title: Biochemical identification of new proteins involved in splicing repression at the Drosophila P-element exonic splicing silencer.
abstract: Splicing of the Drosophila P-element third intron (IVS3) is repressed in somatic tissues due to the function of an exonic splicing silencer (ESS) complex present on the 5' exon RNA. To comprehensively characterize the mechanisms of this alternative splicing regulation, we used biochemical fractionation and affinity purification to isolate the silencer complex assembled in vitro and identify the constituent proteins by mass spectrometry. Functional assays using splicing reporter minigenes identified the proteins hrp36 and hrp38 and the cytoplasmic poly(A)-binding protein PABPC1 as novel functional components of the splicing silencer. hrp48, PSI, and PABPC1 have high-affinity RNA-binding sites on the P-element IVS3 5' exon, whereas hrp36 and hrp38 proteins bind with low affinity to the P-element silencer RNA. RNA pull-down and immobilized protein assays showed that hrp48 protein binding to the silencer RNA can recruit hrp36 and hrp38. These studies identified additional components that function at the P-element ESS and indicated that proteins with low-affinity RNA-binding sites can be recruited in a functional manner through interactions with a protein bound to RNA at a high-affinity binding site. These studies have implications for the role of heterogeneous nuclear ribonucleoproteins (hnRNPs) in the control of alternative splicing at cis-acting regulatory sites.
authors.affiliation: Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA;
authors.forename: Lucas
authors.initials: L
authors.lastname: Horan
authors.affiliation: Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA;
authors.forename: Jiro C
authors.initials: JC
authors.lastname: Yasuhara
authors.affiliation: California Institute for Quantitative Biosciences (QB3), University of California at Berkeley, Berkeley, California 94720, USA;
authors.forename: Lori A
authors.initials: LA
authors.lastname: Kohlstaedt
authors.affiliation: Department of Molecular and Cell Biology, University of California at Berkeley, Berkeley, California 94720, USA; California Institute for Quantitative Biosciences (QB3), University of California at Berkeley, Berkeley, California 94720, USA; Center for RNA Systems Biology, University of California at Berkeley, Berkeley, California 94720, USA.
authors.forename: Donald C
authors.initials: DC
authors.lastname: Rio
mesh_terms: D000818:Animals; D029721:Drosophila Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000235:genetics* / Q000378:metabolism*; D005091:Exons / Q000235:genetics; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D034441:Heterogeneous-Nuclear Ribonucleoproteins / Q000378:metabolism; D013058:Mass Spectrometry; D009687:Nuclear Proteins; D011485:Protein Binding; D034622:RNA Interference; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing / Q000235:genetics / Q000502:physiology*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism; D037941:Silencer Elements, Transcriptional / Q000235:genetics
chemical_list: D029721:Drosophila Proteins; C097984:HRB98DE protein, Drosophila; D034441:Heterogeneous-Nuclear Ribonucleoproteins; C487427:Hrb27C protein, Drosophila; C402657:Hrb87F protein, Drosophila; D009687:Nuclear Proteins; D012322:RNA Precursors; D011994:Recombinant Proteins; D012261:Ribonucleoproteins
keywords: exonic splicing silencers; pre-mRNA splicing; splicing repression