scholarly journals Phosphorylation Regulates In Vivo Interaction and Molecular Targeting of Serine/Arginine-rich Pre-mRNA Splicing Factors

1999 ◽  
Vol 145 (3) ◽  
pp. 447-455 ◽  
Author(s):  
Joanne M. Yeakley ◽  
Hélène Tronchère ◽  
James Olesen ◽  
Jacqueline A. Dyck ◽  
Huan-You Wang ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Mammalian Cells ◽  
Sr Proteins ◽  
Molecular Targeting ◽  
Splicing Factors ◽  
Binding Studies ◽  
Sr Protein ◽  
Domain Interactions

The SR superfamily of splicing factors and regulators is characterized by arginine/serine (RS)-rich domains, which are extensively modified by phosphorylation in cells. In vitro binding studies revealed that RS domain–mediated protein interactions can be differentially affected by phosphorylation. Taking advantage of the single nonessential SR protein–specific kinase Sky1p in Saccharomyces cerevisiae, we investigated RS domain interactions in vivo using the two-hybrid assay. Strikingly, all RS domain–mediated interactions were abolished by SKY1 deletion and were rescuable by yeast or mammalian SR protein–specific kinases, indicating that phosphorylation has a far greater impact on RS domain interactions in vivo than in vitro. To understand this dramatic effect, we examined the localization of SR proteins and found that SC35 was shifted to the cytoplasm in sky1Δ yeast, although this phenomenon was not obvious with ASF/SF2, indicating that nuclear import of SR proteins may be differentially regulated by phosphorylation. Using a transcriptional repression assay, we further showed that most LexA-SR fusion proteins depend on Sky1p to efficiently recognize the LexA binding site in a reporter, suggesting that molecular targeting of RS domain–containing proteins within the nucleus was also affected. Together, these results reveal multiple phosphorylation-dependent steps for SR proteins to interact with one another efficiently and specifically, which may ultimately determine the splicing activity and specificity of these factors in mammalian cells.

scholarly journals The Protein Kinase Clk/Sty Directly Modulates SR Protein Activity: Both Hyper- and Hypophosphorylation Inhibit Splicing

1999 ◽  
Vol 19 (10) ◽  
pp. 6991-7000 ◽  
Author(s):  
Jayendra Prasad ◽  
Karen Colwill ◽  
Tony Pawson ◽  
James L. Manley
Keyword(s):  
Site Selection ◽  
Sr Proteins ◽  
Splicing Factors ◽  
Protein Splicing ◽  
Protein Activity ◽  
Splice Site Selection ◽  
Sr Protein ◽  
Constitutive Splicing

ABSTRACT The splicing of mammalian mRNA precursors requires both protein phosphorylation and dephosphorylation, likely involving modification of members of the SR protein family of splicing factors. Several kinases have been identified that can phosphorylate SR proteins in vitro, and transfection assays have provided evidence that at least one of these, Clk/Sty, can modulate splicing in vivo. But evidence that a specific kinase can directly affect the splicing activity of SR proteins has been lacking. Here, by using purified recombinant Clk/Sty, a catalytically inactive mutant, and individual SR proteins, we show that Clk/Sty directly affects the activity of SR proteins, but not other essential splicing factors, in reconstituted splicing assays. We also provide evidence that both hyper- and hypophosphorylation inhibit SR protein splicing activity, repressing constitutive splicing and switching alternative splice site selection. These findings indicate that Clk/Sty directly and specifically influences the activity of SR protein splicing factors and, importantly, show that both under- and overphosphorylation of SR proteins can modulate splicing.

scholarly journals Roles for SR Proteins and hnRNP A1 in the Regulation of c-src Exon N1

2003 ◽  
Vol 23 (6) ◽  
pp. 1874-1884 ◽  
Author(s):  
Nanette Rooke ◽  
Vadim Markovtsov ◽  
Esra Cagavi ◽  
Douglas L. Black
Keyword(s):  
Regulatory Element ◽  
Sr Proteins ◽  
Hnrnp A1 ◽  
Sr Protein ◽  
Enhancer Element ◽  
Splicing Regulators ◽  
In Vitro Splicing ◽  
Hnrnp F

