Co-transcriptional RNA cleavage provides a failsafe termination mechanism for yeast RNA polymerase I

Abstract

Ribosomal RNA, transcribed by RNA polymerase (Pol) I, accounts for most cellular RNA. Since Pol I transcribes rDNA repeats with high processivity and polymerase density, transcription termination is a critical process. Early in vitro studies proposed polymerase pausing by Reb1 and transcript release at the T-rich element T1 determined transcription termination. However recent in vivo studies revealed a 'torpedo' mechanism for Pol I termination: co-transcriptional RNA cleavage by Rnt1 provides an entry site for the 5'-3' exonuclease Rat1 that degrades Pol I-associated transcripts destabilizing the transcription complex. Significantly Rnt1 inactivation in vivo reveals a second co-transcriptional RNA cleavage event at T1 which provides Pol I with an alternative termination pathway. An intact Reb1-binding site is also required for Rnt1-independent termination. Consequently our results reconcile the original Reb1-mediated termination pathway as part of a failsafe mechanism for this essential transcription process.

Figure 1.

3′-extended 25S rRNA is produced in rnt1Δ cells. (A) Schematic of a S. cerevisiae rDNA repeat. In addition to the sequence encoding 18S, 5.8S and 25S rRNA (gray rectangles), the Pol I transcription unit includes External and Internal Transcribed Sequences (ETS and ITS); the 35S primary transcript is shown as a dashed line. Gray ovals represent binding sites for Reb1, triangle Rnt1 cleavage site and vertical arrows denote the T-rich elements of the terminator. 5S rDNA, transcribed by Pol III in opposite orientation, and Autonomously Replicating Sequence (ARS) are shown. The 3′-labeled probe used in S1 protection and size of the expected bands are indicated below. (B) S1 protection on total RNA from rpa12Δ, rnt1Δ and isogenic WT. S1+ and S1− controls show the probe alone after incubation with or without S1 nuclease. Arrows on the right indicate the position of mature 25S rRNA and transcripts extending to T1 or T2 terminator elements. Longer exposure is shown in the right hand panel.

Figure 2.

Co-transcriptional RNA cleavage maps at the T1 element of the terminator. (A) Schematic of a rDNA repeat, as in Figure 1A, showing the position of the single stranded M13 DNA probes (2–7) used for the TRO assay (thin lines) and of the biotinylated probe used for transcripts hybrid selection (thick line). (B) Left: Hybrid Selection-TRO assay performed in WT and rnt1Δ. Transcription was performed in permeabilized cells in presence of α-³²P-UTP; the extracted RNA was aliquoted in two parts and hybridized to the filter directly (Total) or after fractionation with streptavidin-coated beads and the biotinylated probe shown in A (Selected and Supernatant). M = M13 (negative control), A = Actin (positive control). Right: quantitation of the experiment shown on the left. Data normalized to probe 2 = 100%. Average of three independent experiments is shown, error bars indicate SD. (C) Hybrid Selection Circular-RACE detecting transcripts 3′-ends in WT and rnt1Δ. Hybrid selection followed by RNase H treatment selects the transcripts that extend beyond the Rnt1 cleavage site. The sequence of the rDNA terminator is shown. This includes the 3′-end of 25S rRNA sequence, the Rnt1 cleavage site region, the position of the probe used for hybrid selection and the oligo used for transcript release by RNase H treatment. Underlined are the T-rich element T1 and Reb1-binding site. Vertical arrows indicate the detected transcript 3′-ends in WT (black) and rnt1Δ (white) cells.

Figure 3.

A Pol I minigene to study transcription termination. (A) Top: Scheme of the ribosomal minigene, including Pol I promoter plus upstream intergenic sequence (gray), selection fragment derived from the human β-globin gene (black) and Pol I terminator fragment (white) including Rnt1 cleavage site (triangle), T1 and Reb1-binding site (oval). Sizes in base pairs are indicated below. A and B show position of the PCR products for the RT–qPCR analyses in Figure 4B–D. Bottom: schematic of the terminator mutants incorporated into the minigene. (B) Primer extension showing authentic Pol I 5′-ends are produced from the minigene. Arrow indicates a single primer extension product corresponding to correctly initiated Pol I transcripts. (C) TSS detection on the Pol I minigene by RT–qPCR. The analysis was conducted in WT (left) or rat1-1 sen1-1 (right) cells, transformed with the indicated constructs. Reverse transcription was primed with an oligo selective for the plasmid-encoded transcripts, PCR with a communal reverse primer and different forward primers to generate products 1, 2, 3 and 4, shown in the scheme below. PCR efficiency was normalized to T3 transcript produced in vitro (T3 promoter is indicated).

Figure 4.

Co-transcriptional RNA cleavage and failsafe termination pathway in rnt1Δ cells. (A) Transcript cleavage analysis at the Rnt1 and T1 sites. cDNA was produced with a specific primer (B-rev, see Supplementary Table S2) downstream to the terminator fragment and subjected to PCR with oligos across Rnt1 cleavage site (1 + 2) or T1 element (3 + 4), as indicated. Left: RNA is cleaved by Rnt1 in WT but not rnt1Δ. Data are normalized to ΔRnt1 + RZmut (no cleavage). Right: signal across T1 is detectable in rnt1Δ. Cleavage is dependent on the presence of the T-rich element. The data are normalized towards the total amount of cDNA, detected downstream of the cleavage site. WT and mutant ribozyme provide positive and negative controls. An average of three independent experiments is shown, where error bars indicate SD. (B) Termination efficiency in different minigene mutants (outlined in Figure 3A) in WT and rnt1Δ cells. Termination was measured by RT–qPCR, and plotted relative to the construct lacking the whole terminator (100% termination defect). An average of three independent experiments is shown and error bars indicate SD. Raw data, not normalized toward ΔTF, are provided in Supplementary Figure S1. In WT (grey), termination is efficient so long as cleavage at the Rnt1 site is provided. In rnt1Δ (black), alternative cleavage at T1 and presence of an intact Reb1-binding site are crucial to terminate transcription efficiently. (C) Rat1 and Sen1 effect on termination in different minigene mutants. Termination defect plotted as in Figure 4B. rat1-1 sen1-1 shows higher than WT transcriptional read-through also in ΔRnt1 + RZmut where only T1 cleavage takes place. Data for the WT construct are magnified in the square above for better visualization. (D) Reb1 depletion effect on termination. pGAL-REB1 cells were grown in galactose + glucose or switched to glucose for approximately five generations to repress REB1 transcription. Termination was assessed as described for Figure 4B. Reb1 depletion causes a partial defect in ΔRnt1 + RZmut, where no Rnt1 cleavage takes place.

Figure 5.

Model. Two co-transcriptional cleavage events take place at the Pol I terminator. Cleavage of nascent RNA by Rnt1 provides the initial entry site for the ‘torpedo’ Rat1 to promote Pol I transcription termination. In the absence of Rnt1, the downstream transcript is stabilized and an additional cleavage event occurs at T1, providing an alternative entry site for Rat1. In this situation the specific interaction of Reb1 (or alternative DNA-binding protein) with the Reb1-binding site is required to pause the polymerase and promote transcription termination.