The impact of mRNA structure on guide RNA targeting in kinetoplastid RNA editing.

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 surroundi...

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Autores principales:	Larissa Reifur, Laura E Yu, Jorge Cruz-Reyes, Michelle Vanhartesvelt, Donna J Koslowsky
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2010
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/7424945d2c3e47e2abf1425dec09d8d0
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spelling	oai:doaj.org-article:7424945d2c3e47e2abf1425dec09d8d02021-11-18T06:35:58ZThe impact of mRNA structure on guide RNA targeting in kinetoplastid RNA editing.1932-620310.1371/journal.pone.0012235https://doaj.org/article/7424945d2c3e47e2abf1425dec09d8d02010-08-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/20808932/?tool=EBIhttps://doaj.org/toc/1932-6203Mitochondrial 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.Larissa ReifurLaura E YuJorge Cruz-ReyesMichelle VanhartesveltDonna J KoslowskyPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 5, Iss 8, p e12235 (2010)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Larissa Reifur Laura E Yu Jorge Cruz-Reyes Michelle Vanhartesvelt Donna J Koslowsky The impact of mRNA structure on guide RNA targeting in kinetoplastid RNA editing.
description	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.
format	article
author	Larissa Reifur Laura E Yu Jorge Cruz-Reyes Michelle Vanhartesvelt Donna J Koslowsky
author_facet	Larissa Reifur Laura E Yu Jorge Cruz-Reyes Michelle Vanhartesvelt Donna J Koslowsky
author_sort	Larissa Reifur
title	The impact of mRNA structure on guide RNA targeting in kinetoplastid RNA editing.
title_short	The impact of mRNA structure on guide RNA targeting in kinetoplastid RNA editing.
title_full	The impact of mRNA structure on guide RNA targeting in kinetoplastid RNA editing.
title_fullStr	The impact of mRNA structure on guide RNA targeting in kinetoplastid RNA editing.
title_full_unstemmed	The impact of mRNA structure on guide RNA targeting in kinetoplastid RNA editing.
title_sort	impact of mrna structure on guide rna targeting in kinetoplastid rna editing.
publisher	Public Library of Science (PLoS)
publishDate	2010
url	https://doaj.org/article/7424945d2c3e47e2abf1425dec09d8d0
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The impact of mRNA structure on guide RNA targeting in kinetoplastid RNA editing.

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