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PubMed2003

Inhibition of spliceosome assembly by the cell cycle-regulated protein kinase MELK and involvement of splicing factor NIPP1.

NIPP1 is a ubiquitous nuclear protein that is required for spliceosome assembly. We report here that the phosphothreonine-binding Forkhead-associated domain of NIPP1 interacts with the cell cycle-regulated protein Ser/Thr kinase MELK (maternal embryonic leucine zipper kinase). The NIPP1-MELK interaction was critically dependent on the phosphorylaton of Thr-478 of MELK and was increased in lysates from mitotically arrested cells. Recombinant MELK was a potent inhibitor of an early step of spliceosome assembly in nuclear extracts. This splicing defect was also seen with a kinase-dead mutant but was absent after mutation (T478A) of the NIPP1 binding site of MELK, indicating a mediatory role for NIPP1. Our data suggest that MELK has a role in the cell cycle-regulated control of pre-mRNA splicing.

Animals↗

PubMed2004

Protein stoichiometry of a multiprotein complex, the human spliceosomal U1 small nuclear ribonucleoprotein: absolute quantification using isotope-coded tags and mass spectrometry.

The human U1 snRNP (small nuclear ribonucleoprotein), which is a part of the spliceosome, consists of U1 snRNA and ten different proteins: seven Sm proteins B/B', D1, D2, D3, E, F, and G and the three U1-specific proteins U1-70 K, U1-A, U1-C. To determine the stoichiometry of all ten proteins, the complex was denatured, digested completely with an endoproteinase and labeled with an amine-specific tag. Corresponding peptides were synthesized and labeled with the same tag containing heavier isotopes. The digest was then spiked with defined amounts of the synthetic peptides, and the resulting isotopic peptide pairs were analyzed quantitatively by mass spectrometry. The mass spectra provided information about the absolute amount of each component in the starting protein mixture. The use of the isotope-coded, amine-specific reagents propionyl-N-oxysuccinimide and nicotinoyl-N-oxysuccinimide was evaluated for stoichiometry determination; the nicotinoyl reagent was found to be advantageous because of its greater mass spectrometric sensitivity. Absolute quantities of all ten proteins were measured, showing equal numbers of all ten proteins in the U1 spliceosomal snRNP. These data demonstrate that quantitative mass spectrometry has great potential for the determination of the stoichiometry of multiprotein complexes.

Chromatography, Liquid↗

PubMed2006

Identification of nuclear spliceosomal antigens targeted by NOD mouse antibodies following sodium iodide intake.

The non-obese diabetic (NOD) mouse spontaneously develops a range of autoreactive responses including an autoantibody response to nuclear antigens. As elevated dietary iodine has been shown to increase thyroid autoimmune pathology in NOD mice, the effect of sodium iodide (NaI) on the development of anti-nuclear antibodies (ANA) was assessed. Interestingly, the NaI symporter is expressed in both thyroid and salivary glands. Elevated dietary iodine was found to increase the percentage of male NOD mice developing autoantibodies. Specifically, the nuclear autoantibodies that develop in NOD mice were shown to target specific spliceosomal components. The target specificity of the autoantibodies was determined using recombinant spliceosomal proteins and shown to include U1A, U170K, U2B'', U2A', as well as the Sm proteins D1, D2, and B. The autoantibody isotypes most consistently represented were IgG2a and IgG2b.

Amino Acid Sequence↗

PubMed1994

A 69-kD protein that associates reversibly with the Sm core domain of several spliceosomal snRNP species.

