Previous Article | Next Article 
Molecular and Cellular Biology, February 2001, p. 884-892, Vol. 21, No. 3
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.3.884-892.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Trypanosome RNA Editing: Simple Guide RNA Features
Enhance U Deletion 100-Fold
Jorge
Cruz-Reyes,
Alevtina
Zhelonkina,
Laura
Rusche,
and
Barbara
Sollner-Webb*
Department of Biological Chemistry, The Johns
Hopkins University School of Medicine, Baltimore, Maryland 21205
Received 21 July 2000/Returned for modification 13 September
2000/Accepted 7 November 2000
Trypanosome RNA editing is a massive processing of mRNA by U
deletion and U insertion, directed by trans-acting guide
RNAs (gRNAs). A U deletion cycle and a U insertion cycle have been reproduced in vitro using synthetic ATPase (A6)
pre-mRNA and gRNA. Here we examine which gRNA features are
important for this U deletion. We find that, foremost, this editing
depends critically on the single-stranded character of a few gRNA
and a few mRNA residues abutting the anchor duplex, a feature not
previously appreciated. That plus any base-pairing sequence to tether
the upstream mRNA are all the gRNA needs to direct unexpectedly
efficient in vitro U deletion, using either the purified editing
complex or whole extract. In fact, our optimized gRNA constructs
support faithful U deletion up to 100 times more efficiently than the
natural gRNA, and they can edit the majority of mRNA
molecules. This is a marked improvement of in vitro U deletion, in
which previous artificial gRNAs were no more active than
natural gRNA and the editing efficiencies were at most a few
percent. Furthermore, this editing is not stimulated by most other
previously noted gRNA features, including its potential ligation
bridge, 3' OH moiety, any U residues in the tether, the conserved
structure of the central region, or proteins that normally bind these
regions. Our data also have implications about evolutionary forces
active in RNA editing.
*
Corresponding author. Mailing address: Department of
Biological Chemistry, The Johns Hopkins University School of Medicine, 725 North Wolfe St., Baltimore, MD 21205. Phone: (410) 955-6278. Fax:
(410) 955-0192. E-mail: bsw{at}jhmi.edu.

Present address: Department of Molecular and Cell Biology,
University of California at Berkeley, Berkeley, CA
94720.
Molecular and Cellular Biology, February 2001, p. 884-892, Vol. 21, No. 3
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.3.884-892.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Guo, X., Ernst, N. L., Stuart, K. D.
(2008). The KREPA3 Zinc Finger Motifs and OB-Fold Domain Are Essential for RNA Editing and Survival of Trypanosoma brucei. Mol. Cell. Biol.
28: 6939-6953
[Abstract]
[Full Text]
-
Reifur, L., Koslowsky, D. J.
(2008). Trypanosoma brucei ATPase subunit 6 mRNA bound to gA6-14 forms a conserved three-helical structure. RNA
14: 2195-2211
[Abstract]
[Full Text]
-
Alatortsev, V. S., Cruz-Reyes, J., Zhelonkina, A. G., Sollner-Webb, B.
(2008). Trypanosoma brucei RNA Editing: Coupled Cycles of U Deletion Reveal Processive Activity of the Editing Complex. Mol. Cell. Biol.
28: 2437-2445
[Abstract]
[Full Text]
-
Carnes, J., Trotter, J. R., Peltan, A., Fleck, M., Stuart, K.
(2008). RNA Editing in Trypanosoma brucei Requires Three Different Editosomes. Mol. Cell. Biol.
28: 122-130
[Abstract]
[Full Text]
-
Babbarwal, V. K., Fleck, M., Ernst, N. L., Schnaufer, A., Stuart, K.
(2007). An essential role of KREPB4 in RNA editing and structural integrity of the editosome in Trypanosoma brucei. RNA
13: 737-744
[Abstract]
[Full Text]
-
Cifuentes-Rojas, C., Pavia, P., Hernandez, A., Osterwisch, D., Puerta, C., Cruz-Reyes, J.
(2007). Substrate Determinants for RNA Editing and Editing Complex Interactions at a Site for Full-round U Insertion. J. Biol. Chem.
