Mobilization of a splicing factor through a nuclear kinase–kinase complex

Aubol, Brandon E.; Keshwani, Malik M.; Fattet, Laurent; Adams, Joseph A.

doi:10.1042/bcj20170672

Cited by 28 publications

(48 citation statements)

References 44 publications

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“…New light has been shed on this by two recent studies that show that SRPK1, after being localized to the nucleus, facilitates the dissociation of SRSF1 from the nuclear speckles by the formation of an SRPK1-CLK1 complex. The presence of SRPK1 facilitates the release of SRSF1 from CLK1 and enhances further phosphorylation of SP dipeptides, resulting in the mobilization of SRSF1 from nuclear speckles to nucleoplasm (50,51). In this study, we observed that SRPK2 also plays a role in the mobilization of SRSF3 from the speckles to nucleoplasm.…”

Section: Phosphorylation Mechanisms Of Sr Proteinssupporting

confidence: 55%

Distinct mechanisms govern the phosphorylation of different SR protein splicing factors

Long

Sou

Yung

et al. 2019

Journal of Biological Chemistry

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Serine-arginine (SR) proteins are essential splicing factors containing a canonical RNA recognition motif (RRM), sometimes followed by a pseudo-RRM, and a C-terminal arginine/ serine-rich (RS) domain that undergoes multisite phosphorylation. Phosphorylation regulates the localization and activity of SR proteins, and thus may provide insight into their differential biological roles. The phosphorylation mechanism of the prototypic SRSF1 by serine-arginine protein kinase 1 (SRPK1) has been well-studied, but little is known about the phosphorylation of other SR protein members. In the present study, interaction and kinetic assays unveiled how SRSF1 and the single RRMcontaining SRSF3 are phosphorylated by SRPK2, another member of the SRPK family. We showed that a conserved SRPKspecific substrate-docking groove in SRPK2 impacts the binding and phosphorylation of both SR proteins, and the localization of SRSF3. We identified a nonconserved residue within the groove that affects the kinase processivity. We demonstrated that, in contrast to SRSF1, for which SRPK-mediated phosphorylation is confined to the N-terminal region of the RS domain, SRSF3 phosphorylation sites are spread throughout its entire RS domain in vitro. Despite this, SRSF3 appears to be hypophosphorylated in cells at steady state. Our results suggest that the absence of a pseudo-RRM renders the single RRM-containing SRSF3 more susceptible to dephosphorylation by phosphatase. These findings suggest that the single RRM-and two RRMcontaining SR proteins represent two subclasses of phosphoproteins in which phosphorylation statuses are maintained by unique mechanisms, and pose new directions to explore the distinct roles of SR proteins in vivo.

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Section: Phosphorylation Mechanisms Of Sr Proteinssupporting

confidence: 55%

Distinct mechanisms govern the phosphorylation of different SR protein splicing factors

Long

Sou

Yung

et al. 2019

Journal of Biological Chemistry

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“…We now wished to identify regions in the RS domain that may be responsible for this change in subnuclear mobility. Because we showed previously that CLK1-dependent phosphorylation of several Ser-Pro dipeptides in the C-terminal polypeptide segment of the RS domain (RS2) induces a hyperphosphorylation state that releases SRSF1 from speckles (16,17), we speculated that dephosphorylation of serines at the other end of the RS domain (RS1) (Fig. 6A) might be responsible for the hypophosphorylated state that also releases the SR protein from speckles (Fig.…”

Section: Several Serines In the Rs Domain Regulate Srsf1 Speckle Occumentioning

confidence: 95%

“…Cho et al (4) demonstrated that RRM1 of SRSF1 interacts with an RRM from the U1-70K subunit of U1 snRNP, thus facilitating assembly of the early spliceosome. In a recent study, RRM1 was shown to serve as a platform for PP1 binding and activity control, although the molecular contacts for this interaction were not defined (17). Because these findings highlight the importance of RRM1 as a protein interaction module, we performed experiments to define the molecular nature of the PP1-RRM interface within an SR protein and uncover potential links to mRNA processing events.…”

Section: Pp1 Binding To Splicing Factor Srsf1mentioning

confidence: 99%

“…All forms of GST-SRSF1 and GST-PP1␥ were expressed and purified from a PGEX6P-1 vector in BL21(DE3), as previously described (35). For confocal imaging experiments, GFP-SRSF1 constructs were expressed from pcDNA3.1ϩN-eGFP vectors in HeLa cells (17).…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

“…The latter transition is critical for SR protein mobilization to the spliceosome for splicing function (15). We showed that although CLK1, one of four isoforms, phosphorylates a broad swath of Arg-Ser dipeptides, specific modification of three Ser-Pro dipeptides toward the C terminus of the RS domain triggers bulk movement of SRSF1 from speckles to the nucleoplasm (16,17) (Fig. 1A).…”

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confidence: 90%

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

Aubol

Serrano

Fattet

et al. 2018

Journal of Biological Chemistry

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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.

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Purification of SRSF1 from E. coli for Biophysical and Biochemical Assays

Zhang,

Zhang

2024

Current Protocols

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The Ser/Arg‐rich splicing factors (SR proteins) constitute a crucial protein family in alternative splicing, comprising twelve members characterized by unique repetitive Arg‐Ser dipeptide sequences (RS) and one to two RNA‐recognition motifs (RRM). The RS regions of SR proteins undergo variable phosphorylation, resulting in unphosphorylated, partially phosphorylated, or hyper‐phosphorylated states based on functional requirements. Despite the identification of the SR protein family over 30 years ago, the purification of native SR proteins in soluble form at large quantities has presented challenges due to their low solubility. This protocol delineates a method for acquiring soluble, full‐length, unphosphorylated, hypo‐ and hyper‐phosphorylated SRSF1, a prototypical SR family member. Notably, this protocol facilitates the purification of SRSF1 in ample quantities suitable for NMR, as well as various biophysical and biochemical studies. The methodologies and principles outlined herein are expected to extend beyond SRSF1 protein production and can be adapted for purifying other SR protein family members or SR‐related proteins, such as snRNP70 and U2AF‐35. Given the involvement of these proteins in numerous essential biological processes, this protocol will prove beneficial to researchers in related fields. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.Basic Protocol 1: Purification of SRSF1 from E. coliSupport Protocol: Purification of ULP1Basic Protocol 2: Purification of hypo‐phosphorylated SRSF1 from E. coliBasic Protocol 3: Purification of hyper‐phosphorylated SRSF1 from E. coli

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Mobilization of a splicing factor through a nuclear kinase–kinase complex

Cited by 28 publications

References 44 publications

Distinct mechanisms govern the phosphorylation of different SR protein splicing factors

Distinct mechanisms govern the phosphorylation of different SR protein splicing factors

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

Purification of SRSF1 from E. coli for Biophysical and Biochemical Assays

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