ABSTRACT The splicing of the c-src exon N1 is controlled by an intricate combination of positive and negative RNA elements. Most previous work on these sequences focused on intronic elements found upstream and downstream of exon N1. However, it was demonstrated that the 5′ half of the N1 exon itself acts as a splicing enhancer in vivo. Here we examine the function of this regulatory element in vitro. We show that a mutation in this sequence decreases splicing of the N1 exon in vitro. Proteins binding to this element were identified as hnRNP A1, hnRNP H, hnRNP F, and SF2/ASF by site-specific cross-linking and immunoprecipitation. The binding of these proteins to the RNA was eliminated by a mutation in the exonic element. The activities of hnRNP A1 and SF2/ASF on N1 splicing were examined by adding purified protein to in vitro splicing reactions. SF2/ASF and another SR protein, SC35, are both able to stimulate splicing of c-src pre-mRNA. However, splicing activation by SF2/ASF is dependent on the N1 exon enhancer element whereas activation by SC35 is not. In contrast to SF2/ASF and in agreement with other systems, hnRNP A1 repressed c-src splicing in vitro. The negative activity of hnRNP A1 on splicing was compared with that of PTB, a protein previously demonstrated to repress splicing in this system. Both proteins repress exon N1 splicing, and both counteract the enhancing activity of the SR proteins. Removal of the PTB binding sites upstream of N1 prevents PTB-mediated repression but does not affect A1-mediated repression. Thus, hnRNP A1 and PTB use different mechanisms to repress c-src splicing. Our results link the activity of these well-known exonic splicing regulators, SF2/ASF and hnRNP A1, to the splicing of an exon primarily controlled by intronic factors.

scholarly journals Adenovirus Protein VII Condenses DNA, Represses Transcription, and Associates with Transcriptional Activator E1A

2004 ◽  
Vol 78 (12) ◽  
pp. 6459-6468 ◽  
Author(s):  
Jeffrey S. Johnson ◽  
Yvonne N. Osheim ◽  
Yuming Xue ◽  
Margaux R. Emanuel ◽  
Peter W. Lewis ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Function Analysis ◽  
Specific Protein ◽  
In Vitro Translation ◽  
Major Protein ◽  
Lampbrush Chromosomes ◽  
Binding Studies

ABSTRACT Adenovirus protein VII is the major protein component of the viral nucleoprotein core. It is highly basic, and an estimated 1070 copies associate with each viral genome, forming a tightly condensed DNA-protein complex. We have investigated DNA condensation, transcriptional repression, and specific protein binding by protein VII. Xenopus oocytes were microinjected with mRNA encoding HA-tagged protein VII and prepared for visualization of lampbrush chromosomes. Immunostaining revealed that protein VII associated in a uniform manner across entire chromosomes. Furthermore, the chromosomes were significantly condensed and transcriptionally silenced, as judged by the dramatic disappearance of transcription loops characteristic of lampbrush chromosomes. During infection, the protein VII-DNA complex may be the initial substrate for transcriptional activation by cellular factors and the viral E1A protein. To investigate this possibility, mRNAs encoding E1A and protein VII were comicroinjected into Xenopus oocytes. Interestingly, whereas E1A did not associate with chromosomes in the absence of protein VII, expression of both proteins together resulted in significant association of E1A with lampbrush chromosomes. Binding studies with proteins produced in bacteria or human cells or by in vitro translation showed that E1A and protein VII can interact in vitro. Structure-function analysis revealed that an N-terminal region of E1A is responsible for binding to protein VII. These studies define the in vivo functions of protein VII in DNA binding, condensation, and transcriptional repression and indicate a role in E1A-mediated transcriptional activation of viral genes.

