The Experts below are selected from a list of 231 Experts worldwide ranked by ideXlab platform

John Abelson - One of the best experts on this subject based on the ideXlab platform.

  • Purification of the yeast U4/U6.U5 Small Nuclear Ribonucleoprotein particle and identification of its proteins.
    Proceedings of the National Academy of Sciences of the United States of America, 1999
    Co-Authors: Scott W. Stevens, John Abelson
    Abstract:

    The yeast U4/U6⋅U5 pre-mRNA splicing Small Nuclear Ribonucleoprotein (snRNP) is a 25S Small Nuclear Ribonucleoprotein particle similar in size, composition, and morphology to its counterpart in human cells. The yeast U4/U6⋅U5 snRNP complex has been purified to near homogeneity by affinity chromatography and preparative glycerol gradient sedimentation. We show that there are at least 24 proteins stably associated with this particle and performed mass spectrometry microsequencing to determine their identities. In addition to the seven canonical core Sm proteins, there are a set of U6 snRNP specific Sm proteins, eight previously described U4/U6⋅U5 snRNP proteins, and four novel proteins. Two of the novel proteins have likely RNA binding properties, one has been implicated in the cell cycle, and one has no identifiable sequence homologues or functional motifs. The purification of the low abundance U4/U6⋅U5 snRNP from yeast and the powerful sequencing methodologies using Small amounts of protein make possible the rapid identification of novel and previously unidentified components of large, low-abundance macromolecular machines from any genetically manipulable organism.

  • purification of the yeast u4 u6 u5 Small Nuclear Ribonucleoprotein particle and identification of its proteins
    Proceedings of the National Academy of Sciences of the United States of America, 1999
    Co-Authors: Scott W. Stevens, John Abelson
    Abstract:

    The yeast U4/U6⋅U5 pre-mRNA splicing Small Nuclear Ribonucleoprotein (snRNP) is a 25S Small Nuclear Ribonucleoprotein particle similar in size, composition, and morphology to its counterpart in human cells. The yeast U4/U6⋅U5 snRNP complex has been purified to near homogeneity by affinity chromatography and preparative glycerol gradient sedimentation. We show that there are at least 24 proteins stably associated with this particle and performed mass spectrometry microsequencing to determine their identities. In addition to the seven canonical core Sm proteins, there are a set of U6 snRNP specific Sm proteins, eight previously described U4/U6⋅U5 snRNP proteins, and four novel proteins. Two of the novel proteins have likely RNA binding properties, one has been implicated in the cell cycle, and one has no identifiable sequence homologues or functional motifs. The purification of the low abundance U4/U6⋅U5 snRNP from yeast and the powerful sequencing methodologies using Small amounts of protein make possible the rapid identification of novel and previously unidentified components of large, low-abundance macromolecular machines from any genetically manipulable organism.

Scott W. Stevens - One of the best experts on this subject based on the ideXlab platform.

  • Purification of the yeast U4/U6.U5 Small Nuclear Ribonucleoprotein particle and identification of its proteins.
    Proceedings of the National Academy of Sciences of the United States of America, 1999
    Co-Authors: Scott W. Stevens, John Abelson
    Abstract:

    The yeast U4/U6⋅U5 pre-mRNA splicing Small Nuclear Ribonucleoprotein (snRNP) is a 25S Small Nuclear Ribonucleoprotein particle similar in size, composition, and morphology to its counterpart in human cells. The yeast U4/U6⋅U5 snRNP complex has been purified to near homogeneity by affinity chromatography and preparative glycerol gradient sedimentation. We show that there are at least 24 proteins stably associated with this particle and performed mass spectrometry microsequencing to determine their identities. In addition to the seven canonical core Sm proteins, there are a set of U6 snRNP specific Sm proteins, eight previously described U4/U6⋅U5 snRNP proteins, and four novel proteins. Two of the novel proteins have likely RNA binding properties, one has been implicated in the cell cycle, and one has no identifiable sequence homologues or functional motifs. The purification of the low abundance U4/U6⋅U5 snRNP from yeast and the powerful sequencing methodologies using Small amounts of protein make possible the rapid identification of novel and previously unidentified components of large, low-abundance macromolecular machines from any genetically manipulable organism.

