Figure 1.
Pre-rRNA processing pathways in budding yeast.
(A) Structure of the 35S pre-rRNA, showing the location of cleavage site A2. (B) Pre-rRNA processing pathways via nascent transcript cleavage (NTC) and released transcript cleavage (RTC). The points at which Rat1 functions as a 5′ exonuclease are indicated.
Table 1.
Plasmids used in this project.
Table 2.
Parameters used for modeled curves shown in Figure 2.
Table 3.
Parameters used for modeled curves shown in Figure 4.
Figure 2.
Comparison of predicted labeling curves with different levels of cotranscriptional pre-rRNA cleavage.
(A) Modeled incorporation into 35S and 20S pre-rRNA with high (70%) cotranscriptional cleavage. (B) Modeled incorporation into 35S and 20S pre-rRNA with low (30%) cotranscriptional cleavage. Purple lines represent the NTC population. Orange lines represent the RTC population. The black line is the sum of the NTC and RTC populations.
Figure 3.
Time courses of phenotypes in strains depleted for Rat1.
(A) OD600 of the cultures at the time points indicated. Non-depleted cells, growing in the absence of methionine, were maintained in exponential growth phase by frequent dilution with pre-warmed medium. Rat1-depleted cells, growing in the presence of methionine, were diluted at the same times and to the same extent as non-depleted cells. OD600 values (cell density) for the non-depleted strain at the time points indicated are shown in orange. OD600 values for the Rat1-depleted strain at the same time points are indicated in purple. Doubling time for the non-depleted strain was ∼100 min. (B) Relative abundances of 5.8S(L) (Rat1-independent) and 5.8S(S) (Rat1-dependent), with total 5.8S rRNA abundance set to 100% at each time point. Graphs show the averages of three independent experiments.
Figure 4.
Depletion of Rat1 inhibits NTC.
Kinetics of pre-rRNA labeling in PMET3::RAT1 strains carrying the plasmid expressing Rat1 (shown in purple) or the empty plasmid (shown in orange), following growth in the presence of methionine for 8 h to repress expression of chromosomal Rat1. (A) Incorporation into 35S pre-rRNA. (B) Incorporation into 20S pre-rRNA. (C) Incorporation into 27SA pre-rRNA. (D) Incorporation into 27SB pre-rRNA. The three points shown for each time point represent the values obtained from three independent experiments. Solid lines represent the modeled response using the values from Table 3. Values for Rat1-expression are shown in purple and values for Rat1-depletion in orange.
Figure 5.
Expression of catalytically inactive Rat1D235A increases 35S pre-rRNA accumulation.
Kinetics of pre-rRNA labeling in PMET::RAT1 strains carrying the empty plasmid (shown in orange) or the plasmid expressing Rat1D235A (shown in purple) following growth in the presence of methionine for 8h to repress expression of chromosomal Rat1. (A) Incorporation into 35S pre-rRNA. A higher plateau for 35S was observed in the strain expressing Rat1D235A. (B–D) Incorporation into 20S, 27SA and 27SB pre-rRNAs, respectively. Differences in labeling kinetics for 20S, 27SA and 27SB pre-rRNAs were not significant (p>0.05) as shown by a student’s T-test. The T-test determines the probability that two samples come from the same population, and was performed based on a two-tailed distribution.
Figure 6.
Miller chromatin spreads of rDNA from Rat1 depletion strains.
(A–C; left) Representative EM images showing a single rDNA gene with typical efficient NTC (A), low NTC (B), and no NTC (C). Genes are orientated with the direction of transcription from left to right. Arrows indicate examples of SSU processomes, and the bracket indicates a gene region with mostly cleaved transcripts. (A–C; right) Simplified cartoon versions of the EM images. The branching structures visible in the images are nascent pre-rRNA transcripts, emanating from the central rDNA strand. The black balls are SSU processome complexes, which assemble cotranscriptionally and compact the 5′ regions of the nascent transcript. The balls are lost from pre-rRNA transcripts at the 3′ end of the rDNA (on right of images) that have undergone NTC, as best shown in panel A. (D) Fraction of genes showing co-transcriptional cleavage. More than 75 genes were analyzed per yeast strain. In the presence of methionine, the fraction of genes with NTC decreased significantly in the empty plasmid strain over the Rat1 plasmid strain (P = 1.4×10−6), with further reduction in the Rat1D235A strain (Rat1D235A to empty plasmid P = 0.008; Rat1D235A to Rat1 wild-type P = 5.1×10−13).