scholarly journals Human Autoimmune Sera as Molecular Probes for the Identification of an Autoantigen Kinase Signaling Pathway

2002 ◽  
Vol 196 (9) ◽  
pp. 1213-1226 ◽  
Author(s):  
Makoto Kamachi ◽  
Truc M. Le ◽  
Susan J. Kim ◽  
Meghan E. Geiger ◽  
Paul Anderson ◽  
...  
Keyword(s):  
Topoisomerase I ◽  
Molecular Probes ◽  
Splicing Factors ◽  
Apoptotic Cells ◽  
Sr Protein ◽  
Small Nuclear Ribonucleoprotein ◽  
Alternative Mrna Splicing ◽  
U1 Snrnp

Using human autoimmune sera as molecular probes, we previously described the association of phosphorylated serine/arginine splicing factors (SR splicing factors) with the U1-small nuclear ribonucleoprotein (U1-snRNP) and U3-small nucleolar RNP (snoRNP) in apoptotic cells. SR proteins are highly conserved autoantigens whose activity is tightly regulated by reversible phosphorylation of serine residues by at least eight different SR protein kinase kinases (SRPKs), including SRPK1, SRPK2, and the scleroderma autoantigen topoisomerase I. In this report, we demonstrate that only one of the known SRPKs, SRPK1, is associated with the U1-snRNP autoantigen complex in healthy and apoptotic cells. SRPK1 is activated early during apoptosis, followed by caspase-mediated proteolytic inactivation at later time points. SRPKs are cleaved in vivo after multiple apoptotic stimuli, and cleavage can be inhibited by overexpression of bcl-2 and bcl-xL, and by exposure to soluble peptide caspase inhibitors. Incubation of recombinant caspases with in vitro–translated SRPKs demonstrates that SRPK1 and SRPK2 are in vitro substrates for caspases-8 and -9, respectively. In contrast, topoisomerase I is cleaved by downstream caspases (-3 and -6). Since each of these SRPKs sits at a distinct checkpoint in the caspase cascade, SRPKs may serve an important role in signaling pathways governing apoptosis, alternative mRNA splicing, SR protein trafficking, RNA stability, and possibly the generation of autoantibodies directed against splicing factors.

scholarly journals Biochemical and Genetic Conservation of Fission Yeast Dsk1 and Human SR Protein-Specific Kinase 1

2000 ◽  
Vol 20 (3) ◽  
pp. 816-824 ◽  
Author(s):  
Zhaohua Tang ◽  
Tiffany Kuo ◽  
Jenny Shen ◽  
Ren-Jang Lin
Keyword(s):  
Fission Yeast ◽  
Mammalian Cells ◽  
Specific Protein ◽  
Biochemical Properties ◽  
Growth Defect ◽  
Sr Protein ◽  
Kinase Gene ◽  
Control Circuits

ABSTRACT Arginine/serine-rich (RS) domain-containing proteins and their phosphorylation by specific protein kinases constitute control circuits to regulate pre-mRNA splicing and coordinate splicing with transcription in mammalian cells. We present here the finding that similar SR networks exist in Schizosaccharomyces pombe. We previously showed that Dsk1 protein, originally described as a mitotic regulator, displays high activity in phosphorylating S. pombe Prp2 protein (spU2AF59), a homologue of human U2AF65. We now demonstrate that Dsk1 also phosphorylates two recently identified fission yeast proteins with RS repeats, Srp1 and Srp2, in vitro. The phosphorylated proteins bear the same phosphoepitope found in mammalian SR proteins. Consistent with its substrate specificity, Dsk1 forms kinase-competent complexes with those proteins. Furthermore,dsk1 + gene determines the phenotype ofprp2 + overexpression, providing in vivo evidence that Prp2 is a target for Dsk1. The dsk1-null mutant strain became severely sick with the additional deletion of a related kinase gene. Significantly, human SR protein-specific kinase 1 (SRPK1) complements the growth defect of the double-deletion mutant. In conjunction with the resemblance of dsk1 + andSRPK1 in sequence homology, biochemical properties, and overexpression phenotypes, the complementation result indicates that SRPK1 is a functional homologue of Dsk1. Collectively, our studies illustrate the conserved SR networks in S. pombe consisting of RS domain-containing proteins and SR protein-specific kinases and thus establish the importance of the networks in eucaryotic organisms.

