title: Preferential interaction of a 25kDa protein with an A6 pre-mRNA substrate for RNA editing in Trypanosoma brucei.
abstract: Mitochondrial gene expression in kinetoplastids is controlled after transcription, potentially at the levels of RNA maturation, stability and translation. Among these processes, RNA editing by U-insertion/deletion catalysed by multi-subunit editing complexes is best characterised at the molecular level. Nevertheless, mitochondrial RNA metabolism overall remains poorly understood, including the potential regulatory factors that may interact with the relevant catalytic molecular machines and/or RNA substrates. Here we report on a approximately 25kDa polypeptide in mitochondrial extracts that exhibits a preferential "zero-distance" photo-crosslinking interaction with an A6 pre-mRNA model substrate for RNA editing containing a single [(32)P] at the first editing site. The approximately 25kDa polypeptide purified away from editosomes upon ion-exchange chromatography and glycerol gradient sedimentation. Competition assays with homologous and heterologous transcripts suggest that the preferential recognition of the A6 substrate is based on relatively low-specificity RNA-protein contacts. Our mapping and substrate truncation analyses suggest that the crosslinking activity primarily targeted a predicted stem-loop region containing the first editing sites. Consistent with the notion that pre-mRNA folding may be required, pre-annealing with guide RNA abolished crosslinking. Interestingly, this preferential protein interaction with the A6 substrate seemed to require adenosine 5'-triphosphate but not hydrolysis. As in other biological systems, fine regulation in vivo may be brought about by transient networks of relatively low-specificity interactions in which multiple auxiliary factors bind to mRNAs and/or editing complexes in unique higher-order assemblies.
authors.affiliation: Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, TX 77843, USA.
authors.forename: Kari
authors.initials: K
authors.lastname: Halbig
authors.forename: Anastasia
authors.initials: A
authors.lastname: Sacharidou
authors.forename: Monica
authors.initials: M
authors.lastname: De Nova-Ocampo
authors.forename: Jorge
authors.initials: J
authors.lastname: Cruz-Reyes
mesh_terms: D000255:Adenosine Triphosphate / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D003432:Cross-Linking Reagents; D005786:Gene Expression Regulation / Q000235:genetics; D008928:Mitochondria / Q000235:genetics; D024101:Mitochondrial Proteins / Q000235:genetics; D008970:Molecular Weight; D009690:Nucleic Acid Conformation; D015800:Protozoan Proteins / Q000235:genetics*; D012313:RNA / Q000235:genetics; D017393:RNA Editing / Q000235:genetics*; D012322:RNA Precursors / Q000235:genetics*; D017394:RNA, Guide, Kinetoplastida / Q000235:genetics; D012332:RNA, Heterogeneous Nuclear / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan / Q000235:genetics*; D014346:Trypanosoma brucei brucei / Q000235:genetics*
chemical_list: D003432:Cross-Linking Reagents; D024101:Mitochondrial Proteins; D015800:Protozoan Proteins; D012322:RNA Precursors; D012332:RNA, Heterogeneous Nuclear; D012333:RNA, Messenger; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan; D012313:RNA; D000255:Adenosine Triphosphate; D017394:RNA, Guide, Kinetoplastida