Reconstitution of Uridine-Deletion Precleaved RNA Editing with Two Recombinant Enzymes

Uridine insertion/deletion RNA editing in trypanosomatid mitochondria is a posttranscriptional RNA modification phenomenon required for translation of mitochondrial mRNAs. This process involves guide RNA-mediated cleavage at a specific site, insertion or deletion of Us from the 3′ end of the 5′ mRNA...

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Published inProceedings of the National Academy of Sciences - PNAS Vol. 102; no. 4; pp. 1017 - 1022
Main Authors Kang, Xuedong, Rogers, Kestrel, Gao, Guanghan, Falick, Arnold M., Zhou, Sharleen, Simpson, Larry, Eisenberg, David S.
Format Journal Article
LanguageEnglish
Published United States National Academy of Sciences 25.01.2005
National Acad Sciences
Subjects
Animals
Biochemistry
Biological Sciences
Carbon-Oxygen Ligases - physiology
Chemical compounds
Down regulation
Enzymes
Gels
Genetic vectors
Guide RNA
Leishmania major
Ligation
Messenger RNA
Mitochondrial Proteins - physiology
Proteins
Recombinant Proteins - pharmacology
Ribonucleic acid
RNA
RNA Editing
Trypanosoma brucei
Trypanosoma brucei brucei - genetics
Uridine - metabolism
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Summary:Uridine insertion/deletion RNA editing in trypanosomatid mitochondria is a posttranscriptional RNA modification phenomenon required for translation of mitochondrial mRNAs. This process involves guide RNA-mediated cleavage at a specific site, insertion or deletion of Us from the 3′ end of the 5′ mRNA fragment, and ligation of the two mRNA fragments. The Leishmania major RNA ligase-containing complex protein 2 expressed in insect cells has a 3′-5′ exoribonuclease activity and was therefore renamed RNA editing exonuclease 1 (REX1). Recombinant REX1 specifically trims 3′ overhanging Us and stops at a duplex region. Evidence is presented that REX1 is responsible for deletion of the 3′ overhanging Us from the bridged mRNA 5′ cleavage fragment and that RNA editing ligase 1 is responsible for the ligation of the two mRNA cleavage fragments in U-deletion editing. The evidence involves both in vivo down-regulation of REX1 expression in Trypanosoma brucei by RNA interference and the reconstitution of precleaved U-deletion in vitro editing with only two recombinant enzymes: recombinant REX1 and recombinant RNA editing ligase 1.
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Communicated by David S. Eisenberg, University of California, Los Angeles, CA, December 13, 2004
Abbreviations: gRNA, guide RNA; L-complex, RNA ligase-containing complex; REL1/2, RNA editing ligase 1/2; TUTase, terminal uridylyltransferase; RET1/2, RNA editing 3′ TUTase 1/2; REX1/2, RNA editing exonuclease 1/2; r, recombinant; TAP, tandem affinity purification; CBP, calmodulin binding protein; RNAi, RNA interference; PPi, pyrophosphate.
Author contributions: X.K., K.R., and L.S. designed research; X.K., K.R., G.G., A.M.F., and S.Z. performed research; X.K., G.G., A.M.F., and S.Z. contributed new reagents/analytic tools; X.K., K.R., G.G., A.M.F., and L.S. analyzed data; and L.S. wrote the paper.
To whom correspondence should be addressed. E-mail: simpson@kdna.ucla.edu.
ISSN:0027-8424
1091-6490
DOI:10.1073/pnas.0409275102