Fig. 6
From: Nuclear fate of yeast snoRNA is determined by co-transcriptional Rnt1 cleavage
Lack of snoRNA 5ʹprocessing affects 2'-O-methylation of rRNA. a Diagram showing 25S rRNA methylation sites and amplicon locations for RTLN-qP analysis (R1–4). b Principles of RTLN-qP technique showing that reverse transcriptase pauses and terminates upstream of modified nucleotide at low dNTP concentration. c Delay in RTLN-qP threshold cycle (Ct) of amplicons located over the 25S rRNA methylation clusters in WT versus rnt1Δ strains reflecting cDNA levels. The R2–R3 Ct were normalized to the delay of RTLN-qP reaction over non-methylated R1 giving a ΔCt value as graphically presented. Average from three independent biological replicates is shown. Error bars represent standard deviation. d Diagram illustrating DNAzyme-dependent cleavage assay. e Analysis of site-specific 18S and 25S rRNA methylation using DNAzyme-dependent assay. RNA was visualised by EtBr staining in a denaturing agarose gel. Cleavage products cA and cB are marked by asterisks. f Kinetics of snR13-dependent 25S rRNA methylation mediated by WT snR13 and 5ʹextended snR13e revealed by Northern blot of DNAzyme-dependent cleavage. Upper panels show methylene blue staining of RNA transferred on the membrane while lower panels are Northern blot using oligo probe located downstream of the snR13-dependent methylation sites. Arrows indicate 25S and 18S rRNA positions as well as products of the DNAzyme-dependent cleavage of 25S rRNA (fragments cA and cB). Quantification of the Northern blot indicating percent of cleaved mature 25S rRNA is shown below
