Blom7 alpha Is a Novel Heterogeneous Nuclear Ribonucleoprotein K Homology Domain Protein Involved in Pre-mRNA Splicing That Interacts with SNEVPrp19-Pso4

Johannes Grillari, Marlies Löscher , Marco Denegri, Kiseok Lee, Klaus Fortschegger, Frank Eisenhaber, Paul Ajuh, Angus I. Lamond, Hermann Katinger, Regina Grillari-Voglauer

    Research output: Contribution to journalArticle

    7 Citations (Scopus)

    Abstract

    The removal of introns from pre-mRNA is performed by the spliceosome that stepwise assembles on the pre-mRNA before performing two catalytic steps. The spliceosome-associated CDC5L-SNEVPrp19-Pso4 complex is implicated in activation of the second catalytic step of pre-mRNA splicing, and one of its members, SNEVPrp19-Pso4, is also implicated in spliceosome assembly. To identify interaction partners of SNEVPrp19-Pso4, we have performed yeast two-hybrid screenings. Among the putative binding partners was a so far uncharacterized protein carrying two heterogeneous nuclear ribonucleoprotein K homology domains that we termed Blom7 alpha. Blom7 alpha is expressed in all tissues tested, and at least three splice variants exist. After confirming direct and physical interaction of SNEV and Blom7 alpha, we investigated if it plays a functional role during pre-mRNA splicing. Indeed, Blom7 alpha co-localizes and co-precipitates with splicing factors and pre-mRNA and is present in affinity-purified spliceosomes. More importantly, addition of Blom7 alpha to HeLa nuclear extracts increased splicing activity in a dose-dependent manner. Furthermore, we tested if Blom7 alpha influences splice site selection using two different minigene constructs. Indeed, both 5'- as well as 3'-site selection was altered upon Blom7 alpha overexpression. Thus we suggest that Blom7 alpha is a novel splicing factor of the K homology domain family that might be implicated in alternative splicing by helping to position the CDC5L-SNEVPrp19-Pso4 complex at the splice sites.

    Original languageEnglish
    Pages (from-to)29193-29204
    Number of pages12
    JournalJournal of Biological Chemistry
    Volume284
    Issue number42
    DOIs
    Publication statusPublished - 16 Oct 2009

    Keywords

    • FRAGILE-X-SYNDROME
    • BINDING-PROTEIN
    • IN-VIVO
    • MASS-SPECTROMETRY
    • DNA-REPAIR
    • FUNCTIONAL-ANALYSIS
    • ENDOTHELIAL-CELLS
    • LIPID DROPLETS
    • B COMPLEXES
    • HUMAN PSO4

    Cite this

    Grillari, Johannes ; Löscher , Marlies ; Denegri, Marco ; Lee, Kiseok ; Fortschegger, Klaus ; Eisenhaber, Frank ; Ajuh, Paul ; Lamond, Angus I. ; Katinger, Hermann ; Grillari-Voglauer, Regina. / Blom7 alpha Is a Novel Heterogeneous Nuclear Ribonucleoprotein K Homology Domain Protein Involved in Pre-mRNA Splicing That Interacts with SNEVPrp19-Pso4. In: Journal of Biological Chemistry. 2009 ; Vol. 284, No. 42. pp. 29193-29204.
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    abstract = "The removal of introns from pre-mRNA is performed by the spliceosome that stepwise assembles on the pre-mRNA before performing two catalytic steps. The spliceosome-associated CDC5L-SNEVPrp19-Pso4 complex is implicated in activation of the second catalytic step of pre-mRNA splicing, and one of its members, SNEVPrp19-Pso4, is also implicated in spliceosome assembly. To identify interaction partners of SNEVPrp19-Pso4, we have performed yeast two-hybrid screenings. Among the putative binding partners was a so far uncharacterized protein carrying two heterogeneous nuclear ribonucleoprotein K homology domains that we termed Blom7 alpha. Blom7 alpha is expressed in all tissues tested, and at least three splice variants exist. After confirming direct and physical interaction of SNEV and Blom7 alpha, we investigated if it plays a functional role during pre-mRNA splicing. Indeed, Blom7 alpha co-localizes and co-precipitates with splicing factors and pre-mRNA and is present in affinity-purified spliceosomes. More importantly, addition of Blom7 alpha to HeLa nuclear extracts increased splicing activity in a dose-dependent manner. Furthermore, we tested if Blom7 alpha influences splice site selection using two different minigene constructs. Indeed, both 5'- as well as 3'-site selection was altered upon Blom7 alpha overexpression. Thus we suggest that Blom7 alpha is a novel splicing factor of the K homology domain family that might be implicated in alternative splicing by helping to position the CDC5L-SNEVPrp19-Pso4 complex at the splice sites.",
    keywords = "FRAGILE-X-SYNDROME, BINDING-PROTEIN, IN-VIVO, MASS-SPECTROMETRY, DNA-REPAIR, FUNCTIONAL-ANALYSIS, ENDOTHELIAL-CELLS, LIPID DROPLETS, B COMPLEXES, HUMAN PSO4",
    author = "Johannes Grillari and Marlies L{\"o}scher and Marco Denegri and Kiseok Lee and Klaus Fortschegger and Frank Eisenhaber and Paul Ajuh and Lamond, {Angus I.} and Hermann Katinger and Regina Grillari-Voglauer",
    year = "2009",
    month = "10",
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    doi = "10.1074/jbc.M109.036632",
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    volume = "284",
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    Grillari, J, Löscher , M, Denegri, M, Lee, K, Fortschegger, K, Eisenhaber, F, Ajuh, P, Lamond, AI, Katinger, H & Grillari-Voglauer, R 2009, 'Blom7 alpha Is a Novel Heterogeneous Nuclear Ribonucleoprotein K Homology Domain Protein Involved in Pre-mRNA Splicing That Interacts with SNEVPrp19-Pso4', Journal of Biological Chemistry, vol. 284, no. 42, pp. 29193-29204. https://doi.org/10.1074/jbc.M109.036632

