title: Arabidopsis PTB1 and PTB2 proteins negatively regulate splicing of a mini-exon splicing reporter and affect alternative splicing of endogenous genes differentially.
abstract: This paper examines the function of Arabidopsis thaliana AtPTB1 and AtPTB2 as plant splicing factors. The effect on splicing of overexpression of AtPTB1 and AtPTB2 was analysed in an in vivo protoplast transient expression system with a novel mini-exon splicing reporter. A range of mutations in pyrimidine-rich sequences were compared with and without AtPTB and NpU2AF65 overexpression. Splicing analyses of constructs in protoplasts and RNA from overexpression lines used high-resolution reverse transcription polymerase chain reaction (RT-PCR). AtPTB1 and AtPTB2 reduced inclusion/splicing of the potato invertase mini-exon splicing reporter, indicating that these proteins can repress plant intron splicing. Mutation of the polypyrimidine tract and closely associated Cytosine and Uracil-rich (CU-rich) sequences, upstream of the mini-exon, altered repression by AtPTB1 and AtPTB2. Coexpression of a plant orthologue of U2AF65 alleviated the splicing repression of AtPTB1. Mutation of a second CU-rich upstream of the mini-exon 3' splice site led to a decline in mini-exon splicing, indicating the presence of a splicing enhancer sequence. Finally, RT-PCR of AtPTB overexpression lines with c. 90 known alternative splicing (AS) events showed that AtPTBs significantly altered AS of over half the events. AtPTB1 and AtPTB2 are splicing factors that influence alternative splicing. This occurs in the potato invertase mini-exon via the polypyrimidine tract and associated pyrimidine-rich sequence.
authors.affiliation: Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK.
authors.forename: Craig G
authors.initials: CG
authors.lastname: Simpson
authors.affiliation: Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK.
authors.forename: Dominika
authors.initials: D
authors.lastname: Lewandowska
authors.affiliation: Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK.
authors.forename: Michele
authors.initials: M
authors.lastname: Liney
authors.affiliation: Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK.
authors.forename: Diane
authors.initials: D
authors.lastname: Davidson
authors.affiliation: Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK.
authors.forename: Sean
authors.initials: S
authors.lastname: Chapman
authors.affiliation: Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK.
authors.forename: John
authors.initials: J
authors.lastname: Fuller
authors.affiliation: Biomathematics and Statistics Scotland, Invergowrie, Dundee, DD2 5DA, UK.
authors.forename: Jim
authors.initials: J
authors.lastname: McNicol
authors.affiliation: Information and Computational Sciences, The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK.
authors.forename: Paul
authors.initials: P
authors.lastname: Shaw
authors.affiliation: Cell and Molecular Sciences, The James Hutton Institute, Invergowrie, Dundee, DD2 5DA, UK.|Division of Plant Sciences, University of Dundee at JHI, Invergowrie, Dundee, DD2 5DA, UK.
authors.forename: John W S
authors.initials: JWS
authors.lastname: Brown
mesh_terms: D017398:Alternative Splicing; D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism*; D005091:Exons; D018506:Gene Expression Regulation, Plant; D017930:Genes, Reporter; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics; D030821:Plants, Genetically Modified; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D012261:Ribonucleoproteins / Q000235:genetics; D000072668:Splicing Factor U2AF; D014026:Nicotiana / Q000235:genetics; D043324:beta-Fructofuranosidase / Q000235:genetics
chemical_list: D029681:Arabidopsis Proteins; D009687:Nuclear Proteins; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; D000072668:Splicing Factor U2AF; D043324:beta-Fructofuranosidase; D002238:Carbohydrate Epimerases; C022553:ribulosephosphate 3-epimerase
keywords: alternative splicing; mini-exons; splicing enhancers; splicing regulation; splicing repressors
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