Mapping contacts between gRNA and mRNA in trypanosome RNA editing

All guide RNAs (gRNAs) identified to date have defined 5′ anchor sequences, guiding sequences and a nonencoded 3′ uridylate tail. The 5′ anchor is required for in vitro editing and is thought to be responsible for selection and binding to the pre-edited mRNA. Little is known, however, about how the...

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Bibliographic Details
Published in:Nucleic acids research 1999-02, Vol.27 (3), p.778-787
Main Authors: Leung, S.S, Koslowsky, D.J
Format: Article
Language:English
Subjects:
Animals
Base Sequence
binding
binding sites
Chromosome Mapping
messenger RNA
molecular conformation
Molecular Sequence Data
Nucleic Acid Conformation
Photoaffinity Labels
pre-edited messenger rna
protein secondary structure
RNA
RNA Editing
RNA, Guide - chemistry
RNA, Guide - metabolism
RNA, Messenger - chemistry
RNA, Messenger - metabolism
RNA, Protozoan - chemistry
RNA, Protozoan - metabolism
Sequence Alignment
Trypanosoma
Trypanosoma brucei
Trypanosoma brucei brucei - genetics
u tail
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Summary:All guide RNAs (gRNAs) identified to date have defined 5′ anchor sequences, guiding sequences and a nonencoded 3′ uridylate tail. The 5′ anchor is required for in vitro editing and is thought to be responsible for selection and binding to the pre-edited mRNA. Little is known, however, about how the gRNAs are used to direct RNA editing. Utilizing the photo-reactive crosslinking agent, azidophenacyl (APA), attached to the 5′- or 3′-terminus of the gRNA, we have begun to map the structural relationships between the different defined regions of the gRNA with the pre-edited mRNA. Analyses of crosslinked conjugates produced with a 5′-terminal APA group confirm that the anchor of the gRNA is correctly positioning the interacting molecules. 3′ Crosslinks (X-linker placed at the 3′-end of a U10 tail) have also been mapped for three different gRNA/mRNA pairs. In all cases, analyses indicate that the U-tail can interact with a range of nucleotides located upstream of the first edited site. It appears that the U-tail prefers purine-rich sites, close to the first few editing sites. These results suggest that the U-tail may act in concert with the anchor to melt out secondary structure in the mRNA in the immediate editing domain, possibly increasing the accessibility of the editing complex to the proper editing sites.
ISSN:0305-1048
1362-4962
DOI:10.1093/nar/27.3.778