The biogenesis of the spliceosomal small nuclear ribonucleoproteins (snRNPs) U1, U2, U4, and U5 involves: (a) migration of the snRNA molecules from the nucleus to the cytoplasm; (b) assembly of a group of common proteins (Sm proteins) and their binding to a region on the snRNAs called the Sm-binding site; and (c) translocation of the RNP back to the nucleus. A first prerequisite for understanding the assembly pathway and nuclear transport of the snRNPs in more detail is the knowledge of all the snRNP proteins that play essential roles in these processes. We have recently observed a previously undetected 69-kD protein in 12S U1 snRNPs isolated from HeLa nuclear extracts under non-denaturing conditions that is clearly distinct from the U1-70K protein. The following evidence indicates that the 69-kD protein is a common, rather than a U1-specific, protein, possibly associating with the snRNP core particles by protein-protein interaction. (a) Antibodies raised against the 69-kD protein, which did not cross-react with any of the Sm proteins B'-G, precipitated not only U1 snRNPs, but also the other spliceosomal snRNPs U2, U4/U6 and U5, albeit to a lower extent. (b) U1, U2, and U5 core RNP particles reconstituted in vitro contain the 69-kD protein. (c) Xenopus laevis oocytes contain an immunologically related homologue of the human 69-kD protein. When U1 snRNA as well as a mutant U1 snRNA, that can bind the Sm core proteins but lacks the capacity to bind the U1-specific proteins 70K, A, and C, were injected into Xenopus oocytes to allow assembly in vivo, they were recognized by antibodies specific against the 69-kD protein in the ooplasm and in the nucleus. The 69-kD protein is under-represented, if present at all, in purified 17S U2 and in 25S [U4/U6.U5] tri-snRNPs, isolated from HeLa nuclear extracts. Our results are consistent with the working hypothesis that this protein may either play a role in the cytoplasmic assembly of the core domain of the snRNPs and/or in the nuclear transport of the snRNPs. After transport of the snRNPs into the nucleus, it may dissociate from the particles as for example in the case of the 17S U2 or the 25S [U4/U6.U5] tri-snRNP, which bind more than 10 different snRNP specific proteins each in the nucleus.

Animals↗

PubMed1995

Immunoglobulin epitope spreading and autoimmune disease after peptide immunization: Sm B/B'-derived PPPGMRPP and PPPGIRGP induce spliceosome autoimmunity.

Autoantibodies from many patients with systemic lupus erythematosus bind the Sm autoantigen B/B' polypeptide. The binding of serial serum specimens to the 233 overlapping octapeptides of Sm B/B' have shown that of the B/B'-derived octapeptides, PPPGMRPP and PPPGIRGP are early targets of the autoimmune response in some lupus patients. Rabbits immunized with PPPGMRPP and PPPGIRGP develop antibodies which not only bind these octapeptides, but also subsequently bind many other octapeptides of Sm B/B'. Eventually, the rabbits immunized with one octapeptide develop autoantibodies that bind other spliceosomal proteins including D, 70K, A, and C. Any mechanisms that operate to maintain tolerance or anergy for the spliceosome are thus overcome. Features considered typical of human systemic lupus erythematosus are also found in these peptide-immunized animals, such as antinuclear antibodies, anti-Sm precipitins, anti-double-stranded DNA, thrombocytopenia, seizures, and proteinuria. This disease model provides access to a mechanism for the development of humoral autoimmunity and may provide a basis to explain the immunopathogenesis of lupus in humans.

Amino Acid Sequence↗

PubMed2003

Generation of a monoclonal antibody against the mouse Sf3b1 (SAP155) gene product for U2 snRNP component of spliceosome.

SAP155 is a subunit of the U2 snRNP, and plays an important role for prespliceosome assembly and splicing catalysis of the major spliceosome. Recently, it was reported that SAP155 was also a subunit of the minor spliceosome. These suggest that SAP155 is essential for the removal of any type of intron. More recently, a homolog of SAP155 cDNA, designated Sf3b1, was isolated from mouse. In this study, we report the generation of a monoclonal antibody (MAb) against murine Sf3b1 protein. This MAb recognizes endogenous Sf3b1 gene product by Western blotting, but less efficiently by immunoprecipitation.

Animals↗

PubMed1998

The yeast U2A'/U2B complex is required for pre-spliceosome formation.

Human U2 snRNP contains two specific proteins, U2A' and U2B", that interact with U2 snRNA stem-loop IV. In Saccharomyces cerevisiae, only the counterpart of human U2B", Yib9p, has been identified. Database searches revealed a gene potentially coding for a protein with striking similarities to human U2A', henceforth called LEA1 (looks exceptionally like U2A'). We demonstrate that Lea1p is a specific component of the yeast U2 snRNP. In addition, we show that Lea1p interacts directly with Yib9p. In vivo association of Lea1p with U2 snRNA requires Yib9p. Reciprocally, Yib9p binds to the U2 snRNA only in the presence of Lea1p in vivo, even though it has been previously shown to associate on its own with the U2 snRNA stem-loop IV in vitro. Strains lacking LEA1 and/or YIB9 grow slowly, are temperature sensitive and contain reduced levels of U2 snRNA. Pre-mRNA splicing is strongly impaired in these cells. In vitro studies demonstrate that spliceosome assembly is blocked prior to addition of U2 snRNP. This phenotype can be rescued partially, but specifically, by addition of the corresponding recombinant protein(s). This demonstrates a specific role for the yeast U2 snRNP specific proteins during formation of the pre-spliceosome.