282: 4265-4276
[Abstract]
[Full Text]
-
Law, J. A., O'Hearn, S., Sollner-Webb, B.
(2007). In Trypanosoma brucei RNA Editing, TbMP18 (Band VII) Is Critical for Editosome Integrity and for both Insertional and Deletional Cleavages. Mol. Cell. Biol.
27: 777-787
[Abstract]
[Full Text]
-
Kang, X., Gao, G., Rogers, K., Falick, A. M., Zhou, S., Simpson, L.
(2006). Reconstitution of full-round uridine-deletion RNA editing with three recombinant proteins. Proc. Natl. Acad. Sci. USA
103: 13944-13949
[Abstract]
[Full Text]
-
Sacharidou, A., Cifuentes-Rojas, C., Halbig, K., Hernandez, A., Dangott, L. J., De Nova-Ocampo, M., Cruz-Reyes, J.
(2006). RNA editing complex interactions with a site for full-round U deletion in Trypanosoma brucei. RNA
12: 1219-1228
[Abstract]
[Full Text]
-
Miller, M. M., Halbig, K., Cruz-Reyes, J., Read, L. K.
(2006). RBP16 stimulates trypanosome RNA editing in vitro at an early step in the editing reaction. RNA
12: 1292-1303
[Abstract]
[Full Text]
-
Panigrahi, A. K., Ernst, N. L., Domingo, G. J., Fleck, M., Salavati, R., Stuart, K. D.
(2006). Compositionally and functionally distinct editosomes in Trypanosoma brucei. RNA
12: 1038-1049
[Abstract]
[Full Text]
-
Yu, L. E., Koslowsky, D. J.
(2006). Interactions of mRNAs and gRNAs involved in trypanosome mitochondrial RNA editing: Structure probing of a gRNA bound to its cognate mRNA. RNA
12: 1050-1060
[Abstract]
[Full Text]
-
ZHELONKINA, A. G., O'HEARN, S. F., LAW, J. A., CRUZ-REYES, J., HUANG, C. E., ALATORTSEV, V. S., SOLLNER-WEBB, B.
(2006). T. brucei RNA editing: Action of the U-insertional TUTase within a U-deletion cycle.. RNA
12: 476-487
[Abstract]
[Full Text]
-
Cifuentes-Rojas, C., Halbig, K., Sacharidou, A., De Nova-Ocampo, M., Cruz-Reyes, J.
(2005). Minimal pre-mRNA substrates with natural and converted sites for full-round U insertion and U deletion RNA editing in trypanosomes. Nucleic Acids Res
33: 6610-6620
[Abstract]
[Full Text]
-
Carnes, J., Trotter, J. R., Ernst, N. L., Steinberg, A., Stuart, K.
(2005). An essential RNase III insertion editing endonuclease in Trypanosoma brucei. Proc. Natl. Acad. Sci. USA
102: 16614-16619
[Abstract]
[Full Text]
-
Law, J. A., Huang, C. E., O'Hearn, S. F., Sollner-Webb, B.
(2005). In Trypanosoma brucei RNA Editing, Band II Enables Recognition Specifically at Each Step of the U Insertion Cycle. Mol. Cell. Biol.
25: 2785-2794
[Abstract]
[Full Text]
-
Vondruskova, E., van den Burg, J., Zikova, A., Ernst, N. L., Stuart, K., Benne, R., Lukes, J.
(2005). RNA Interference Analyses Suggest a Transcript-specific Regulatory Role for Mitochondrial RNA-binding Proteins MRP1 and MRP2 in RNA Editing and Other RNA Processing in Trypanosoma brucei. J. Biol. Chem.
280: 2429-2438
[Abstract]
[Full Text]
-
GOLDEN, D. E., HAJDUK, S. L.
(2005). The 3'-untranslated region of cytochrome oxidase II mRNA functions in RNA editing of African trypanosomes exclusively as a cis guide RNA. RNA
11: 29-37
[Abstract]
[Full Text]
-
HALBIG, K., DE NOVA-OCAMPO, M., CRUZ-REYES, J.
