilized SRSF1-RS domain

A) Superposition of the [¹⁵N,¹H]-HSQC spectra of ¹⁵N-labeled SRSF1-RRM2 with (red) and without (black) addition of 1.2 equivalents of GST-RS1. Residues of SRSF1-RRM2 experiencing chemical shifts and/or line broadening upon binding of GST-RS1 are indicated. B) Superposition of the [¹⁵N,¹H]-HSQC spectra of ¹⁵N-labeled SRSF1-RRM2 with (red) and without (black) addition of 1.2 equivalents of phosphorylated GST-RS1. There are no detectable chemical shift perturbations; This spectrum also documents the high purity/homogeneity of the protein preparation. C,D) Front and back surface views of SRSF1-RRM2, with residues affected by the presence of GST-RS1 (see panel A) colored cyan. Some amino acid positions are indicated to guide the eye.

Figure 3. Mapping Electrostatic Contacts Between RRM2 and RS1

A) RRM2 structure with several solvent–accessible, negatively charged residues shown as sticks. B) Pull-down assays for wild-type and several mutant forms of RRM2 using GST-RS1. I=input; PD=pull down. C) Alignment of affected sequences in RRM2’s of several SR proteins. Red boxes highlight residues Glu143, Asp146, and Asp151 in SRSF1 and the positions of conserved residues in four other SR proteins. D) GST-tagged RS1 deletion constructs. E) Pull-down assays for deletion constructs using wild-type RRM2. I=input; PD=pull down. F) Pull-down assays using GST-RS1(N) and RRM2 mutants defective in binding GST-RS1. I=input; PD=pull down. G) Time-dependent incubation of cSR(R210K) and cSR(R218K) with LysC. Rate constants for cSR(R210K) are 0.26 and 0.0062 min⁻¹ and those for cSR(R218K) are 0.91 and 0.018 min⁻¹. H) Model of RRM2 with the RSRSRSRS octapeptide bound. The surface of RRM2 in the same orientation as in Fig. 2C, using the same color code. Residues Glu143, Asp146 and Asp151 found to influence binding affinity are colored red and the non-responsive acidic residues Glu120, Glu166 and Asp167 are grey. The RSRSRSRS octapeptide is presented as a space-filling model of the backbone with carbon, nitrogen and oxygen atoms shown in brown, red and blue, respectively. For clarity the side chain atoms used in the docking model are not shown. The structure was generated with AUTODOCK and the drawing was prepared with pyMOL .

Figure 4. Residues in RRM2 modulate SRPK1 association with SRSF1

A) Steady-state kinetic profiles for SRSF1 and SRSF1_3M. Bar graphs represent kinetic parameters from fitting plots of enzyme-normalized velocity versus substrate. B,C) Effects of 30% sucrose on the steady-state kinetic parameters for SRSF1 (B) and SRSF1_3M (C).

Figure 5. Interactions of RRM2 With RS1 Affect Cytoplasmic-Nuclear Distribution and Dephosphorylation of SRSF1

A–C) Phosphorylation of 1 μM SRSF1 (A), SRSF1_3M (B) and RS (C) by SRPK1 (75 nM) in the absence (●) and presence (▲) of PP1 (300 nM). D) Bar graph showing relative phosphorylation levels of SR proteins in the presence of both SRPK1 and PP1. E) Confocal imaging of GFP-SRSF1 and GFP-SRSF1_3M in HeLa cells. F) Subcellular fractionation of HeLa cells expressing GFP-SRSF1 and GFP-SRSF1_3M. WT=GFP-SRSF1; Mut=GFP-SRSF1_3M.

Figure 6. Residues in RRM2 Regulate Directional Phosphorylation of RS1 in SRSF1

A) Phosphorylation of cl-SRSF1 & cl-SRSF1_3M monitored by autoradiography. A proteolytic fragment of cl-SRSF1_3M migrates at 15 kDa. B) N-terminal His-tagged cleavage substrate, cl-nSRSF1. LysC cleavage generates two fragments splitting RS1 in half. C) SRPK1 phosphorylates cl-nSRSF1 in a C-to-N manner. cl-nSRSF1 is phosphorylated by excess SRPK1 using varying ³²P-ATP and then treated with LysC to generate the N- & C-terminal fragments. The ratios of the N- & C-terminal fragments (N/C) are plotted as a function of the total number of phosphorylation sites in cl-nSRSF1. D) N/Cs ratio of cl-SRSF1_D151A and cl-SRSF1_D143A,E146A phosphorylated with SRPK1 at low ATP (0.2 μM) and treated with LysC. E) Bar graph showing SRPK1 specificity (C/N ratio) for C-terminus of RS1 for cl-SRSF1, cl-SRSF1_D151A, cl-SRSF1_D143A,E146A & cl-nRS.

Figure 7. Model for SRPK1-Dependent Directional Phosphorylation and SRSF1 Nuclear Import

RRM2-RS1 interactions direct SRPK1 to C-terminal end of RS1 for efficient, directional phosphorylation driven by the docking groove. Severing RRM2-RS1 interactions by mutation leads to non-directional phosphorylation, increased accessibility of RS domain to protein phosphatases (PPases) and enhanced protease sensitivity (box).