title: RRP6 from Trypanosoma brucei: crystal structure of the catalytic domain, association with EAP3 and activity towards structured and non-structured RNA substrates.
abstract: RRP6 is a 3'-5' exoribonuclease associated to the eukaryotic exosome, a multiprotein complex essential for various RNA processing and degradation pathways. In Trypanosoma brucei, RRP6 associates with the exosome in stoichiometric amounts and was localized in both cytoplasm and nucleus, in contrast to yeast Rrp6 which is exclusively nuclear. Here we report the biochemical and structural characterization of T. brucei RRP6 (TbRRP6) and its interaction with the so-called T. brucei Exosome Associated Protein 3 (TbEAP3), a potential orthologue of the yeast Rrp6 interacting protein, Rrp47. Recombinant TbEAP3 is a thermo stable homodimer in solution, however it forms a heterodimeric complex with TbRRP6 with 1∶1 stoichiometry. The crystallographic structure of the TbRRP6 catalytic core exposes for the first time the native catalytic site of this RNase and also reveals a disulfide bond linking two helices of the HRDC domain. RNA degradation assays show the distributive exoribonuclease activity of TbRRP6 and novel findings regarding the structural range of its RNA substrates. TbRRP6 was able to degrade single and double-stranded RNAs and also RNA substrates containing stem-loops including those with 3' stem-loop lacking single-stranded extensions. Finally, association with TbEAP3 did not significantly interfere with the TbRRP6 catalytic activity in vitro.
authors.affiliation: Synchrotron SOLEIL, Gif-sur Yvette, France.
authors.forename: Rosicler L
authors.initials: RL
authors.lastname: Barbosa
authors.affiliation: Synchrotron SOLEIL, Gif-sur Yvette, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Legrand
authors.affiliation: Synchrotron SOLEIL, Gif-sur Yvette, France.
authors.forename: Frank
authors.initials: F
authors.lastname: Wien
authors.affiliation: Synchrotron SOLEIL, Gif-sur Yvette, France.
authors.forename: Blandine
authors.initials: B
authors.lastname: Pineau
authors.affiliation: Synchrotron SOLEIL, Gif-sur Yvette, France.
authors.forename: Andrew
authors.initials: A
authors.lastname: Thompson
authors.affiliation: Synchrotron SOLEIL, Gif-sur Yvette, France.
authors.forename: Beatriz G
authors.initials: BG
authors.lastname: Guimarães
mesh_terms: D001483:Base Sequence; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003001:Cloning, Molecular; D024202:Electrophoretic Mobility Shift Assay; D013058:Mass Spectrometry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000235:genetics; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000737:chemistry* / Q000235:genetics; D016415:Sequence Alignment; D014346:Trypanosoma brucei brucei / Q000737:chemistry*
chemical_list: D009841:Oligonucleotides; D015800:Protozoan Proteins