title: The Pentatricopeptide Repeat Protein EMB2654 Is Essential for Trans-Splicing of a Chloroplast Small Ribosomal Subunit Transcript.
abstract: We report the partial complementation and subsequent comparative molecular analysis of two nonviable mutants impaired in chloroplast translation, one (emb2394) lacking the RPL6 protein, and the other (emb2654) carrying a mutation in a gene encoding a P-class pentatricopeptide repeat protein. We show that EMB2654 is required for the trans-splicing of the plastid rps12 transcript and that therefore the emb2654 mutant lacks Rps12 protein and fails to assemble the small subunit of the plastid ribosome, explaining the loss of plastid translation and consequent embryo-lethal phenotype. Predictions of the EMB2654 binding site match a small RNA "footprint" located on the 5' half of the trans-spliced intron that is almost absent in the partially complemented mutant. EMB2654 binds sequence specifically to this target sequence in vitro. Altered patterns in nuclease-protected small RNA fragments in emb2654 show that EMB2654 binding must be an early step in, or prior to, the formation of a large protein-RNA complex covering the free ends of the two rps12 intron halves.
authors.affiliation: Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley 6009 Western Australia, Australia (N.A., H.R., L.V.P.S., L.E., J.D.B., K.A.H., I.S., C.C.d.F.-S.); and.|Institute of Biology, Humboldt-University of Berlin, 10115 Berlin, Germany (H.R.).
authors.forename: Nader
authors.identifier: 0000-0003-4666-9175
authors.initials: N
authors.lastname: Aryamanesh
authors.affiliation: Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley 6009 Western Australia, Australia (N.A., H.R., L.V.P.S., L.E., J.D.B., K.A.H., I.S., C.C.d.F.-S.); and.|Institute of Biology, Humboldt-University of Berlin, 10115 Berlin, Germany (H.R.).
authors.forename: Hannes
authors.identifier: 0000-0002-5576-5807
authors.initials: H
authors.lastname: Ruwe
authors.affiliation: Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley 6009 Western Australia, Australia (N.A., H.R., L.V.P.S., L.E., J.D.B., K.A.H., I.S., C.C.d.F.-S.); and.|Institute of Biology, Humboldt-University of Berlin, 10115 Berlin, Germany (H.R.).
authors.forename: Lilian Vincis Pereira
authors.initials: LV
authors.lastname: Sanglard
authors.affiliation: Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley 6009 Western Australia, Australia (N.A., H.R., L.V.P.S., L.E., J.D.B., K.A.H., I.S., C.C.d.F.-S.); and.|Institute of Biology, Humboldt-University of Berlin, 10115 Berlin, Germany (H.R.).
authors.forename: Leila
authors.identifier: 0000-0001-9922-8985
authors.initials: L
authors.lastname: Eshraghi
authors.affiliation: Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley 6009 Western Australia, Australia (N.A., H.R., L.V.P.S., L.E., J.D.B., K.A.H., I.S., C.C.d.F.-S.); and.|Institute of Biology, Humboldt-University of Berlin, 10115 Berlin, Germany (H.R.).
authors.forename: John D
authors.initials: JD
authors.lastname: Bussell
authors.affiliation: Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley 6009 Western Australia, Australia (N.A., H.R., L.V.P.S., L.E., J.D.B., K.A.H., I.S., C.C.d.F.-S.); and.|Institute of Biology, Humboldt-University of Berlin, 10115 Berlin, Germany (H.R.).
authors.forename: Katharine A
authors.initials: KA
authors.lastname: Howell
authors.affiliation: Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley 6009 Western Australia, Australia (N.A., H.R., L.V.P.S., L.E., J.D.B., K.A.H., I.S., C.C.d.F.-S.); and ian.small@uwa.edu.au catherine.colasdesfrancs-small@uwa.edu.au.|Institute of Biology, Humboldt-University of Berlin, 10115 Berlin, Germany (H.R.) ian.small@uwa.edu.au catherine.colasdesfrancs-small@uwa.edu.au.
authors.forename: Ian
authors.identifier: 0000-0001-5300-1216
authors.initials: I
authors.lastname: Small
authors.affiliation: Australian Research Council Centre of Excellence in Plant Energy Biology, University of Western Australia, Crawley 6009 Western Australia, Australia (N.A., H.R., L.V.P.S., L.E., J.D.B., K.A.H., I.S., C.C.d.F.-S.); and ian.small@uwa.edu.au catherine.colasdesfrancs-small@uwa.edu.au.|Institute of Biology, Humboldt-University of Berlin, 10115 Berlin, Germany (H.R.) ian.small@uwa.edu.au catherine.colasdesfrancs-small@uwa.edu.au.
authors.forename: Catherine Colas
authors.identifier: 0000-0002-6386-5672
authors.initials: CC
authors.lastname: des Francs-Small
mesh_terms: D029681:Arabidopsis Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002736:Chloroplasts / Q000378:metabolism*; D005816:Genetic Complementation Test; D007438:Introns / Q000235:genetics; D008957:Models, Genetic; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D010641:Phenotype; D018087:Plastids / Q000378:metabolism; D011485:Protein Binding; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D054679:Ribosome Subunits, Small / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D020040:Trans-Splicing / Q000235:genetics*
chemical_list: D029681:Arabidopsis Proteins; C000623000:EMB2654 protein, Arabidopsis; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C538752:pentatricopeptide repeat protein, Arabidopsis