In vitro digestion with recombinant exonucleases reveals sites of 2′-phosphorylation. (A) Digestion of 2′-phosphorylated RNA by Xrn1 yields a product that inhibits further degradation. (B) The 2′-phosphate on tRNA-Pro-UGG can be detected by northern blotting for 3′-exon in total RNA from RNA repair strains treated with and without recombinant Xrn1 in vitro. In the lower panel, the same membrane has been stripped and reprobed for 5S rRNA as a loading control. Asterisks correspond to previously described but uncharacterized Pro-TGG splicing intermediates (Spinelli et al. 1997; Cherry et al. 2018). (C) Unmodified, 2′-phosphorylated, and 2′-O-methylated RNA substrates show varying levels of resistance to Xrn1 after a 2 h digestion and imaging on a stained denaturing gel. (D) A 2′-phosphate inhibits decay by the 5′ → 3′ exonucleases Xrn1, Dxo1, and RNase J1. Enzymatic digestion intermediates were interrogated as in C, with multiple exonucleases. The location of the 2′-phosphorylated linkage is indicated by the red “G” in the substrate at right, with inferred stalling sites for each exonuclease marked with arrows. (E) In budding yeast, the unfolded protein response (UPR) initiates a noncanonical splicing event on the mRNA HAC1 during which the endonuclease Ire1 excises the HAC1 intron (thin line), followed by exon ligation by Trl1 (black and gray boxes), generating an internal 2′-phosphate at the splice junction that is subsequently removed by the 2′-phosphotransferase Tpt1. (F) RNA from WT and xrn1Δ tpt1Δ (10×-tRNA) cells was treated with DMSO or tunicamycin (Tm) and left untreated (left panel) or enzymatically digested (right panel) followed by nanopore sequencing. 5′-end alignments of reads are expressed in counts per million reads (CPM). (G) Subtraction of undigested background 5′-end signal in F from the corresponding signal in the Tm-treated, rXrn1-digested libraries in F, yields a predominant single nucleotide peak on HAC1s at the splice junction (yellow box). At right, an enhanced view of this region shows that the major peak in wild-type cells is located precisely at the exon–exon junction (position 727), whereas the end signal in the mutant is 13 nt downstream, an offset consistent with premature termination of base calling when a bona fide 5′ RNA end exits a nanopore. (H) The wild-type rXrn1 enrichment scores above have been subtracted from the mutant scores at each nucleotide position.
