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

2018 ◽  
Vol 475 (3) ◽  
pp. 677-690 ◽  
Author(s):  
Brandon E. Aubol ◽  
Malik M. Keshwani ◽  
Laurent Fattet ◽  
Joseph A. Adams
Keyword(s):  
Protein Kinase ◽  
Signaling Pathway ◽  
Sr Proteins ◽  
Splicing Factor ◽  
Nuclear Speckles ◽  
Down Regulation ◽  
Sr Protein ◽  
Protein Activation

The splicing of mRNA is dependent on serine-arginine (SR) proteins that are mobilized from membrane-free, nuclear speckles to the nucleoplasm by the Cdc2-like kinases (CLKs). This movement is critical for SR protein-dependent assembly of the macromolecular spliceosome. Although CLK1 facilitates such trafficking through the phosphorylation of serine-proline dipeptides in the prototype SR protein SRSF1, an unrelated enzyme known as SR protein kinase 1 (SRPK1) performs the same function but does not efficiently modify these dipeptides in SRSF1. We now show that the ability of SRPK1 to mobilize SRSF1 from speckles to the nucleoplasm is dependent on active CLK1. Diffusion from speckles is promoted by the formation of an SRPK1–CLK1 complex that facilitates dissociation of SRSF1 from CLK1 and enhances the phosphorylation of several serine-proline dipeptides in this SR protein. Down-regulation of either kinase blocks EGF-stimulated mobilization of nuclear SRSF1. These findings establish a signaling pathway that connects SRPKs to SR protein activation through the associated CLK family of kinases.

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

2002 ◽  
Vol 13 (7) ◽  
pp. 2436-2447 ◽  
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.

scholarly journals N-terminus of the protein kinase CLK1 induces SR protein hyperphosphorylation

2014 ◽  
Vol 462 (1) ◽  
pp. 143-152 ◽  
Author(s):  
Brandon E. Aubol ◽  
Ryan M. Plocinik ◽  
Malik M. Keshwani ◽  
Maria L. McGlone ◽  
Jonathan C. Hagopian ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Sr Proteins ◽  
Sr Protein ◽  
N Terminus

The N-terminus of the protein kinase CLK1 induces hyperphosphorylation of SR proteins.

scholarly journals The Acute Myeloid Leukemia-Associated Protein, Dek, Forms a Splicing-Dependent Interaction with Exon-Product Complexes

2000 ◽  
Vol 150 (2) ◽  
pp. 309-320 ◽  
Author(s):  
Tim McGarvey ◽  
Emanuel Rosonina ◽  
Susan McCracken ◽  
Qiyu Li ◽  
Ramy Arnaout ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Chromosomal Translocation ◽  
Sr Proteins ◽  
Splicing Factor ◽  
Nuclear Speckles ◽  
Myeloid Leukemias ◽  
Acute Myeloid ◽  
Dependent Interaction

DEK is an ∼45-kD phosphoprotein that is fused to the nucleoporin CAN as a result of a (6;9) chromosomal translocation in a subset of acute myeloid leukemias (AMLs). It has also been identified as an autoimmune antigen in juvenile rheumatoid arthritis and other rheumatic diseases. Despite the association of DEK with several human diseases, its function is not known. In this study, we demonstrate that DEK, together with SR proteins, associates with the SRm160 splicing coactivator in vitro. DEK is recruited to splicing factor-containing nuclear speckles upon concentration of SRm160 in these structures, indicating that DEK and SRm160 associate in vivo. We further demonstrate that DEK associates with splicing complexes through interactions mediated by SR proteins. Significantly, DEK remains bound to the exon-product RNA after splicing, and this association requires the prior formation of a spliceosome. Thus, DEK is a candidate factor for controlling postsplicing steps in gene expression that are influenced by the prior removal of an intron from pre-mRNA.

