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.

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Regulation of Alternative Splicing by SRrp86 and Its Interacting Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.23.21.7437-7447.2003 ◽

2003 ◽

Vol 23 (21) ◽

pp. 7437-7447 ◽

Cited By ~ 28

Author(s):

Jun Li ◽

Ian C. Hawkins ◽

Christopher D. Harvey ◽

Jennifer L. Jennings ◽

Andrew J. Link ◽

...

Keyword(s):

Alternative Splicing ◽

Sr Proteins ◽

Splicing Factors ◽

Rna Recognition Motif ◽

Rna Recognition ◽

Protein Activity ◽

Splice Site Selection ◽

Sr Protein ◽

Library Screen ◽

Specific Proteins

ABSTRACT SRrp86 is a unique member of the SR protein superfamily containing one RNA recognition motif and two serine-arginine (SR)-rich domains separated by an unusual glutamic acid-lysine (EK)-rich region. Previously, we showed that SRrp86 could regulate alternative splicing by both positively and negatively modulating the activity of other SR proteins and that the unique EK domain could inhibit both constitutive and alternative splicing. These functions were most consistent with the model in which SRrp86 functions by interacting with and thereby modulating the activity of target proteins. To identify the specific proteins that interact with SRrp86, we used a yeast two-hybrid library screen and immunoprecipitation coupled to mass spectrometry. We show that SRrp86 interacts with all of the core SR proteins, as well as a subset of other splicing regulatory proteins, including SAF-B, hnRNP G, YB-1, and p72. In contrast to previous results that showed activation of SRp20 by SRrp86, we now show that SAF-B, hnRNP G, and 9G8 all antagonize the activity of SRrp86. Overall, we conclude that not only does SRrp86 regulate SR protein activity but that it is, in turn, regulated by other splicing factors to control alternative splice site selection.

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A Conserved DrosophilaTransportin-Serine/Arginine-rich (SR) Protein Permits Nuclear Import ofDrosophila SR Protein Splicing Factors and Their Antagonist Repressor Splicing Factor 1

Molecular Biology of the Cell ◽

10.1091/mbc.e02-02-0102 ◽

2002 ◽

Vol 13 (7) ◽

pp. 2436-2447 ◽

Cited By ~ 28

Author(s):

Eric Allemand ◽

Svetlana Dokudovskaya ◽

Rémy Bordonné ◽

Jamal Tazi

Keyword(s):

Nuclear Import ◽

Direct Interaction ◽

Sr Proteins ◽

Splicing Factor ◽

Nuclear Speckles ◽

Sr Protein ◽

Nuclear Factors ◽

Localization Signal ◽

Import Receptors ◽

Splicing Factor 1

Members of the highly conserved serine/arginine-rich (SR) protein family are nuclear factors involved in splicing of metazoan mRNA precursors. In mammals, two nuclear import receptors, transportin (TRN)-SR1 and TRN-SR2, are responsible for targeting SR proteins to the nucleus. Distinctive features in the nuclear localization signal between Drosophila and mammalian SR proteins prompted us to examine the mechanism by whichDrosophila SR proteins and their antagonist repressor splicing factor 1 (RSF1) are imported into nucleus. Herein, we report the identification and characterization of a Drosophilaimportin β-family protein (dTRN-SR), homologous to TRN-SR2, that specifically interacts with both SR proteins and RSF1. dTRN-SR has a broad localization in the cytoplasm and the nucleus, whereas an N-terminal deletion mutant colocalizes with SR proteins in nuclear speckles. Far Western experiments established that the RS domain of SR proteins and the GRS domain of RSF1 are required for the direct interaction with dTRN-SR, an interaction that can be modulated by phosphorylation. Using the yeast model system in which nuclear import of Drosophila SR proteins and RSF1 is impaired, we demonstrate that complementation with dTRN-SR is sufficient to target these proteins to the nucleus. Together, the results imply that the mechanism by which SR proteins are imported to the nucleus is conserved between Drosophila and humans.

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Identification and Characterization of a Novel Serine-Arginine-Rich Splicing Regulatory Protein

Molecular and Cellular Biology ◽

10.1128/mcb.20.9.3049-3057.2000 ◽

2000 ◽

Vol 20 (9) ◽

pp. 3049-3057 ◽

Cited By ~ 36

Author(s):

Daron C. Barnard ◽

James G. Patton

Keyword(s):

Regulatory Protein ◽

Structural Similarity ◽

Sr Proteins ◽

Rna Recognition Motif ◽

Sr Protein ◽

Amino Terminal ◽

Carboxy Terminal ◽

Rs Domains

ABSTRACT We have identified an 86-kDa protein containing a single amino-terminal RNA recognition motif and two carboxy-terminal domains rich in serine-arginine (SR) dipeptides. Despite structural similarity to members of the SR protein family, p86 is clearly unique. It is not found in standard SR protein preparations, does not precipitate in the presence of high magnesium concentrations, is not recognized by antibodies specific for SR proteins, and cannot complement splicing-defective S100 extracts. However, we have found that p86 can inhibit the ability of purified SR proteins to activate splicing in S100 extracts and can even inhibit the in vitro and in vivo activation of specific splice sites by a subset of SR proteins, including ASF/SF2, SC35, and SRp55. In contrast, p86 activates splicing in the presence of SRp20. Thus, it appears that pairwise combination of p86 with specific SR proteins leads to altered splicing efficiency and differential splice site selection. In all cases, such regulation requires the presence of the two RS domains and a unique intervening EK-rich region, which appear to mediate direct protein-protein contact between these family members. Full-length p86, but not a mutant lacking the RS-EK-RS domains, was found to preferentially interact with itself, SRp20, ASF/SF2, SRp55, and, to a slightly lesser extent, SC35. Because of the primary sequence and unique properties of p86, we have named this protein SRrp86 for SR-related protein of 86 kDa.

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