Redirecting SR Protein Nuclear Trafficking through an Allosteric Platform

Abstract

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 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, re-directing 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.

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

“…These results are in keeping with a prior report (18) and show that the apparent binding affinity of RRM1 is at least 40-fold higher than that for RRM2. Because RRM1 is a noncompetitive inhibitor with respect to PNPP, app K I is also equivalent to the true K I (18). However, because this assay depends on functional changes in PP1 and RRM2 might bind to PP1 but not influence catalysis, we decided to perform a competition experiment.…”

“…Because we showed previously that RRM1 in SRSF1 binds and down-regulates PP1, we wished to identify unique residues in this domain that interact with this phosphatase. We first verified previous findings that PP1 binds selectively to RRM1 in SRSF1 (18). In pulldown assays, we showed that GST-PP1 interacts with His-tagged RRM1 but not with His-tagged RRM2 ( Fig.…”

“…1A). We showed previously that such an intermediate phosphorylation state is controlled by a repressive interaction between protein phosphatase 1 (PP1) and SRSF1 (18) (Fig. 1B), but the molecular nature of this interaction and its connection to pre-mRNA splicing are unknown.…”

Distinct mechanisms govern the phosphorylation of different SR protein splicing factors

“…regulates the interaction and dephosphorylation activity of PP1␥ (41). Because SRSF3 lacks both interaction due to the absence of a pseudo-RRM and the conserved RVXF PP1-binding sequence, we speculated that the hypophosphorylation state of SRSF3 is the result of increased sensitivity to dephosphorylation by phosphatases.…”

“…The interaction between an acidic cluster within the pseudo-RRM and the RS domain, in particular, prevents dephosphorylation of the RS domain by phosphatases, suggesting a novel role of SR protein pseudo-RRM in vivo. The same research team later found that the canonical RRM of SRSF1, which contains a putative PP1-binding motif, also plays a regulatory role during its RS domain dephosphorylation by interacting with and allosterically inhibiting PP1␥ (41). SRSF3 does not contain either structural element to protect its RS domain from dephosphorylation.…”