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 Transportin-SR, a Nuclear Import Receptor for SR Proteins

1999 ◽  
Vol 145 (6) ◽  
pp. 1145-1152 ◽  
Author(s):  
Naoyuki Kataoka ◽  
Jennifer L. Bachorik ◽  
Gideon Dreyfuss
Keyword(s):  
Nuclear Import ◽  
Protein Import ◽  
Sequence Similarity ◽  
Sr Proteins ◽  
Amino Acid Sequence Similarity ◽  
Sr Protein ◽  
Localization Signal ◽  
Import Receptors ◽  
Import Receptor

The SR proteins, a group of abundant arginine/serine (RS)-rich proteins, are essential pre-mRNA splicing factors that are localized in the nucleus. The RS domain of these proteins serves as a nuclear localization signal. We found that RS domain–bearing proteins do not utilize any of the known nuclear import receptors and identified a novel nuclear import receptor specific for SR proteins. The SR protein import receptor, termed transportin-SR (TRN-SR), binds specifically and directly to the RS domains of ASF/SF2 and SC35 as well as several other SR proteins. The nuclear transport regulator RanGTP abolishes this interaction. Recombinant TRN-SR mediates nuclear import of RS domain– bearing proteins in vitro. TRN-SR has amino acid sequence similarity to several members of the importin β/transportin family. These findings strongly suggest that TRN-SR is a nuclear import receptor for the SR protein family.

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 Role for SRp54 during Intron Bridging of Small Introns with Pyrimidine Tracts Upstream of the Branch Point

1998 ◽  
Vol 18 (9) ◽  
pp. 5425-5434 ◽  
Author(s):  
Catharine F. Kennedy ◽  
Angela Krämer ◽  
Susan M. Berget
Keyword(s):  
Binding Site ◽  
Branch Point ◽  
Splice Site ◽  
Early Recognition ◽  
Splicing Factor ◽  
Splicing Factors ◽  
Sr Protein ◽  
U2 Snrnp ◽  
Auxiliary Factor ◽  
Splicing Factor 1

ABSTRACT One of the earliest steps in pre-mRNA recognition involves binding of the splicing factor U2 snRNP auxiliary factor (U2AF or MUD2 inSaccharomyces cerevisiae) to the 3′ splice site region. U2AF interacts with a number of other proteins, including members of the serine/arginine (SR) family of splicing factors as well as splicing factor 1 (SF1 or branch point bridging protein in S. cerevisiae), thereby participating in bridging either exons or introns. In vertebrates, the binding site for U2AF is the pyrimidine tract located between the branch point and 3′ splice site. Many small introns, especially those in nonvertebrates, lack a classical 3′ pyrimidine tract. Here we show that a 59-nucleotide Drosophila melanogaster intron contains C-rich pyrimidine tracts between the 5′ splice site and branch point that are needed for maximal binding of both U1 snRNPs and U2 snRNPs to the 5′ and 3′ splice site, respectively, suggesting that the tracts are the binding site for an intron bridging factor. The tracts are shown to bind both U2AF and the SR protein SRp54 but not SF1. Addition of a strong 3′ pyrimidine tract downstream of the branch point increases binding of SF1, but in this context, the upstream pyrimidine tracts are inhibitory. We suggest that U2AF- and/or SRp54-mediated intron bridging may be an alternative early recognition mode to SF1-directed bridging for small introns, suggesting gene-specific early spliceosome assembly.

scholarly journals The Renpenning syndrome–associated protein PQBP1 facilitates the nuclear import of splicing factor TXNL4A through the karyopherin β2 receptor

2020 ◽  
Vol 295 (13) ◽  
pp. 4093-4100
Author(s):  
Xian Liu ◽  
Lin-Xia Dou ◽  
Junhai Han ◽  
Zi Chao Zhang
Keyword(s):  
Intellectual Disability ◽  
Nuclear Import ◽  
Recombinant Protein Expression ◽  
Subcellular Location ◽  
Splicing Factor ◽  
Binding Assays ◽  
Intrinsically Disordered ◽  
Localization Signal ◽  
Functional Nuclear Localization Signal

