biostudies-literatureWhite LKRNA Bioscience Initiativethe Molecular Biology Program, the Bolie Family FoundationNational Institutes of HealthNIGMS NIH HHS847-861https://www.ebi.ac.uk/biostudies/studies/S-EPMC10187680biostudies-literatureUnknown29(6)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 <i>HAC1</i> 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 <i>HAC1</i> 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 on <i>HAC1</i> and tRNAs produce diagnoRNA (New York, N.Y.)Nanopore sequencing of internal 2'-PO₄ modifications installed by RNA repair.PMC10187680T32 GM136444R35 GM119550Strugar SMWhite LKMacFadden AHesselberth JRfalseNanopore sequencing of internal 2'-PO₄ modifications installed by RNA repair.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 <i>HAC1</i> 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 <i>HAC1</i> 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 on <i>HAC1</i> and tRNAs produce diagno2023-01-01T00:00:00Z2023 Jun2026-07-15T20:04:04.692Z2026-07-09T03:10:14.148ZS-EPMC101876803685460810.1261/rna.079290.122