Amino Acid Sequence↗

PubMed1999

Partial purification of the yeast U2 snRNP reveals a novel yeast pre-mRNA splicing factor required for pre-spliceosome assembly.

We have partially purified the U2 snRNP of Saccharomyces cerevisiae. Identification of some proteins consistently found in the purified fractions by nanoelectrospray mass spectrometry indicated the presence of a novel splicing factor named Rse1p. The RSE1 gene is essential and codes for a 148.2 kDa protein. We demonstrated that Rse1p associates specifically with U2 snRNA at low salt concentrations. In addition, we showed that Rse1p is a component of the pre-spliceosome. Depletion of Rse1p and analysis of a conditional mutant indicated that Rse1p was required for efficient splicing in vivo. In vitro Rse1p is required for the formation of pre-spliceosomes. Database searches revealed that Rse1p is conserved in humans and that it belongs to a large protein family that includes polyadenylation factors and DNA repair proteins. The characteristics of Rse1p suggest that its human homologue could be a subunit of the SF3 splicing factor.

Amino Acid Sequence↗

PubMed1999

Phosphorylation of splicing factor SF1 on Ser20 by cGMP-dependent protein kinase regulates spliceosome assembly.

Splicing factor 1 (SF1) functions at early stages of pre-mRNA splicing and contributes to splice site recognition by interacting with the essential splicing factor U2AF65 and binding to the intron branch site. We have identified an 80 kDa substrate of cGMP-dependent protein kinase-I (PKG-I) isolated from rat brain, which is identical to SF1. PKG phosphorylates SF1 at Ser20, which inhibits the SF1-U2AF65 interaction leading to a block of pre-spliceosome assembly. Mutation of Ser20 to Ala or Thr also inhibits the interaction with U2AF65, indicating that Ser20 is essential for binding. SF1 is phosphorylated in vitro by PKG, but not by cAMP-dependent protein kinase A (PKA). Phosphorylation of SF1 also occurs in cultured neuronal cells and is increased on Ser20 in response to a cGMP analogue. These results suggest a new role for PKG in mammalian pre-mRNA splicing by regulating in a phosphorylation-dependent manner the association of SF1 with U2AF65 and spliceosome assembly.

Amino Acid Sequence↗

PubMed2001

A small nucleolar guide RNA functions both in 2'-O-ribose methylation and pseudouridylation of the U5 spliceosomal RNA.

In eukaryotes, two distinct classes of small nucleolar RNAs (snoRNAs), namely the fibrillarin-associated box C/D snoRNAs and the Gar1p-associated box H/ACA snoRNAs, direct the site-specific 2'-O-ribose methylation and pseudouridylation of ribosomal RNAs (rRNAs), respectively. We have identified a novel evolutionarily conserved snoRNA, called U85, which possesses the box elements of both classes of snoRNAs and associates with both fibrillarin and Gar1p. In vitro and in vivo pseudouridylation and 2'-O-methylation experiments provide evidence that the U85 snoRNA directs 2'-O-methylation of the C45 and pseudouridylation of the U46 residues in the invariant loop 1 of the human U5 spliceosomal RNA. The U85 is the first example of a snoRNA that directs modification of an RNA polymerase II-transcribed spliceosomal RNA and that functions both in RNA pseudouridylation and 2'-O-methylation.

Animals↗

PubMed2002

Assisted RNP assembly: SMN and PRMT5 complexes cooperate in the formation of spliceosomal UsnRNPs.

Although spliceosomal Sm proteins can assemble spontaneously onto UsnRNA in vitro, this process requires assisting factors in vivo. SMN, the protein involved in spinal muscular atrophy, is part of a complex that contains the Sm proteins and serves as a critical factor for this reaction. Here, we have reconstituted the SMN-dependent assembly of UsnRNPs in vitro. We demonstrate that the SMN complex is necessary and sufficient for the assembly reaction. The PRMT5 complex, previously implicated in methylation and storage of Sm proteins, interacts with the SMN complex and enhances its activity in an ATP-dependent manner. These data uncover the SMN-PRMT5 complex as a functional entity that promotes the assisted assembly of spliceosomal UsnRNPs, and potentially other, RNA-protein complexes.