Renpenning syndrome belongs to a group of X-linked intellectual disability disorders. The Renpenning syndrome–associated protein PQBP1 (polyglutamine-binding protein 1) is intrinsically disordered, associates with several splicing factors, and is involved in pre-mRNA splicing. PQBP1 uses its C-terminal YxxPxxVL motif for binding to the splicing factor TXNL4A (thioredoxin like 4A), but the biological function of this interaction has yet to be elucidated. In this study, using recombinant protein expression, in vitro binding assays, and immunofluorescence microscopy in HeLa cells, we found that a recently reported X-linked intellectual disability–associated missense mutation, resulting in the PQBP1-P244L variant, disrupts the interaction with TXNL4A. We further show that this interaction is critical for the subcellular location of TXNL4A. In combination with other PQBP1 variants lacking a functional nuclear localization signal required for recognition by the nuclear import receptor karyopherin β2, we demonstrate that PQBP1 facilitates the nuclear import of TXNL4A via a piggyback mechanism. These findings expand our understanding of the molecular basis of the PQBP1–TXNL4A interaction and of the etiology and pathogenesis of Renpenning syndrome and related disorders.

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 SIPP1, a novel pre-mRNA splicing factor and interactor of protein phosphatase-1

2004 ◽  
Vol 378 (1) ◽  
pp. 229-238 ◽  
Author(s):  
Miriam LLORIAN ◽  
Monique BEULLENS ◽  
Isabel ANDRÉS ◽  
Jose-Miguel ORTIZ ◽  
Mathieu BOLLEN
Keyword(s):  
Protein Phosphatase ◽  
Fluorescent Protein ◽  
Splicing Factor ◽  
Protein Phosphatase 1 ◽  
Nuclear Speckles ◽  
Binding Domains ◽  
Nuclear Extracts ◽  
Localization Signal ◽  
Green Fluorescent ◽  
Binding Sequence

We have identified a polypeptide that was already known to interact with polyglutamine-tract-binding protein (PQBP)-1/Npw38 as a novel splicing factor and interactor of protein phosphatase-1, hence the name SIPP1 for splicing factor that interacts with PQBP-1 and PP1 (protein phosphotase 1). SIPP1 was inhibitory to PP1, and its inhibitory potency was increased by phosphorylation with protein kinase CK1. Two-hybrid and co-sedimentation analysis revealed that SIPP1 has two distinct PP1-binding domains and that the binding of SIPP1 with PP1 involves a RVXF (Arg-Val-Xaa-Phe) motif, which functions as a PP1-binding sequence in most interactors of PP1. Enhanced-green-fluorescent-protein-tagged SIPP1 was targeted exclusively to the nucleus and was enriched in the nuclear speckles, which represent storage/assembly sites of splicing factors. We have mapped a nuclear localization signal in the N-terminus of SIPP1, while the proline-rich C-terminal domain appeared to be required for its subnuclear targeting to the speckles. Finally, we found that SIPP1 is also a component of the spliceosomes and that a SIPP1-fragment inhibits splicing catalysis by nuclear extracts independent of its ability to interact with PP1.

scholarly journals Defective nuclear import of Tpr in Progeria reflects the Ran sensitivity of large cargo transport

2013 ◽  
Vol 201 (4) ◽  
pp. 541-557 ◽  
Author(s):  
Chelsi J. Snow ◽  
Ashraf Dar ◽  
Anindya Dutta ◽  
Ralph H. Kehlenbach ◽  
Bryce M. Paschal
Keyword(s):  
Nuclear Import ◽  
Protein Complexes ◽  
Nucleocytoplasmic Transport ◽  
Large Protein ◽  
Localization Signal ◽  
Import Receptors ◽  
Progeria Syndrome ◽  
Hutchinson Gilford Progeria Syndrome ◽  
Major Defect ◽  
Import Receptor

The RanGTPase acts as a master regulator of nucleocytoplasmic transport by controlling assembly and disassembly of nuclear transport complexes. RanGTP is required in the nucleus to release nuclear localization signal (NLS)–containing cargo from import receptors, and, under steady-state conditions, Ran is highly concentrated in the nucleus. We previously showed the nuclear/cytoplasmic Ran distribution is disrupted in Hutchinson-Gilford Progeria syndrome (HGPS) fibroblasts that express the Progerin form of lamin A, causing a major defect in nuclear import of the protein, translocated promoter region (Tpr). In this paper, we show that Tpr import was mediated by the most abundant import receptor, KPNA2, which binds the bipartite NLS in Tpr with nanomolar affinity. Analyses including NLS swapping revealed Progerin did not cause global inhibition of nuclear import. Rather, Progerin inhibited Tpr import because transport of large protein cargoes was sensitive to changes in the Ran nuclear/cytoplasmic distribution that occurred in HGPS. We propose that defective import of large protein complexes with important roles in nuclear function may contribute to disease-associated phenotypes in Progeria.

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.

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