(2004). Complete cycles of bloodstream trypanosome RNA editing in vitro. RNA
10: 914-920
[Abstract]
[Full Text]
-
SIMPSON, L., APHASIZHEV, R., GAO, G., KANG, X.
(2004). Mitochondrial proteins and complexes in Leishmania and Trypanosoma involved in U-insertion/deletion RNA editing. RNA
10: 159-170
[Abstract]
[Full Text]
-
Oppegard, L. M., Hillestad, M., McCarthy, R. T., Pai, R. D., Connell, G. J.
(2003). Cis-acting Elements Stimulating Kinetoplastid Guide RNA-directed Editing. J. Biol. Chem.
278: 51167-51175
[Abstract]
[Full Text]
-
O'Hearn, S. F., Huang, C. E., Hemann, M., Zhelonkina, A., Sollner-Webb, B.
(2003). Trypanosoma brucei RNA Editing Complex: Band II Is Structurally Critical and Maintains Band V Ligase, Which Is Nonessential. Mol. Cell. Biol.
23: 7909-7919
[Abstract]
[Full Text]
-
Domingo, G. J., Palazzo, S. S., Wang, B., Pannicucci, B., Salavati, R., Stuart, K. D.
(2003). Dyskinetoplastic Trypanosoma brucei Contains Functional Editing Complexes. Eukaryot Cell
2: 569-577
[Abstract]
[Full Text]
-
Wang, B., Ernst, N. L., Palazzo, S. S., Panigrahi, A. K., Salavati, R., Stuart, K.
(2003). TbMP44 Is Essential for RNA Editing and Structural Integrity of the Editosome in Trypanosoma brucei. Eukaryot Cell
2: 578-587
[Abstract]
[Full Text]
-
Ogbadoyi, E. O., Robinson, D. R., Gull, K.
(2003). A High-Order Trans-Membrane Structural Linkage Is Responsible for Mitochondrial Genome Positioning and Segregation by Flagellar Basal Bodies in Trypanosomes. Mol. Biol. Cell
14: 1769-1779
[Abstract]
[Full Text]
-
PAI, R. D., OPPEGARD, L. M., CONNELL, G. J.
(2003). Sequence and structural requirements for optimal guide RNA-directed insertional editing within Leishmania tarentolae. RNA
9: 469-483
[Abstract]
[Full Text]
-
SIMPSON, L., SBICEGO, S., APHASIZHEV, R.
(2003). Uridine insertion/deletion RNA editing in trypanosome mitochondria: A complex business. RNA
9: 265-276
[Abstract]
[Full Text]
-
Cruz-Reyes, J., Zhelonkina, A. G., Huang, C. E., Sollner-Webb, B.
(2002). Distinct Functions of Two RNA Ligases in Active Trypanosoma brucei RNA Editing Complexes. Mol. Cell. Biol.
22: 4652-4660
[Abstract]
[Full Text]
-
Huang, C. E., O'Hearn, S. F., Sollner-Webb, B.
(2002). Assembly and Function of the RNA Editing Complex in Trypanosoma brucei Requires Band III Protein. Mol. Cell. Biol.
22: 3194-3203
[Abstract]
[Full Text]
-
Igo, R. P. Jr., Lawson, S. D., Stuart, K.
(2002). RNA Sequence and Base Pairing Effects on Insertion Editing in Trypanosoma brucei. Mol. Cell. Biol.
22: 1567-1576
[Abstract]
[Full Text]
-
Igo, R. P. Jr., Weston, D. S., Ernst, N. L., Panigrahi, A. K., Salavati, R., Stuart, K.
(2002). Role of Uridylate-Specific Exoribonuclease Activity in Trypanosoma brucei RNA Editing. Eukaryot Cell
1: 112-118
[Abstract]
[Full Text]
-
Kabb, A. L., Oppegard, L. M., McKenzie, B. A., Connell, G. J.
(2001). A mRNA determinant of gRNA-directed kinetoplastid editing. Nucleic Acids Res
29: 2575-2580
[Abstract]
[Full Text]