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Direct detection of RNA repair by nanopore sequencing

View ORCID ProfileLaura K. White, View ORCID ProfileSaylor M. Strugar, Andrea MacFadden, View ORCID ProfileJay R. Hesselberth
doi: https://doi.org/10.1101/2022.05.29.493267
Laura K. White
1Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora CO 80045
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Saylor M. Strugar
1Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora CO 80045
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Andrea MacFadden
1Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora CO 80045
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Jay R. Hesselberth
1Department of Biochemistry and Molecular Genetics, RNA Bioscience Initiative, University of Colorado School of Medicine, Aurora CO 80045
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  • For correspondence: jay.hesselberth@cuanschutz.edu
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Abstract

Ligation by plant and fungal RNA ligases yields an internal 2′-phosphate group on each RNA ligation product. In budding yeast, this covalent mark occurs at the splice junction of two targets of ligation: intron-containing tRNAs and the messenger RNA HAC1. The repertoire of RNA molecules repaired by RNA ligation has not been explored due to a lack of unbiased approaches for identifying RNA ligation products. Here, we define several unique signals produced by 2′-phosphorylated RNAs during nanopore sequencing. A 2′-phosphate at the splice junction of HAC1 mRNA inhibits 5′→3′ degradation, enabling detection of decay intermediates in yeast RNA repair mutants by nanopore sequencing. During direct RNA sequencing, intact 2′-phosphorylated RNAs produce diagnostic changes in nanopore current properties and base calling features, including stalls produced as the modified RNA translocates through the nanopore motor protein. These approaches enable directed studies to identify novel RNA repair events from multiple species.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted May 29, 2022.
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Direct detection of RNA repair by nanopore sequencing
Laura K. White, Saylor M. Strugar, Andrea MacFadden, Jay R. Hesselberth
bioRxiv 2022.05.29.493267; doi: https://doi.org/10.1101/2022.05.29.493267
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Direct detection of RNA repair by nanopore sequencing
Laura K. White, Saylor M. Strugar, Andrea MacFadden, Jay R. Hesselberth
bioRxiv 2022.05.29.493267; doi: https://doi.org/10.1101/2022.05.29.493267

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