  • purification of the yeast u4 u6 u5 Small Nuclear Ribonucleoprotein particle and identification of its proteins
    Proceedings of the National Academy of Sciences of the United States of America, 1999
    Co-Authors: Scott W. Stevens, John Abelson
    Abstract:

    The yeast U4/U6⋅U5 pre-mRNA splicing Small Nuclear Ribonucleoprotein (snRNP) is a 25S Small Nuclear Ribonucleoprotein particle similar in size, composition, and morphology to its counterpart in human cells. The yeast U4/U6⋅U5 snRNP complex has been purified to near homogeneity by affinity chromatography and preparative glycerol gradient sedimentation. We show that there are at least 24 proteins stably associated with this particle and performed mass spectrometry microsequencing to determine their identities. In addition to the seven canonical core Sm proteins, there are a set of U6 snRNP specific Sm proteins, eight previously described U4/U6⋅U5 snRNP proteins, and four novel proteins. Two of the novel proteins have likely RNA binding properties, one has been implicated in the cell cycle, and one has no identifiable sequence homologues or functional motifs. The purification of the low abundance U4/U6⋅U5 snRNP from yeast and the powerful sequencing methodologies using Small amounts of protein make possible the rapid identification of novel and previously unidentified components of large, low-abundance macromolecular machines from any genetically manipulable organism.

Rene J. Herrera - One of the best experts on this subject based on the ideXlab platform.

  • differential immunoglobulin class mediated responses to components of the u1 Small Nuclear Ribonucleoprotein particle in systemic lupus erythematosus and mixed connective tissue disease
    Lupus, 2013
    Co-Authors: Annia Mesa, Jason A. Somarelli, Laisel Martinez, Eric L Greidinger, Melissa B Blom, Rene J. Herrera
    Abstract:

    ObjectiveThe objective of this paper is to determine whether patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) possess differential IgM- and IgG-specific reactivity against peptides from the U1 Small Nuclear Ribonucleoprotein particle (U1 snRNP).MethodsThe IgM- and IgG-mediated responses against 15 peptides from subunits of the U1 snRNP were assessed by indirect enzyme linked immunosorbent assays (ELISAs) in sera from patients with SLE and MCTD and healthy individuals (n = 81, 41, and 31, respectively). Additionally, 42 laboratory tests and 40 clinical symptoms were evaluated to uncover potential differences. Binomial logistic regression analyses (BLR) were performed to construct models to support the independent nature of SLE and MCTD. Receiver operating characteristic (ROC) curves corroborated the classification power of the models.ResultsWe analyzed IgM and IgG anti-U1 snRNP titers to classify SLE and MCTD patients. IgG anti-U1 snRNP reactivity segregates SLE a...

  • epitope mapping of the u1 Small Nuclear Ribonucleoprotein particle in patients with systemic lupus erythematosus and mixed connective tissue disease
    Lupus, 2011
    Co-Authors: Jason A. Somarelli, Annia Mesa, Rosa Rodriguez, R Avellan, Laisel Martinez, Yunjuan Zang, Eric L Greidinger, Rene J. Herrera
    Abstract:

    Systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD) are autoimmune illnesses characterized by the presence of high titers of autoantibodies directed against a wide range of ‘self ’ antigens. Proteins of the U1 Small Nuclear Ribonucleoprotein particle (U1 snRNP) are among the most immunogenic molecules in patients with SLE and MCTD. The recent release of a crystallized U1 snRNP provides a unique opportunity to evaluate the effects of tertiary and quaternary structures on autoantigenicity within the U1 snRNP. In the present study, an epitope map was created using the U1 snRNP crystal structure. A total of 15 peptides were tested in a cohort of 68 patients with SLE, 29 with MCTD and 26 healthy individuals and mapped onto the U1 snRNP structure. Antigenic sites were detected in a variety of structures and appear to include RNA binding domains, but mostly exclude regions necessary for protein–protein interactions. These data suggest that while some autoantibodies may target U1 snRNP p...

