Direct visualisation of RNA editing within a Leishmania tarentolae mitochondrial extract.
The coding sequence within several mitochondrial mRNAs of the trypanosomatid protozoa is created through editing by the precise insertion and deletion of U nucleotides. The biochemical characterisation of the editing reaction in the Leishmania genus of the trypanosomatids has been hindered by the lack of a direct in vitro assay. We describe here the first direct assay for the detection of guide RNA-directed editing mediated by a mitochondrial extract prepared from two independent isolates of Leishmania tarentolae. The assay enabled the editing activity within a L. tarentolae mitochondrial extract to be significantly enriched and will facilitate the characterisation of the editing reaction. The results suggest that the difficulty in establishing an assay for the L. tarentolae reaction was not simply a result of the catalytic machinery being limiting but rather reflected the presence of constraints on both the guide RNA and mRNA sequences.
Oppegard
Lisa M
LM
Department of Pharmacology, University of Minnesota, 6-120 Jackson Hall, 321 Church Street S.E., Minneapolis 55455-0347, USA.
Connell
Gregory J
GJ
D000818:Animals; D001483:Base Sequence; D003573:Cytochrome b Group / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D007891:Leishmania / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000235:genetics*; D017393:RNA Editing / Q000235:genetics*; D017394:RNA, Guide, Kinetoplastida / Q000737:chemistry / Q000235:genetics*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics*; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan / Q000737:chemistry / Q000235:genetics*
D003573:Cytochrome b Group; D012333:RNA, Messenger; D000077278:RNA, Mitochondrial; D016053:RNA, Protozoan; D012313:RNA; D017394:RNA, Guide, Kinetoplastida