    Blom7 alpha Is a Novel Heterogeneous Nuclear Ribonucleoprotein K Homology Domain Protein Involved in Pre-mRNA Splicing That Interacts with SNEVPrp19-Pso4. / Grillari, Johannes; Löscher , Marlies; Denegri, Marco; Lee, Kiseok; Fortschegger, Klaus; Eisenhaber, Frank; Ajuh, Paul; Lamond, Angus I.; Katinger, Hermann; Grillari-Voglauer, Regina.

    In: Journal of Biological Chemistry, Vol. 284, No. 42, 16.10.2009, p. 29193-29204.

    Research output: Contribution to journalArticle

    TY - JOUR

    T1 - Blom7 alpha Is a Novel Heterogeneous Nuclear Ribonucleoprotein K Homology Domain Protein Involved in Pre-mRNA Splicing That Interacts with SNEVPrp19-Pso4

    AU - Grillari, Johannes

    AU - Löscher , Marlies

    AU - Denegri, Marco

    AU - Lee, Kiseok

    AU - Fortschegger, Klaus

    AU - Eisenhaber, Frank

    AU - Ajuh, Paul

    AU - Lamond, Angus I.

    AU - Katinger, Hermann

    AU - Grillari-Voglauer, Regina

    PY - 2009/10/16

    Y1 - 2009/10/16

    N2 - The removal of introns from pre-mRNA is performed by the spliceosome that stepwise assembles on the pre-mRNA before performing two catalytic steps. The spliceosome-associated CDC5L-SNEVPrp19-Pso4 complex is implicated in activation of the second catalytic step of pre-mRNA splicing, and one of its members, SNEVPrp19-Pso4, is also implicated in spliceosome assembly. To identify interaction partners of SNEVPrp19-Pso4, we have performed yeast two-hybrid screenings. Among the putative binding partners was a so far uncharacterized protein carrying two heterogeneous nuclear ribonucleoprotein K homology domains that we termed Blom7 alpha. Blom7 alpha is expressed in all tissues tested, and at least three splice variants exist. After confirming direct and physical interaction of SNEV and Blom7 alpha, we investigated if it plays a functional role during pre-mRNA splicing. Indeed, Blom7 alpha co-localizes and co-precipitates with splicing factors and pre-mRNA and is present in affinity-purified spliceosomes. More importantly, addition of Blom7 alpha to HeLa nuclear extracts increased splicing activity in a dose-dependent manner. Furthermore, we tested if Blom7 alpha influences splice site selection using two different minigene constructs. Indeed, both 5'- as well as 3'-site selection was altered upon Blom7 alpha overexpression. Thus we suggest that Blom7 alpha is a novel splicing factor of the K homology domain family that might be implicated in alternative splicing by helping to position the CDC5L-SNEVPrp19-Pso4 complex at the splice sites.

    AB - The removal of introns from pre-mRNA is performed by the spliceosome that stepwise assembles on the pre-mRNA before performing two catalytic steps. The spliceosome-associated CDC5L-SNEVPrp19-Pso4 complex is implicated in activation of the second catalytic step of pre-mRNA splicing, and one of its members, SNEVPrp19-Pso4, is also implicated in spliceosome assembly. To identify interaction partners of SNEVPrp19-Pso4, we have performed yeast two-hybrid screenings. Among the putative binding partners was a so far uncharacterized protein carrying two heterogeneous nuclear ribonucleoprotein K homology domains that we termed Blom7 alpha. Blom7 alpha is expressed in all tissues tested, and at least three splice variants exist. After confirming direct and physical interaction of SNEV and Blom7 alpha, we investigated if it plays a functional role during pre-mRNA splicing. Indeed, Blom7 alpha co-localizes and co-precipitates with splicing factors and pre-mRNA and is present in affinity-purified spliceosomes. More importantly, addition of Blom7 alpha to HeLa nuclear extracts increased splicing activity in a dose-dependent manner. Furthermore, we tested if Blom7 alpha influences splice site selection using two different minigene constructs. Indeed, both 5'- as well as 3'-site selection was altered upon Blom7 alpha overexpression. Thus we suggest that Blom7 alpha is a novel splicing factor of the K homology domain family that might be implicated in alternative splicing by helping to position the CDC5L-SNEVPrp19-Pso4 complex at the splice sites.

    KW - FRAGILE-X-SYNDROME

    KW - BINDING-PROTEIN

    KW - IN-VIVO

    KW - MASS-SPECTROMETRY

    KW - DNA-REPAIR

    KW - FUNCTIONAL-ANALYSIS

    KW - ENDOTHELIAL-CELLS

    KW - LIPID DROPLETS

    KW - B COMPLEXES

    KW - HUMAN PSO4

    U2 - 10.1074/jbc.M109.036632

    DO - 10.1074/jbc.M109.036632

    M3 - Article

    VL - 284

    SP - 29193

    EP - 29204

    JO - Journal of Biological Chemistry

    JF - Journal of Biological Chemistry

    SN - 0021-9258

    IS - 42

    ER -