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

Braglia P, Kawauchi J, Proudfoot NJ - Nucleic Acids Res. (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, UK.

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.

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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.
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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.

Mentions: RNA polymerase (Pol) I is responsible for ribosomal RNA transcription and synthesizes the great majority of RNA in every living cell. Efficient termination of transcription is crucial to coordinate Pol I transcription and allow polymerase recycling. Ribosomal DNA is organized in a tandem array of units comprising the pre-rRNA encoding DNA plus upstream and downstream regulatory elements; a schematic of a yeast rDNA repeat is represented in Figure 1A. The 35S pre-rRNA is transcribed and subsequently processed to produce mature 18S, 5.8S and 25S rRNAs. Each rDNA unit also contains the Pol III-transcribed 5S rRNA gene in the opposite orientation.Figure 1.


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

Braglia P, Kawauchi J, Proudfoot NJ - Nucleic Acids Res. (2010)

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3045592&req=5

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.
Mentions: RNA polymerase (Pol) I is responsible for ribosomal RNA transcription and synthesizes the great majority of RNA in every living cell. Efficient termination of transcription is crucial to coordinate Pol I transcription and allow polymerase recycling. Ribosomal DNA is organized in a tandem array of units comprising the pre-rRNA encoding DNA plus upstream and downstream regulatory elements; a schematic of a yeast rDNA repeat is represented in Figure 1A. The 35S pre-rRNA is transcribed and subsequently processed to produce mature 18S, 5.8S and 25S rRNAs. Each rDNA unit also contains the Pol III-transcribed 5S rRNA gene in the opposite orientation.Figure 1.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford, OX1 3RE, UK.

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.

Show MeSH
Related in: MedlinePlus