Termination of the C/D box small nucleolar RNA snR13 transcript does not require Rrp6.

<p>(A) Graphical representation of strand-specific RNA-seq reads mapped to snR13-<i>YDR473C</i> region. Reads mapped to the positive strand are on top in red, while reads mapped to the negative strand are on the bottom in blue. The location and direction of transcription for all analyzed annotations are diagrammed below the graphs to scale. Processed length of snRNAs and mRNAs are in black, snRNA-<u>e</u>xtended <u>t</u>ranscripts, including pre-snRNAs and termination read-through products, are in green (labeled “ETs”), NUTs are in aqua, and arrows indicate direction of transcription. The dotted black line marks the transcription start site (TSS) of <i>TRS31</i>. (B) Rpb3-FLAG localization as determined by ChIP-exo sequencing reads mapped to the same region and aligned to (A). Wild-type normalized read counts are in black, and <i>rrp6Δ</i> are in orange. Nrd1 binding sites as determined by PAR-CLIP from Creamer <i>et al</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004999#pgen.1004999.ref039" target="_blank">39</a>] are shown for comparison in aqua. (C) Average normalized read counts ± standard deviations for significantly altered transcripts in <i>rrp6Δ</i> versus wild-type (n = 4). Two stars represent a p-value of <0.01 as determined by an unpaired, two-tailed student’s t-test. The colors of the bars in each graph correspond to the color representing the related annotation. (D) Strand-specific northern blot analysis using a 5’ end labeled DNA oligonucleotide probe specific to the processed region of snR13 directly comparing <i>rrp6Δ</i> to mutants known to be defective in Nrd1-dependent termination. The temperature sensitive <i>nrd1Δ151–214</i> strain was grown at 30°C overnight, diluted to an OD₆₀₀ of 0.5, and grown at 37°C for 30 minutes or 60 minutes as indicated. The <i>ssu72 TOV</i> strain has been previously shown by Loya et al. [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004999#pgen.1004999.ref060" target="_blank">60</a>] to bypass Nrd1-dependent termination at the <i>IMD2</i> locus. The 26S and 18S ribosomal RNAs are shown as a loading control (bottom).