Figure 1.
Nuclear depletion of Pol II termination factors.
Live yeast cell imaging of: A. NRD1-FRBGFP, RPB3-TAGRFP; B. YSH1-FRBGFP, RPB3-TAGRFP; C. GALpSEN1-FRBGFP, RPB3-TAGRFP strains. Time after rap treatment is indicated above the pictures. D. Growth of indicated FRB strains on CSM plates with and without rap.
Figure 2.
Ysh1 depletion causes readthrough at pA sites.
A. Mean reads every 10 bp for the 500 most frequently used pA sites with (dotted) and without (solid) Ysh1. B. Plot showing percent readthrough of each of 500 pA terminators as a fraction of the total 500 pA terminators. C and D. Histograms representing normalized reads with Ysh1 (grey) and without Ysh1 (black) at the given genomic locations. The TSS with the direction of transcription is indicated by an arrow. Genes and pA sites are represented below each graph and the length of the genome depicted is given in the lower right hand corner.
Figure 3.
Mapping Nrd1-dependent terminators.
A. Histograms representing of normalized reads for NRD1-FRB with Nrd1 (grey) and without Nrd1 (black) at the given genomic locations. The difference between WT and Nrd1-depleted at every nucleotide is represented as a histogram below each graph. B. Mean reads every 10 bp for 49 snoRNA termination sites showing the highest level of readthrough and 144 CUT termination sites with Nrd1 (dotted) and without Nrd1 (solid). C. Most abundant Nrd1 motif and the most abundant Nab3 motif in the 150 nt 5′ of the snoRNA and CUT terminators, respectively. MEME input parameters were any number of motifs of length 4–15 nt. Results are presented using WebLogo [92].
Figure 4.
NET-Seq data from Churchman and Weissman [53] are shown above PAR-CLIP data from the SNR13-TRS31 locus. The number of NET-seq reads from peaks of Pol II are shown above the lines. The asterisk indicates reads derived from mature snR13 RNA that contaminates the NET-seq library. The calculated SNR13 termination region has been expanded to show U-rich sequences (red) surrounding the termination point. The sequences of several antisense CUT termination regions are shown below the SNR13 sequence.
Figure 5.
Nrd1 increases the stability of the readthrough transcripts.
A. Northern blot and qPCR at different times after addition of rap using the amplicon highlighted in red. B. Histograms representing normalized reads with Nrd1 (grey) and without Nrd1 (black) at the given genomic locations. The Y-axis has been changed to emphasize the differences between treatment with (black) and without rap (grey) for the SNR13-TRS31 locus. C. Similar to A but Schultz et. al. [65] 4tU-seq data is also represented in as a histogram below each graph for the same region of the genome with Nrd1 (grey) and without Nrd1 (black).
Figure 6.
Sen1 depletion causes readthrough at non-pA terminators.
A.–C. Mean reads every 10 bp for the top 500 polyA,144 calculated CUT termination sites, and 49 snoRNA termination sites, with Sen1 (dotted) and without Sen1 (solid). D. Histograms representing normalized reads with Sen1 (grey) and without Sen1 (black) at the given genomic locations. The same region is also represented in as a histogram below each graph with Nrd1 (grey) and without Nrd1 (black). E. Percent readthrough of non-pA terminators (ordered from highest to lowest) represented as a fraction of the total number of calculated non-pA terminators. SEN1-FRB is represented as diamonds and NRD1-FRB is represented as circles.
Figure 7.
Schematic representation of Pol II termination after removal of non-pA and pA termination factors.
Elongating Pol II (green) terminates pA transcripts (A) after an allosteric change (red) that reduces processivity. (B) Depletion of Ysh1 leads to minimally extended readthrough transcripts but does not block the allosteric change in Pol II. (C) Nrd1 and Nab3 binding recruit Sen1 for termination of non-pA transcripts. (D) Pol II elongation complex lacking Nrd1 does not recognize termination sequences in the nascent transcript and thus does not facilitate the allosteric transition in Pol II. This leads to processive readthrough. (E) Nrd1 and Nab3 recognize terminator sequences allowing the allosteric change in Pol II but depletion of Sen1 blocks removal of Pol II from the template.