Human papillomavirus regulation of SR proteins

2010 ◽  
Vol 38 (4) ◽  
pp. 1116-1121 ◽  
Author(s):  
Melanie McFarlane ◽  
Sheila V. Graham
Keyword(s):  
Human Papillomavirus ◽  
Alternative Splicing ◽  
Sr Proteins ◽  
Hpv Infection ◽  
Splicing Factor ◽  
Sr Protein ◽  
Splicing Regulators ◽  
Constitutive Splicing ◽  
Rna Biogenesis ◽  
Specific Alternative

Splicing is a cellular process essential for mRNA biogenesis. There are two types of splicing: constitutive and alternative splicing. During constitutive splicing, non-coding intron sequences are removed and exonic coding sequences are spliced together to form mature mRNAs. Alternative splicing can maximize the coding capacity of the genome by specific alternative selection of exons from multi-exon metazoan pre-mRNAs. Splicing is a tightly regulated process, so when control is lost disease may occur. SR proteins (serine/arginine-rich proteins) are a family of highly conserved splicing regulators that are also involved in other steps in RNA biogenesis and expression. Many viruses have evolved to utilize the cellular splicing machinery to enhance their proteome from a limited number of genes. HPV (human papillomavirus) is an example of one such virus. The HPV transcription/replication factor E2 (early 2) specifically up-regulates expression of the SR proteins SF2/ASF (splicing factor 2/alternative splicing factor), SRp20 and SC35 in infected epithelial cells. These SR proteins are essential for viral RNA processing. SF2/ASF is a proto-oncogene that is also up-regulated in a number of cancers. For example, SF2/ASF, together with SRp20 and SC35 is selectively up-regulated in cervical tumours caused by persistent oncogenic HPV infection. However, the mode of SR protein up-regulation in tumours is different to the E2-directed transcriptional regulation in normal transient HPV infection. SR proteins could provide excellent targets for HPV antiviral therapy as well as anticancer therapy.

scholarly journals Molecular Mechanism of SR Protein Kinase 1 Inhibition by the Herpes Virus Protein ICP27

mBio ◽  
2019 ◽  
Vol 10 (5) ◽  
Author(s):  
Richard B. Tunnicliffe ◽  
William K. Hu ◽  
Michele Y. Wu ◽  
Colin Levy ◽  
A. Paul Mould ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Alternative Splicing ◽  
Herpes Simplex ◽  
Protein Kinase ◽  
Molecular Mechanism ◽  
Sr Proteins ◽  
Sr Protein ◽  
High Affinity ◽  
Simplex Virus

ABSTRACT Serine-arginine (SR) protein kinase 1 (SRPK1) catalyzes the phosphorylation of SR proteins, which are a conserved family of splicing factors that contain a domain rich in arginine and serine repeats. SR proteins play important roles in constitutive pre-mRNA splicing and are also important regulators of alternative splicing. During herpes simplex virus infection, SRPK1 is inactivated and its cellular distribution is markedly altered by interaction with the viral protein ICP27, resulting in hypophosphorylation of SR proteins. Mutational analysis previously showed that the RGG box motif of ICP27 is required for interaction with SRPK1; however, the mechanism for the inhibition and the exact role of the RGG box was unknown. Here, we used solution nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC) to demonstrate that the isolated peptide comprising the RGG box of ICP27 binds to SRPK1 with high affinity, competing with a native substrate, the SR repeat region of SR protein SRSF1. We determined the crystal structure of the complex between SRPK1 and an RGG box peptide, which revealed that the viral peptide binds to the substrate docking groove, mimicking the interactions of SR repeats. Site-directed mutagenesis within the RGG box further confirmed the importance of selected arginine residues for interaction, relocalization, and inhibition of SRPK1 in vivo. Together these data reveal the molecular mechanism of the competitive inhibition of cellular SRPK1 by viral ICP27, which modulates SRPK1 activity. IMPORTANCE Serine arginine (SR) proteins are a family of mRNA regulatory proteins that can modulate spliceosome association with different splice sites and therefore regulate alternative splicing. Phosphorylation within SR proteins is necessary for splice-site recognition, and this is catalyzed by SR protein kinase 1 (SRPK1). The herpes simplex virus (HSV-1) protein ICP27 has been shown previously to interact with and downregulate SRPK1 activity in vivo; however, the molecular mechanism for this interaction and inhibition was unknown. Here, we demonstrate that the isolated peptide fragment of ICP27 containing RGG box binds to SRPK1 with high affinity, and competes with a native cellular substrate. Elucidation of the SRPK1-RGG box crystal structure further showed that a short palindromic RGRRRGR sequence binds in the substrate docking groove of SRPK1, mimicking the binding of SR repeats of substrates. These data reveal how the viral protein ICP27 inactivates SRPK1, promoting hypophosphorylation of proteins regulating splicing.

