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

Abstract

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.

Proteasome inhibitor-induced modulation reveals the spliceosome as a specific therapeutic vulnerability in multiple myeloma

“…However, a much larger fraction of SA mutant is trapped in the cytosol despite NLS tagging. Consistent with prior work 31,32 , this finding suggests that RS phosphorylation is a major requirement for entry into the nucleus.…”

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

“…To accomplish this, we used a form of SRSF1 that is defective in entering the nucleus because of mutations in its NLS. In a previous study, we demonstrated that replacing 8 serines in the N terminus of the RS domain generates a mutant SRSF1 (GFP-SRSF1 mut ) resistant to SRPK1-dependent nuclear transport (29) (Fig. 5A).…”

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

“…We next wished to determine whether mutation of the CLK1 binding residues impacts the phosphorylation state of SRSF1 by observing the migration of GFP‐SRSF1 on 10% SDS/PAGE. We showed previously that Ser‐Pro phosphorylation (hyper‐phosphorylation) can be monitored by reduced migration of GFP‐SRSF1 on low percentage SDS/PAGE [21,32]. We found that the ratio of slow‐migrating species compared to fast‐migrating species was much greater with HA‐SRPK1 than HA‐SRPK1 mut expression suggesting that the binding surface induces enhanced hyper‐phosphorylation in GFP‐SRSF1 (Fig.…”

“…(D) Phosphorylation of SRSF1 by CLK1 with and without SRPK1 under steady-state conditions. CLK1 (250 nM), 32…”