Animals↗

PubMed2000

Suppressors of a cold-sensitive mutation in yeast U4 RNA define five domains in the splicing factor Prp8 that influence spliceosome activation.

The highly conserved splicing factor Prp8 has been implicated in multiple stages of the splicing reaction. However, assignment of a specific function to any part of the 280-kD U5 snRNP protein has been difficult, in part because Prp8 lacks recognizable functional or structural motifs. We have used a large-scale screen for Saccharomyces cerevisiae PRP8 alleles that suppress the cold sensitivity caused by U4-cs1, a mutant U4 RNA that blocks U4/U6 unwinding, to identify with high resolution five distinct regions of PRP8 involved in the control of spliceosome activation. Genetic interactions between two of these regions reveal a potential long-range intramolecular fold. Identification of a yeast two-hybrid interaction, together with previously reported results, implicates two other regions in direct and indirect contacts to the U1 snRNP. In contrast to the suppressor mutations in PRP8, loss-of-function mutations in the genes for two other splicing factors implicated in U4/U6 unwinding, Prp44 (Brr2/Rss1/Slt22/Snu246) and Prp24, show synthetic enhancement with U4-cs1. On the basis of these results we propose a model in which allosteric changes in Prp8 initiate spliceosome activation by (1) disrupting contacts between the U1 snRNP and the U4/U6-U5 tri-snRNP and (2) orchestrating the activities of Prp44 and Prp24.

Amino Acid Sequence↗

PubMed1999

Essential role for the tudor domain of SMN in spliceosomal U snRNP assembly: implications for spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a neurodegenerative disease of spinal motor neurons caused by reduced levels of functional survival of motor neurons (SMN) protein. SMN is part of a macromolecular complex that contains the SMN-interacting protein 1 (SIP1) and spliceosomal Sm proteins. Although it is clear that SIP1 as a component of this complex is essential for spliceosomal uridine-rich small ribonucleoprotein (U snRNP) assembly, the role of SMN and its functional interactions with SIP1 and Sm proteins are poorly understood. Here we show that the central region of SMN comprising a tudor domain facilitates direct binding to Sm proteins. Strikingly, the SMA-causing missense mutation E134K within the tudor domain severely reduced the ability of SMN to interact with Sm proteins. Moreover, antibodies directed against the tudor domain prevent Sm protein binding to SMN and abolish assembly of U snRNPs in vivo. Thus, our data show that SMN is an essential U snRNP assembly factor and establish a direct correlation between defects in the biogenesis of U snRNPs and SMA.

Animals↗

PubMed2000

Characterization of a nuclear 20S complex containing the survival of motor neurons (SMN) protein and a specific subset of spliceosomal Sm proteins.

Spinal muscular atrophy (SMA) is a neurodegenerative disease of motor neurons caused by reduced levels of functional survival of motor neurons (SMN) protein. Cytoplasmic SMN directly interacts with spliceosomal Sm proteins and facilitates their assembly onto U snRNAs. Nuclear SMN, in contrast, mediates recycling of pre-mRNA splicing factors. In this study, we have addressed the function of SMN in the nucleus. We show that a monoclonal antibody directed against SMN inhibits pre-mRNA splicing. Interestingly, the mode of inhibition suggests a novel role for SMN in splicing that occurs prior to, or in addition to, its role in recycling. Using biochemical fractionation and anti-SMN immunoaffinity chromatography, we identified two distinct nuclear SMN complexes termed NSC1 and NSC2. The biochemical properties and protein composition of NSC1 were determined in detail. NSC1 migrates in sucrose gradients as a U snRNA-free 20S complex containing at least 10 proteins. In addition to SMN, these include the SMN-interacting protein 1 (SIP-1), the putative helicase dp103/Gemin3, the novel dp103/Gemin3-interacting protein GIP1/Gemin4 and three additional proteins with apparent masses of 43, 33 and 18 kDa, respectively. Most surprisingly, NSC1 also contains a specific subset of spliceosomal Sm proteins. This shows that the SMN-Sm protein interaction is not restricted to the cytoplasm. Our data imply that nuclear SMN affects splicing by modulating the Sm protein composition of U snRNPs.

Amino Acid Sequence↗

PubMed1992

Analysis of spliceosome assembly and the structure of a regulated intron in Drosophila in vitro splicing extracts.

