Nanopore sequencing of internal 2′-PO4modifications installed by RNA repair

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 RNAHAC1. 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 ofHAC1mRNA 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 onHAC1and tRNAs 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 in other contexts.