Functional Complementation of Trypanosoma brucei RNA in vitro Editing with Recombinant RNA Ligase

The ≈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 ≈16 proteins, including the two RNA-editing ligase...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 102; no. 13; pp. 4712 - 4717
Main Authors Gao, Guanghan, Simpson, Agda M., Kang, Xuedong, Rogers, Kestrel, Nebohacova, Martina, Li, Feng, Simpson, Larry, Borst, P.
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 29.03.2005
National Acad Sciences
Subjects
Animals
Baculoviridae
Baculovirus
Biological Sciences
Carbon-Oxygen Ligases - metabolism
Complementation
Down regulation
Electrophoresis
Enzymes
Escherichia coli
Gels
Genetic Complementation Test
Genetic recombination
Genetic Vectors
Leishmania - metabolism
Leishmania tarentolae
Ligases
Ligation
Mitochondria
Mitochondria - metabolism
Mitochondria - physiology
Mitochondrial DNA
Mitochondrial Proteins - metabolism
Oligonucleotides
Proteins
Recombinant proteins
Recombinant Proteins - metabolism
Ribonucleic acid
RNA
RNA editing
RNA Editing - physiology
RNA Interference
Trypanosoma brucei
Trypanosoma brucei brucei - metabolism
Trypanosoma brucei brucei - physiology
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Summary:The ≈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 ≈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.
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This paper was submitted directly (Track II) to the PNAS office.
Author contributions: G.G. and L.S. designed research; G.G., A.M.S., X.K., K.R., M.N., and F.L. performed research; A.M.S., and M.N. contributed new reagents/analytic tools; G.G., A.M.S., and L.S. analyzed data; and G.G. and L.S. wrote the paper.
Edited by P. Borst, The Netherlands Cancer Institute, Amsterdam, The Netherlands, and approved February 16, 2005
Abbreviations: TUTase, terminal uridylyl transferase; RET, RNA-editing TUTase; REL, RNA-editing ligase; rREL, recombinant REL; TAP, tandem affinity purification; L-complex, ligase-containing complex; Lt, Leishmania tarentolae; Tb, Trypanosoma brucei; RNAi, RNA interference; CBP, calmodulin-binding peptide; brRNA, bridge RNA.
To whom correspondence should be addressed at: Department of Microbiology, Immunology, and Molecular Genetics, University of California, 6780 MRL, 675 Charles E. Young Drive South, Los Angeles, CA 90095. E-mail: simpson@kdna.ucla.edu.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0500553102