We characterize spliceosome assembly in Drosophila embryonic nuclear extracts. Further, we show that these extracts contain high levels of a 5' to 3' exoribonuclease activity allowing rapid, convenient protection mapping of 5' splice site and branchpoint sequences. We use this assay to show, for the first time, that a regulated arthropod intron uses a remote branchpoint strikingly similar in structure to those observed previously in regulated vertebrate introns. These results provide new evidence that both regulated and constitutive splicing are similar in detail in vertebrates and arthropods indicating that the powerful genetic systems for analysis of splicing regulation in Drosophila are likely to be directly informative for regulated splicing throughout metazoa. In addition, we report formation of a novel class of intron-dependent complexes. Behavior of these complexes indicates that they represent a mutually exclusive, kinetically competing pathway with spliceosome assembly. We propose that this competition represents the basis for a kinetic proofreading mechanism enhancing fidelity of intron recognition. We also discuss possible implications of this model for regulated splicing.

Animals↗

PubMed1997

Association of U2 snRNP with the spliceosomal complex E.

In metazoans, the E complex is operationally defined as an ATP-independent spliceosomal complex that elutes as a single peak on a gel filtration column and can be chased into spliced products in the presence of an excess of competitor pre-mRNA. The A complex is the first ATP-dependent functional spliceosomal complex. U1 snRNP first binds tightly to the 5'splice site in the E complex and U2 snRNP first binds tightly to the branch site in the A complex. In this study, we have generated and characterized a monoclonal antibody (mAb 4G8) directed against SAP 62, a component of U2 snRNP and a subunit of the essential mammalian splicing factor SF3a. We show that this antibody is highly specific for SAP 62, detecting only SAP 62 on Western blots and immunoprecipitating only SAP 62 from nuclear extracts. The anti-SAP 62 antibody also immunoprecipitates U2 snRNP and the A complex. Significantly, however, we find that the E complex is also efficiently immunoprecipitated by the anti-SAP 62 antibody. This antibody does not cross-react with any E complex-specific components, indicating that SAP 62 itself is associated with the E complex. To determine whether other U2 snRNP components are associated with the E complex, we used antibodies to the U2 snRNP proteins B"and SAP 155. These antibodies also specifically immunoprecipitate the E complex. These observations indicate that U2 snRNP is associated with the E complex. However, we find that U2 snRNP is not as tightly bound in the E complex as it is in the A complex. The possible significance of the weak association of U2 snRNP with the E complex is discussed.

Animals↗

PubMed1998

U2 and U6 snRNA genes in the microsporidian Nosema locustae: evidence for a functional spliceosome.

The removal of introns from pre-messenger RNA is mediated by the spliceosome, a large complex composed of many proteins and five small nuclear RNAs (snRNAs). Of the snRNAs, the U6 and U2 snRNAs are the most conserved in sequence, as they interact extensively with each other and also with the intron, in several base pairings that are necessary for splicing. We have isolated and sequenced the genes encoding both U6 and U2 snRNAs from the intracellularly parasitic microsporidian Nosema locustae . Both genes are expressed. Both RNAs can be folded into secondary structures typical of other known U6 and U2 snRNAs. In addition, the N.locustae U6 and U2 snRNAs have the potential to base pair in the functional intermolecular interactions that have been characterized by extensive analyses in yeast and mammalian systems. These results indicate that the N.locustae U6 and U2 snRNAs may be functional components of an active spliceosome, even though introns have not yet been found in microsporidian genes.

Animals↗

PubMed1997

Polycistronic expression and RNA-binding specificity of the C. elegans homologue of the spliceosome-associated protein SAP49.

Splicing of mRNA precursors (pre-mRNAs) occurs in a multimolecular complex, termed spliceosome, which is comprised of pre-mRNA, small nuclear ribonucleoprotein particles (snRNPs), and other protein factors including spliceosome-associated proteins (SAPs). SAP49 is thought to be a subunit of the essential splicing factor SF3b and is involved in U2 snRNP function in mammalian cells. We have isolated a Caenorhabditis elegans cDNA encoding an RNA-binding protein with two RNA recognition motifs (RRMs) which shows extensive similarity to the human SAP49. The primary transcript for this C. elegans SAP49 homologue (cSAP49) seems to contain at least two additional cistrons and can be processed into three different mature mRNAs by trans-splicing. The cSAP49 mRNA, like other mRNAs in the same polycistronic unit, is expressed in most of the developmental stages, consistent with its putative essential function for mRNA splicing. By means of an in vitro RNA selection system, we demonstrate that cSAP49 possesses specific RNA-binding activity which resides in its second RRM.

Amino Acid Sequence↗