scholarly journals The distribution of phosphorylated SR proteins and alternative splicing are regulated by RANBP2

2012 ◽  
Vol 23 (6) ◽  
pp. 1115-1128 ◽  
Author(s):  
Noriko Saitoh ◽  
Chiyomi Sakamoto ◽  
Masatoshi Hagiwara ◽  
Lourdes T. Agredano-Moreno ◽  
Luis Felipe Jiménez-García ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Mammalian Cells ◽  
Nuclear Architecture ◽  
Sr Proteins ◽  
Nucleocytoplasmic Transport ◽  
Dependent Manner ◽  
Small Interfering Rnas ◽  
Nuclear Speckles ◽  
Sr Protein ◽  
Interchromatin Granule

The mammalian cell nucleus is functionally compartmentalized into various substructures. Nuclear speckles, also known as interchromatin granule clusters, are enriched with SR splicing factors and are implicated in gene expression. Here we report that nuclear speckle formation is developmentally regulated; in certain cases phosphorylated SR proteins are absent from the nucleus and are instead localized at granular structures in the cytoplasm. To investigate how the nuclear architecture is formed, we performed a phenotypic screen of HeLa cells treated with a series of small interfering RNAs. Depletion of Ran-binding protein 2 induced cytoplasmic intermediates of nuclear speckles in G1 phase. Detailed analyses of these structures suggested that a late step in the sequential nuclear entry of mitotic interchromatin granule components was disrupted and that phosphorylated SR proteins were sequestered in an SR protein kinase–dependent manner. As a result, the cells had an imbalanced subcellular distribution of phosphorylated and hypophosphorylated SR proteins, which affected alternative splicing patterns. This study demonstrates that the speckled distribution of phosphorylated pre-mRNA processing factors is regulated by the nucleocytoplasmic transport system in mammalian cells and that it is important for alternative splicing.

scholarly journals Differential effects of hyperphosphorylation on splicing factor SRp55

2003 ◽  
Vol 371 (3) ◽  
pp. 937-945 ◽  
Author(s):  
Ming-Chih LAI ◽  
Ru-Inn LIN ◽  
Woan-Yuh TARN
Keyword(s):  
Rna Polymerase Ii ◽  
Rna Binding ◽  
Sr Proteins ◽  
Binding Activity ◽  
Protein Family ◽  
Nuclear Speckles ◽  
Sr Protein ◽  
Rich Domain ◽  
First Time ◽  
Amino Acid Segment

Members of the serine/arginine-rich (SR) protein family play an important role in both constitutive and regulated splicing of precursor mRNAs. Phosphorylation of the arginine/serine dipeptide-rich domain (RS domain) can modulate the activity and the subcellular localization of SR proteins. However, whether the SR protein family members are individually regulated and how this is achieved remain unclear. In this report we show that 5,6-dichloro-1β-d-ribofuranosyl-benzimidazole (DRB), an inhibitor of RNA polymerase II-dependent transcription, specifically induced hyperphosphorylation of SRp55 but not that of any other SR proteins tested. Hyperphosphorylation of SRp55 occurs at the RS domain and appears to require the RNA-binding activity. Upon DRB treatment, hyperphosphorylated SRp55 relocates to enlarged nuclear speckles. Intriguingly, SRp55 is specifically targeted for degradation by the proteasome upon overexpression of the SR protein kinase Clk/Sty. Although a destabilization signal is mapped within the C-terminal 43-amino acid segment of SRp55, its adjacent lysine/serine-rich RS domain is nevertheless critical for the Clk/Sty-mediated degradation. We report for the first time that SRp55 can be hyperphosphorylated under different circumstances whereby its fate is differentially influenced.

