The impact of mRNA structure on guide RNA targeting in kinetoplastid RNA editing
| Autor: | Larissa Reifur, Laura E. Yu, Michelle vanHartesvelt, Jorge Cruz-Reyes, Donna J. Koslowsky |
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| Rok vydání: | 2010 |
| Předmět: |
Science
Molecular Sequence Data Molecular Biology/Molecular Evolution Biology 03 medical and health sciences 0302 clinical medicine Gene expression Biochemistry/RNA Structure Guide RNA RNA Messenger Microbiology/Parasitology Nucleic acid structure Binding site Kinetoplastida Molecular Biology Cell Biology/Gene Expression 030304 developmental biology Genetics 0303 health sciences Messenger RNA Multidisciplinary Binding Sites Base Sequence Infectious Diseases/Protozoal Infections RNA Enzyme structure Cell biology Kinetics RNA editing Medicine Nucleic Acid Conformation RNA Editing 030217 neurology & neurosurgery RNA Guide Kinetoplastida Research Article |
| Zdroj: | PLoS ONE PLoS ONE, Vol 5, Iss 8, p e12235 (2010) |
| ISSN: | 1932-6203 |
| Popis: | Mitochondrial mRNA editing in Trypanosoma brucei requires the specific interaction of a guide RNA with its cognate mRNA. Hundreds of gRNAs are involved in the editing process, each needing to target their specific editing domain within the target message. We hypothesized that the structure surrounding the mRNA target may be a limiting factor and involved in the regulation process. In this study, we selected four mRNAs with distinct target structures and investigated how sequence and structure affected efficient gRNA targeting. Two of the mRNAs, including the ATPase subunit 6 and ND7-550 (5′ end of NADH dehydrogenase subunit 7) that have open, accessible anchor binding sites show very efficient gRNA targeting. Electrophoretic mobility shift assays indicate that the cognate gRNA for ND7-550 had 10-fold higher affinity for its mRNA than the A6 pair. Surface plasmon resonance studies indicate that the difference in affinity was due to a four-fold faster association rate. As expected, mRNAs with considerable structure surrounding the anchor binding sites were less accessible and had very low affinity for their cognate gRNAs. In vitro editing assays indicate that efficient pairing is crucial for gRNA directed cleavage. However, only the A6 substrate showed gRNA-directed cleavage at the correct editing site. This suggests that different gRNA/mRNA pairs may require different “sets” of accessory factors for efficient editing. By characterizing a number of different gRNA/mRNA interactions, we may be able to define a “bank” of RNA editing substrates with different putative chaperone and other co-factor requirements. This will allow the more efficient identification and characterization of transcript specific RNA editing accessory proteins. |
| Databáze: | OpenAIRE |
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