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

2000 ◽  
Vol 20 (9) ◽  
pp. 3049-3057 ◽  
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.

scholarly journals SRPK2: A Differentially Expressed SR Protein-specific Kinase Involved in Mediating the Interaction and Localization of Pre-mRNA Splicing Factors in Mammalian Cells

1998 ◽  
Vol 140 (4) ◽  
pp. 737-750 ◽  
Author(s):  
Huan-You Wang ◽  
Wen Lin ◽  
Jacqueline A. Dyck ◽  
Joanne M. Yeakley ◽  
Zhou Songyang ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mrna Splicing ◽  
Splicing Factors ◽  
Splicing Regulation ◽  
Phosphorylation Sites ◽  
Sr Protein ◽  
Random Peptide ◽  
Selection For

Abstract. Reversible phosphorylation plays an important role in pre-mRNA splicing in mammalian cells. Two kinases, SR protein-specific kinase (SRPK1) and Clk/Sty, have been shown to phosphorylate the SR family of splicing factors. We report here the cloning and characterization of SRPK2, which is highly related to SRPK1 in sequence, kinase activity, and substrate specificity. Random peptide selection for preferred phosphorylation sites revealed a stringent preference of SRPK2 for SR dipeptides, and the consensus derived may be used to predict potential phosphorylation sites in candidate arginine and serine-rich (RS) domain–containing proteins. Phosphorylation of an SR protein (ASF/SF2) by either SRPK1 or 2 enhanced its interaction with another RS domain–containing protein (U1 70K), and overexpression of either kinase induced specific redistribution of splicing factors in the nucleus. These observations likely reflect the function of the SRPK family of kinases in spliceosome assembly and in mediating the trafficking of splicing factors in mammalian cells. The biochemical and functional similarities between SRPK1 and 2, however, are in contrast to their differences in expression. SRPK1 is highly expressed in pancreas, whereas SRPK2 is highly expressed in brain, although both are coexpressed in other human tissues and in many experimental cell lines. Interestingly, SRPK2 also contains a proline-rich sequence at its NH2 terminus, and a recent study showed that this NH2-terminal sequence has the capacity to interact with a WW domain protein in vitro. Together, our studies suggest that different SRPK family members may be uniquely regulated and targeted, thereby contributing to splicing regulation in different tissues, during development, or in response to signaling.

scholarly journals Regulated Cellular Partitioning of SR Protein-specific Kinases in Mammalian Cells

2006 ◽  
Vol 17 (2) ◽  
pp. 876-885 ◽  
Author(s):  
Jian-Hua Ding ◽  
Xiang-Yang Zhong ◽  
Jonathan C. Hagopian ◽  
Marissa M. Cruz ◽  
Gourisankar Ghosh ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Mammalian Cells ◽  
Nuclear Translocation ◽  
Nuclear Export Signal ◽  
Sr Proteins ◽  
Splicing Factors ◽  
Dependent Manner ◽  
Sr Protein ◽  
Catalytic Core ◽  
Induced Aggregation

Reversible phosphorylation of the SR family of splicing factors plays an important role in pre-mRNA processing in the nucleus. Interestingly, the SRPK family of kinases specific for SR proteins is localized in the cytoplasm, which is critical for nuclear import of SR proteins in a phosphorylation-dependent manner. Here, we report molecular dissection of the mechanism involved in partitioning SRPKs in the cytoplasm. Common among all SRPKs, the bipartite kinase catalytic core is separated by a unique spacer sequence. The spacers in mammalian SRPK1 and SRPK2 share little sequence homology, but they function interchangeably in restricting the kinases in the cytoplasm. Removal of the spacer in SRPK1 had little effect on the kinase activity, but it caused a quantitative translocation of the kinase to the nucleus and consequently induced aggregation of splicing factors in the nucleus. Rather than carrying a nuclear export signal as suggested previously, we found multiple redundant signals in the spacer that act together to anchor the kinase in the cytoplasm. Interestingly, a cell cycle signal induced nuclear translocation of the kinase at the G2/M boundary. These findings suggest that SRPKs may play an important role in linking signaling to RNA metabolism in higher eukaryotic cells.

