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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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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.
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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.

Mentions: We provide a unifying model for termination of transcription by Pol I and identify a new co-transcriptional cleavage activity associated with the rDNA terminator transcript (Figure 5). We have mapped this cleavage sequence to T1 in the 3′-ETS and demonstrate that it provides an alternative pathway for termination in the absence of Rnt1. Rat1 and Sen1 activities are involved in this pathway, suggesting that this secondary cleavage is an alternative or ‘failsafe’ entry site for the torpedo Rat1, to promote Pol I termination. We cannot determine whether this cleavage activity works together with or only independently of Rnt1. In WT cells it is not detectable since Rnt1 cleavage provides an entry site for rapid degradation of the downstream transcript by the exonuclease Rat1. In any case T1 co-transcriptional cleavage appears to be dispensable for termination in a WT situation where the transcript is cut at the upstream position. We have also shown that RNA cleavage at T1 generates an upstream 3′-end that is not a substrate for normal exosome processing, resulting in an extended 25S rRNA (Figure 1). The transcript ends detectable at T1 could arise from either RNA cleavage or from Pol I termination. However the hsTRO and Pol I minigene cleavage assays imply a cleavage process as they both detect read-through transcripts downstream of T1, arguing that Pol I is still engaged with the template. On the other hand the presence of an intact Reb1-binding site is required for efficient termination but does not seem to influence RNA cleavage in the rnt1Δ strain. This supports a combined termination mechanism, involving ‘torpedo’ and ‘pausing/release’. Early studies that suggested the pausing/release mechanism for Pol I termination (1) were conducted in vitro and therefore missed the connection between transcription and rRNA processing, in particular RNA cleavage by Rnt1. Here we show a clear defect in termination when we mutate the Reb1-binding site in rnt1Δ and partially reproduce the defect by depletion of Reb1. It is therefore puzzling that ChIP analysis fails to detect the presence of Reb1 over the Pol I terminator (8). We also performed Reb1 ChIP in rnt1Δ cells and obtained the same negative results (data not shown). A possible explanation for these results is the involvement of an additional DNA-binding protein with sequence specificity similar to Reb1. We propose that polymerase pausing by Reb1 (or an alternative DNA-binding protein) works in concert with co-transcriptional cleavage at T1, thereby enhancing an otherwise inefficient or kinetically slow event.Figure 5.


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

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

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

License
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getmorefigures.php?uid=PMC3045592&req=5

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.
Mentions: We provide a unifying model for termination of transcription by Pol I and identify a new co-transcriptional cleavage activity associated with the rDNA terminator transcript (Figure 5). We have mapped this cleavage sequence to T1 in the 3′-ETS and demonstrate that it provides an alternative pathway for termination in the absence of Rnt1. Rat1 and Sen1 activities are involved in this pathway, suggesting that this secondary cleavage is an alternative or ‘failsafe’ entry site for the torpedo Rat1, to promote Pol I termination. We cannot determine whether this cleavage activity works together with or only independently of Rnt1. In WT cells it is not detectable since Rnt1 cleavage provides an entry site for rapid degradation of the downstream transcript by the exonuclease Rat1. In any case T1 co-transcriptional cleavage appears to be dispensable for termination in a WT situation where the transcript is cut at the upstream position. We have also shown that RNA cleavage at T1 generates an upstream 3′-end that is not a substrate for normal exosome processing, resulting in an extended 25S rRNA (Figure 1). The transcript ends detectable at T1 could arise from either RNA cleavage or from Pol I termination. However the hsTRO and Pol I minigene cleavage assays imply a cleavage process as they both detect read-through transcripts downstream of T1, arguing that Pol I is still engaged with the template. On the other hand the presence of an intact Reb1-binding site is required for efficient termination but does not seem to influence RNA cleavage in the rnt1Δ strain. This supports a combined termination mechanism, involving ‘torpedo’ and ‘pausing/release’. Early studies that suggested the pausing/release mechanism for Pol I termination (1) were conducted in vitro and therefore missed the connection between transcription and rRNA processing, in particular RNA cleavage by Rnt1. Here we show a clear defect in termination when we mutate the Reb1-binding site in rnt1Δ and partially reproduce the defect by depletion of Reb1. It is therefore puzzling that ChIP analysis fails to detect the presence of Reb1 over the Pol I terminator (8). We also performed Reb1 ChIP in rnt1Δ cells and obtained the same negative results (data not shown). A possible explanation for these results is the involvement of an additional DNA-binding protein with sequence specificity similar to Reb1. We propose that polymerase pausing by Reb1 (or an alternative DNA-binding protein) works in concert with co-transcriptional cleavage at T1, thereby enhancing an otherwise inefficient or kinetically slow event.Figure 5.

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