Correlation between Dbp2p binding sites, Sen1p binding sites, and R-loops on snoRNAs. (A) Representative traces of Dbp2p iCLIP, Sen1p PAR-CLIP (Jamonnak et al. 2011), and rnh1Δrnh201ΔS1-DRIP-Seq (R-loops) (Wahba et al. 2016) for the H/ACA-box snR9. Bars for Dbp2p represent the 5′ end of the iCLIP reads. Bars for Sen1p show the nucleotide conversion in the PAR-CLIP reads. R-loops are shown as complete reads. (B) Representative traces of Dbp2p iCLIP, Sen1p PAR-CLIP (Jamonnak et al. 2011), and rnh1Δrnh201ΔS1-DRIP-Seq (Wahba et al. 2016) for the C/D-box snR40. Data are presented as in panel A. (C) Metagene plots for Dbp2p iCLIP, Sen1p PAR-CLIP, and rnh1Δrnh201ΔS1-DRIP-Seq for H/ACA-box snoRNAs (n = 28), expressed as fractions of binding per bin (Dbp2p and Sen1p) and fraction of read density per bin (R-loops). R, Pearson correlation coefficients. P-values were calculated by a two-tailed t-test. (D) Metagene plots for Dbp2p iCLIP, Sen1p PAR-CLIP, and rnh1Δrnh201ΔS1-DRIP-Seq for C/D-box snoRNAs (n = 44). Data are presented as in panel C. (E) Proximity of Sen1p binding sites and regions of R-loop formation to Dbp2p binding sites on H/ACA-box RNAs (n = 25, three transcripts without clear 3′ peaks were excluded from the analysis). Metagene plot of normalized fraction per nucleotide of Sen1p binding and R-loop formation averaged for all H/ACA-box snoRNAs, in relation to the Dbp2p binding site, indicated by the gray line at zero. The background signal per nucleotide was subtracted by randomizing binding peaks over the RNAs three times and calculating the mean. (F) Proximity of Sen1p binding sites and regions of R-loop formation to Dbp2p binding sites on C/D-box RNAs (n = 43, one transcript without clear 3′ peak was excluded from the analysis). Data are shown as in panel E. (G) Experimental scheme for Dbp2p-Sen1p coimmunoprecipitation. Measurements were performed in the presence of RNases. (H) Representative western blot for Sen1p-Ded1p co-IP. The membrane was probed with streptavidin antibody for Dbp2p-HTBH. Experiments were performed four times for biological replicates with virtually indistinguishable results.