  • A three‐dimensional model of the U1 Small Nuclear Ribonucleoprotein particle
    Entomological Research, 2010
    Co-Authors: Jason A. Somarelli, Annia Mesa, Ambrish Roy, Yang Zhang, Rene J. Herrera
    Abstract:

    Most of the pre-mRNAs in the eukaryotic cell are comprised of protein-coding exons and non-protein-coding introns. The introns are removed and the exons are ligated together, or spliced, by a large, macromolecular complex known as the spliceosome. This RNA-protein assembly is made up of five uridine-rich Small Nuclear RNAs (U1-, U2-, U4-, U5- and U6-snRNA) as well over 300 proteins, which form Small Nuclear Ribonucleoprotein particles (snRNPs). Initial recognition of the 5′ exon/intron splice site is mediated by the U1 snRNP, which is composed of the U1 snRNA as well as at least ten proteins. By combining structural informatics tools with the available biochemical and crystallographic data, we attempted to simulate a complete, three dimensional U1 snRNP from the silk moth, Bombyx mori. Comparison of our model with empirically derived crystal structures and electron micrographs pinpoints both the strengths and weaknesses in the in silico determination of macromolecular complexes. One of the most striking differences between our model and experimentally generated structures is in the positioning of the U1 snRNA stem-loops. This highlights the continuing difficulties in generating reliable, complex RNA structures; however, three-dimensional modeling of individual protein subunits by threading provided models of biological significance and the use of both automated and manual docking strategies generated a complex that closely reflects the assembly found in nature.Yet, without utilizing experimentally-derived contacts to select the most likely docking scenario, ab initio docking would fall short of providing a reliable model. Our work shows that the combination of experimental data with structural informatics tools can result in generation of near-native macromolecular complexes.

Matthias Mann - One of the best experts on this subject based on the ideXlab platform.

  • identification of the proteins of the yeast u1 Small Nuclear Ribonucleoprotein complex by mass spectrometry
    Proceedings of the National Academy of Sciences of the United States of America, 1997
    Co-Authors: Gitte Neubauer, Reinhard Luhrmann, Alexander Gottschalk, Patrizia Fabrizio, Bertrand Seraphin, Matthias Mann
    Abstract:

    Here we report the rapid identification of the proteins of the spliceosomal U1 Small Nuclear Ribonucleoprotein (snRNP) from the yeast Saccharomyces cerevisiae by searching mass spectrometric data in genomic sequence databases. The U1 snRNP, containing a histidine-tagged 70K protein, was isolated from cell extracts by anti m3G-cap immunoaffinity and subsequent nickel nitrilotriacetic acid chromatography. A U1 snRNP fraction containing 20 proteins was obtained. Further purification by glycerol gradient centrifugation identified nine U1 snRNP specific and six common proteins. The U1 snRNP proteins were partially sequenced by nanoelectrospray mass spectrometry, and their genes were identified in the data base via multiple peptide sequence tags. Apart from the already known common proteins D1, D3, F, and G, the D2 and E homologs were also identified. The same six common proteins were detected in core U2 snRNP, which was purified and analyzed separately. The biochemical association of these six proteins with yeast snRNPs is shown here for the first time. Intriguingly, the Sm B/B′ homolog was not detected. In addition to the well characterized yeast U1 specific proteins [U1-70K (Snp1p), U1-A (Mud1p), Prp39p, and Prp40p] the homolog of the U1-C protein was identified together with four additional novel U1 specific proteins, which are not found in mammalian U1. This is the first time that the components of a multiprotein complex from an organism with a sequenced genome have been characterized by mass spectrometry. The technique should be applicable to any protein complex that can be biochemically purified from an organism whose genome is known.

  • identification of the proteins of the yeast u1 Small Nuclear Ribonucleoprotein complex by mass spectrometry
    Proceedings of the National Academy of Sciences of the United States of America, 1997
    Co-Authors: Gitte Neubauer, Reinhard Luhrmann, Alexander Gottschalk, Patrizia Fabrizio, Bertrand Seraphin, Matthias Mann
    Abstract:

    Here we report the rapid identification of the proteins of the spliceosomal U1 Small Nuclear Ribonucleoprotein (snRNP) from the yeast Saccharomyces cerevisiae by searching mass spectrometric data in genomic sequence databases. The U1 snRNP, containing a histidine-tagged 70K protein, was isolated from cell extracts by anti m3G-cap immunoaffinity and subsequent nickel nitrilotriacetic acid chromatography. A U1 snRNP fraction containing 20 proteins was obtained. Further purification by glycerol gradient centrifugation identified nine U1 snRNP specific and six common proteins. The U1 snRNP proteins were partially sequenced by nanoelectrospray mass spectrometry, and their genes were identified in the data base via multiple peptide sequence tags. Apart from the already known common proteins D1, D3, F, and G, the D2 and E homologs were also identified. The same six common proteins were detected in core U2 snRNP, which was purified and analyzed separately. The biochemical association of these six proteins with yeast snRNPs is shown here for the first time. Intriguingly, the Sm B/B′ homolog was not detected. In addition to the well characterized yeast U1 specific proteins [U1-70K (Snp1p), U1-A (Mud1p), Prp39p, and Prp40p] the homolog of the U1-C protein was identified together with four additional novel U1 specific proteins, which are not found in mammalian U1. This is the first time that the components of a multiprotein complex from an organism with a sequenced genome have been characterized by mass spectrometry. The technique should be applicable to any protein complex that can be biochemically purified from an organism whose genome is known.

Reinhard Luhrmann - One of the best experts on this subject based on the ideXlab platform.

  • Small Nuclear Ribonucleoprotein remodeling during catalytic activation of the spliceosome
    Science, 2002
    Co-Authors: Evgeny M Makarov, Cindy L Will, Olga V Makarova, Marc Gentzel, Matthias Wilm, Reinhard Luhrmann
    Abstract:

    Major structural changes occur in the spliceosome during its activation just before catalyzing the splicing of pre-messenger RNAs (pre-mRNAs). Whereas changes in Small Nuclear RNA (snRNA) conformation are well documented, little is known about remodeling of Small Nuclear Ribonucleoprotein (snRNP) structures during spliceosome activation. Here, human 45S activated spliceosomes and a previously unknown 35S U5 snRNP were isolated by immunoaffinity selection and were characterized by mass spectrometry. Comparison of their protein components with those of other snRNP and spliceosomal complexes revealed a major change in protein composition during spliceosome activation. Our data also suggest that the U5 snRNP is dramatically remodeled at this stage, with the Prp19 complex and other factors tightly associating, possibly in exchange for other U5 proteins, and suggest that after catalysis the remodeled U5 is eventually released from the postsplicing complex as a 35S snRNP particle.

  • Arrangement of RNA and proteins in the spliceosomal U1 Small Nuclear Ribonucleoprotein particle
    Nature, 2001
    Co-Authors: Holger Stark, Reinhard Luhrmann, Prakash Dube, Berthold Kastner
    Abstract:

    In eukaryotic cells, freshly synthesized messenger RNA (pre-mRNA) contains stretches of non-coding RNA that must be excised before the RNA can be translated into protein. Their removal is catalysed by the spliceosome, a large complex formed when a number of Small Nuclear Ribonucleoprotein particles (snRNPs) bind sequentially to the pre-mRNA. The first snRNP to bind is called U1; other snRNPs (U2, U4/U6 and U5) follow. Here we describe the three-dimensional structure of human U1 snRNP, determined by single-particle electron cryomicroscopy at 10 A resolution. The reconstruction reveals a doughnut-shaped central element that accommodates the seven Sm proteins common to all snRNPs, supporting a proposed model of circular Sm protein arrangement. By taking earlier biochemical results into account, we were able to assign the remaining density of the map to the other known components of U1 snRNP, deriving a structural model that describes the three-dimensional arrangement of proteins and RNA in U1 snRNP.

  • identification of the proteins of the yeast u1 Small Nuclear Ribonucleoprotein complex by mass spectrometry
    Proceedings of the National Academy of Sciences of the United States of America, 1997
    Co-Authors: Gitte Neubauer, Reinhard Luhrmann, Alexander Gottschalk, Patrizia Fabrizio, Bertrand Seraphin, Matthias Mann
    Abstract:

