PT  - JOURNAL ARTICLE
AU  - Laura K. White
AU  - Saylor M. Strugar
AU  - Andrea MacFadden
AU  - Jay R. Hesselberth
TI  - Direct detection of RNA repair by nanopore sequencing
AID  - 10.1101/2022.05.29.493267
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.05.29.493267
4099  - http://biorxiv.org/content/early/2022/05/29/2022.05.29.493267.short
4100  - http://biorxiv.org/content/early/2022/05/29/2022.05.29.493267.full
AB  - 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 StatementThe authors have declared no competing interest.