scholarly journals Regulation and Substrate Specificity of the SR Protein Kinase Clk/Sty

2003 ◽  
Vol 23 (12) ◽  
pp. 4139-4149 ◽  
Author(s):  
Jayendra Prasad ◽  
James L. Manley
Keyword(s):  
Protein Kinase ◽  
Substrate Specificity ◽  
Cell Types ◽  
Sr Proteins ◽  
Splicing Factors ◽  
Sr Protein ◽  
Opposite Pattern ◽  
Founding Member ◽  
Dual Specificity ◽  
Different Cell Types

ABSTRACT SR proteins constitute a family of splicing factors that play key roles in both constitutive and regulated splicing in metazoan organisms. The proteins are extensively phosphorylated, and kinases capable of phosphorylating them have been identified. However, little is known about how these kinases function, for example, whether they target specific SR proteins or whether the kinases themselves are regulated. Here we describe properties of one such kinase, Clk/Sty, the founding member of the Clk/Sty family of dual-specificity kinases. Clk/Sty is autophosphorylated on both Ser/Thr and Thr residues, and using both direct kinase assays and SR protein-dependent splicing assays, we have analyzed the effects of each type of modification. We find not only that the pattern of phosphorylation on a specific SR protein substrate, ASF/SF2, is modulated by autophosphorylation but also that the ability of Clk/Sty to recognize different SR proteins is influenced by the extent and nature of autophosphorylation. Strikingly, phosphorylation of ASF/SF2 is sensitive to changes in Tyr, but not Ser/Thr, autophosphorylation while that of SC35 displays the opposite pattern. In contrast, phosphorylation of a third SR protein, SRp40, is unaffected by autophosphorylation. We also present biochemical data indicating that as expected for a factor directly involved in splicing control (but in contrast to recent reports), Clk/Sty is found in the nucleus of several different cell types.

scholarly journals Hypophosphorylated SR splicing factors transiently localize around active nucleolar organizing regions in telophase daughter nuclei

2004 ◽  
Vol 167 (1) ◽  
pp. 51-63 ◽  
Author(s):  
Paula A. Bubulya ◽  
Kannanganattu V. Prasanth ◽  
Thomas J. Deerinck ◽  
Daniel Gerlich ◽  
Joel Beaudouin ◽  
...  
Keyword(s):  
Rna Polymerase Ii ◽  
Sr Proteins ◽  
Splicing Factors ◽  
Nuclear Speckles ◽  
Sr Protein ◽  
Nucleolar Organizing Regions ◽  
Transcription Sites ◽  
Nuclear Regions ◽  
Rna Polymerase Ii Transcription

Upon completion of mitosis, daughter nuclei assemble all of the organelles necessary for the implementation of nuclear functions. We found that upon entry into daughter nuclei, snRNPs and SR proteins do not immediately colocalize in nuclear speckles. SR proteins accumulated in patches around active nucleolar organizing regions (NORs) that we refer to as NOR-associated patches (NAPs), whereas snRNPs were enriched at other nuclear regions. NAPs formed transiently, persisting for 15–20 min before dissipating as nuclear speckles began to form in G1. In the absence of RNA polymerase II transcription, NAPs increased in size and persisted for at least 2 h, with delayed localization of SR proteins to nuclear speckles. In addition, SR proteins in NAPs are hypophosphorylated, and the SR protein kinase Clk/STY colocalizes with SR proteins in NAPs, suggesting that phosphorylation releases SR proteins from NAPs and their initial target is transcription sites. This work demonstrates a previously unrecognized role of NAPs in splicing factor trafficking and nuclear speckle biogenesis.

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