Fig. 6
From: Common mechanism of transcription termination at coding and noncoding RNA genes in fission yeast
The endonucleolytic activity of Ysh1 is necessary for termination of snoRNA transcription. a–c Normalized ChIP-seq signal of total RNAPII (Rbp1) in WT (top) and ysh1 mutant (bottom) cells across the fba1 mRNA (a), the snR99 snoRNA (b), and the snu5 snRNA (c) genes grown in rapamycin-treated minimal medium for 4 h. The dashed-line rectangles highlight transcriptional read-through at fba1 and snR99 genes in Ysh1-deficient cells. d, e Average ChIP-seq profile of RNAPII (Rpb1) in WT (solid lines) and Ysh1-deficient (dotted lines) cells across 4755 mRNA (d) and 24 monocistronic snoRNA (e) genes grown in rapamycin-treated minimal medium for 4 h. f, g RNAPII ChIP-qPCR analysis on the fba1 (f) and snR99 (g) genes using extracts prepared from either wild-type (WT) or ysh1 anchor-away (ysh1-AA) strains containing chromosomally integrated constructs that express the indicated versions of FLAG-tagged Ysh1 (WT, H403F, and H165F) as well as an empty vector (EV) control. Bars above the fba1 and snR99 genes show the positions of PCR products used for ChIP-qPCR analyses. Cells were grown in the presence of rapamycin for 4 h to deplete endogenous Ysh1 from the nucleus. ChIP signals (percent of input) were normalized to region 1. Error bars indicate SD. n = 3 biological replicates from independent cultures. h–j Northern blot analysis of fba1 (h), snR3 (i), and snR99 (j) genes using total RNA prepared from using total RNA prepared from the same WT (lane 1) and ysh1 anchor-away (ysh1-AA, lanes 2–5) strains as in f, g. The position of read-through (RT) transcripts is indicated on the right. The 18S rRNA was used as a loading control
