Functional complementation of Trypanosoma brucei RNA in vitro editing with recombinant RNA ligase.
The approximately 20S RNA ligase-containing complex (L-complex) in trypanosomatid mitochondria interacts by means of RNA linkers with at least two other multiprotein complexes to mediate the editing of mitochondrial cryptogene transcripts. The L-complex contains approximately 16 proteins, including the two RNA-editing ligases (RELs), REL1 and REL2. Leishmania tarentolae REL1 and REL2 and Trypanosoma brucei REL1 were expressed as enzymatically active tandem affinity purification-tagged proteins in a Baculovirus system. When these proteins were added to mitochondrial lysates from T. brucei procyclic cells that were depleted of the cognate endogenous ligase by RNA interference down-regulation of expression, the added proteins were integrated into the L-complex, and, in the case of REL1, there was a complementation of in vitro-precleaved U-insertion and U-deletion editing activities of the 20S L-complex. Integration of the recombinant proteins did not occur or occurred at a very low level with noncognate ligase-depleted L-complex or with wild-type L-complex. A C-terminal region of the T. brucei recombinant REL1 downstream of the catalytic domain was identified as being involved in integration into the L-complex. The ability to perform functional complementation in vitro provides a powerful tool for molecular dissection of the editing reaction.
Gao
Guanghan
G
Department of Microbiology, Immunology, and Molecular Genetics, University of California, Los Angeles, CA 90095, USA.
Simpson
Agda M
AM
Kang
Xuedong
X
Rogers
Kestrel
K
Nebohacova
Martina
M
Li
Feng
F
Simpson
Larry
L
D000818:Animals; D016367:Baculoviridae; D019729:Carbon-Oxygen Ligases / Q000378:metabolism*; D004586:Electrophoresis; D004926:Escherichia coli; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D007891:Leishmania / Q000378:metabolism*; D008025:Ligases; D008928:Mitochondria / Q000378:metabolism* / Q000502:physiology; D024101:Mitochondrial Proteins / Q000378:metabolism*; D009841:Oligonucleotides; D017393:RNA Editing / Q000502:physiology*; D034622:RNA Interference; D011994:Recombinant Proteins / Q000378:metabolism*; D014346:Trypanosoma brucei brucei / Q000378:metabolism* / Q000502:physiology
D024101:Mitochondrial Proteins; D009841:Oligonucleotides; D011994:Recombinant Proteins; D008025:Ligases; D019729:Carbon-Oxygen Ligases; C476580:REL1 RNA ligase, Trypanosoma brucei