scholarly journals Interactions among SR proteins, an exonic splicing enhancer, and a lentivirus Rev protein regulate alternative splicing.

1996 ◽  
Vol 16 (5) ◽  
pp. 2325-2331 ◽  
Author(s):  
R R Gontarek ◽  
D Derse
Keyword(s):  
Alternative Splicing ◽  
Exon Skipping ◽  
Sr Proteins ◽  
Splicing Factors ◽  
Exonic Splicing Enhancer ◽  
Exon Inclusion ◽  
Alternative Mrna Splicing ◽  
Splicing Enhancer

We examine here the roles of cellular splicing factors and virus regulatory proteins in coordinately regulating alternative splicing of the tat/rev mRNA of equine infectious anemia virus (EIAV). This bicistronic mRNA contains four exons; exons 1 and 2 encode Tat, and exons 3 and 4 encode Rev. In the absence of Rev expression, the four-exon mRNA is synthesized exclusively, but when Rev is expressed, exon 3 is skipped to produce an mRNA that contains only exons 1, 2, and 4. We identify a purine-rich exonic splicing enhancer (ESE) in exon 3 that promotes exon inclusion. Similar to other cellular ESEs that have been identified by other laboratories, the EIAV ESE interacted specifically with SR proteins, a group of serine/arginine-rich splicing factors that function in constitutive and alternative mRNA splicing. Substitution of purines with pyrimidines in the ESE resulted in a switch from exon inclusion to exon skipping in vivo and abolished binding of SR proteins in vitro. Exon skipping was also induced by expression of EIAV Rev. We show that Rev binds to exon 3 RNA in vitro, and while the precise determinants have not been mapped, Rev function in vivo and RNA binding in vitro indicate that the RNA element necessary for Rev responsiveness overlaps or is adjacent to the ESE. We suggest that EIAV Rev promotes exon skipping by interfering with SR protein interactions with RNA or with other splicing factors.

Vacuoles Induced in Mammalian Cells by Helicobacter Cytotoxin are Acidic Organelles

1990 ◽  
Vol 48 (3) ◽  
pp. 168-169
Author(s):  
M. H. Chestnut ◽  
C. E. Catrenich
Keyword(s):  
Acridine Orange ◽  
Mammalian Cells ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Ulcer Disease ◽  
Gastroduodenal Disease ◽  
H Pylori

Helicobacter pylori is a non-invasive, Gram-negative spiral bacterium first identified in 1983, and subsequently implicated in the pathogenesis of gastroduodenal disease including gastritis and peptic ulcer disease. Cytotoxic activity, manifested by intracytoplasmic vacuolation of mammalian cells in vitro, was identified in 55% of H. pylori strains examined. The vacuoles increase in number and size during extended incubation, resulting in vacuolar and cellular degeneration after 24 h to 48 h. Vacuolation of gastric epithelial cells is also observed in vivo during infection by H. pylori. A high molecular weight, heat labile protein is believed to be responsible for vacuolation and to significantly contribute to the development of gastroduodenal disease in humans. The mechanism by which the cytotoxin exerts its effect is unknown, as is the intracellular origin of the vacuolar membrane and contents. Acridine orange is a membrane-permeant weak base that initially accumulates in low-pH compartments. We have used acridine orange accumulation in conjunction with confocal laser scanning microscopy of toxin-treated cells to begin probing the nature and origin of these vacuoles.

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