    Here we report the rapid identification of the proteins of the spliceosomal U1 Small Nuclear Ribonucleoprotein (snRNP) from the yeast Saccharomyces cerevisiae by searching mass spectrometric data in genomic sequence databases. The U1 snRNP, containing a histidine-tagged 70K protein, was isolated from cell extracts by anti m3G-cap immunoaffinity and subsequent nickel nitrilotriacetic acid chromatography. A U1 snRNP fraction containing 20 proteins was obtained. Further purification by glycerol gradient centrifugation identified nine U1 snRNP specific and six common proteins. The U1 snRNP proteins were partially sequenced by nanoelectrospray mass spectrometry, and their genes were identified in the data base via multiple peptide sequence tags. Apart from the already known common proteins D1, D3, F, and G, the D2 and E homologs were also identified. The same six common proteins were detected in core U2 snRNP, which was purified and analyzed separately. The biochemical association of these six proteins with yeast snRNPs is shown here for the first time. Intriguingly, the Sm B/B′ homolog was not detected. In addition to the well characterized yeast U1 specific proteins [U1-70K (Snp1p), U1-A (Mud1p), Prp39p, and Prp40p] the homolog of the U1-C protein was identified together with four additional novel U1 specific proteins, which are not found in mammalian U1. This is the first time that the components of a multiprotein complex from an organism with a sequenced genome have been characterized by mass spectrometry. The technique should be applicable to any protein complex that can be biochemically purified from an organism whose genome is known.

  • identification of the proteins of the yeast u1 Small Nuclear Ribonucleoprotein complex by mass spectrometry
    Proceedings of the National Academy of Sciences of the United States of America, 1997
    Co-Authors: Gitte Neubauer, Reinhard Luhrmann, Alexander Gottschalk, Patrizia Fabrizio, Bertrand Seraphin, Matthias Mann
    Abstract:

    Here we report the rapid identification of the proteins of the spliceosomal U1 Small Nuclear Ribonucleoprotein (snRNP) from the yeast Saccharomyces cerevisiae by searching mass spectrometric data in genomic sequence databases. The U1 snRNP, containing a histidine-tagged 70K protein, was isolated from cell extracts by anti m3G-cap immunoaffinity and subsequent nickel nitrilotriacetic acid chromatography. A U1 snRNP fraction containing 20 proteins was obtained. Further purification by glycerol gradient centrifugation identified nine U1 snRNP specific and six common proteins. The U1 snRNP proteins were partially sequenced by nanoelectrospray mass spectrometry, and their genes were identified in the data base via multiple peptide sequence tags. Apart from the already known common proteins D1, D3, F, and G, the D2 and E homologs were also identified. The same six common proteins were detected in core U2 snRNP, which was purified and analyzed separately. The biochemical association of these six proteins with yeast snRNPs is shown here for the first time. Intriguingly, the Sm B/B′ homolog was not detected. In addition to the well characterized yeast U1 specific proteins [U1-70K (Snp1p), U1-A (Mud1p), Prp39p, and Prp40p] the homolog of the U1-C protein was identified together with four additional novel U1 specific proteins, which are not found in mammalian U1. This is the first time that the components of a multiprotein complex from an organism with a sequenced genome have been characterized by mass spectrometry. The technique should be applicable to any protein complex that can be biochemically purified from an organism whose genome is known.

  • Polypeptide components of Drosophila Small Nuclear Ribonucleoprotein particles
    Nucleic acids research, 1991
    Co-Authors: Trevor Paterson, Jean D. Beggs, David J. Finnegan, Reinhard Luhrmann
    Abstract:

    Abstract In eukaryotes splicing of pre-mRNAs is mediated by the spliceosome, a dynamic complex of Small Nuclear Ribonucleoprotein particles (snRNPs) that associate transiently during spliceosome assembly and the splicing reaction. We have purified snRNPs from Nuclear extracts of Drosophila cells by affinity chromatography with an antibody specific for the trimethylguanosine (m3G) cap structure of snRNAs U1-U5. The polypeptide components of Drosophila snRNPs have been characterized and shown to consist of a number of proteins shared by all the snRNPs, and some proteins which appear to be specific to individual snRNP particles. On the basis of their apparent molecular weight and antigenicity many of these common and particle specific Drosophila snRNP proteins are remarkably conserved between Drosophila and human spliceosomes. By probing western blots of the Drosophila snRNP polypeptides with a number of antisera raised against human snRNP proteins, Drosophila polypeptides equivalent to many of the HeLa snRNP-common proteins have been identified, as well as candidates for a number of U1, U2 and U5-specific proteins.