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Interactions of mRNAs and gRNAs involved in trypanosome mitochondrial RNA editing: Structure probing of an mRNA bound to its cognate gRNA

Published online by Cambridge University Press:  11 January 2002

SHELDON S. LEUNG
Affiliation:
Interdepartmental Graduate Program in Genetics, Michigan State University, East Lansing, Michigan 48824, USA
DONNA J. KOSLOWSKY
Affiliation:
Interdepartmental Graduate Program in Genetics, Michigan State University, East Lansing, Michigan 48824, USA Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, Michigan 48824, USA
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Abstract

Posttranscriptional editing of trypanosome mitochondrial messenger RNA is directed by small guide RNAs (gRNAs). Using crosslinking techniques, we have previously shown that the gRNA base pairs to the mRNA via a 5′ anchor, whereas its 3′ U-tail interacts with upstream purine-rich mRNA sequences. The incorporation of crosslinking data into RNA folding programs produced similar structure predictions for all gRNA/mRNA pairs examined. This suggests that gRNA/mRNA pairs can form common secondary structure motifs that may be important for recognition by the editing complex. In this study, the structure of CYb mRNA crosslinked to gCYb-558 was examined using solution-probing techniques. The mRNA/gRNA crosslinked molecules are efficient substrates for gRNA-directed cleavage. In addition, when the cleavage assay is performed in the presence or absence of additional UTP, the activities of both the U-specific exonuclease and terminal uridylyl transferase (tutase) can be detected. These results indicate that a partial editing complex can assemble and function on these substrates suggesting that the crosslink captured the molecules in a biologically relevant interaction. The structure probing data directly show that the U-tail protects several mRNA bases predicted to be involved in the U-tail-mRNA duplex. In combination with our previous studies, these new data provide additional support for the predicted secondary structure of interacting gRNA/mRNA pairs.

Type
Research Article
Information
RNA , Volume 7 , Issue 12 , December 2001 , pp. 1803 - 1816
Copyright
2001 RNA Society

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