Cargando…
Kinetic Analysis Demonstrates a Requirement for the Rat1 Exonuclease in Cotranscriptional Pre-rRNA Cleavage
During yeast ribosome synthesis, three early cleavages generate the 20S precursor to the 18S rRNA component of the 40S subunits. These cleavages can occur either on the nascent transcript (nascent transcript cleavage; NTC) or on the 35S pre-rRNA that has been fully transcribed and released from the...
Autores principales: | , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2014
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911906/ https://www.ncbi.nlm.nih.gov/pubmed/24498264 http://dx.doi.org/10.1371/journal.pone.0085703 |
_version_ | 1782302011328299008 |
---|---|
author | Axt, Konstantin French, Sarah L. Beyer, Ann L. Tollervey, David |
author_facet | Axt, Konstantin French, Sarah L. Beyer, Ann L. Tollervey, David |
author_sort | Axt, Konstantin |
collection | PubMed |
description | During yeast ribosome synthesis, three early cleavages generate the 20S precursor to the 18S rRNA component of the 40S subunits. These cleavages can occur either on the nascent transcript (nascent transcript cleavage; NTC) or on the 35S pre-rRNA that has been fully transcribed and released from the rDNA (released transcript cleavage; RTC). These alternative pathways cannot be assessed by conventional RNA analyses, since the pre-rRNA products of NTC and RTC are identical. They can, however, be distinguished kinetically by metabolic labeling and quantified by modeling of the kinetic data. The aim of this work was to use these approaches as a practical tool to identify factors that mediate the decision between utilization of NTC and RTC. The maturation pathways of the 40S and 60S ribosomal subunits are largely distinct. However, depletion of some early-acting 60S synthesis factors, including the 5′-exonuclease Rat1, leads to accumulation of the 35S pre-rRNA and delayed 20S pre-rRNA synthesis. We speculated that this might reflect the loss of NTC. Rat1 acts catalytically in 5.8S and 25S rRNA processing but binds to the pre-rRNA prior to these activities. Kinetic data for strains depleted of Rat1 match well with the modeled effects of strongly reduced NTC. This was confirmed by EM visualization of “Miller” chromatin spreads of nascent pre-rRNA transcripts. Modeling further indicates that NTC takes place in a limited time window, when the polymerase has transcribed ∼1.5Kb past the A2 cleavage site. We speculate that assembly of early-acting 60S synthesis factors is monitored as a quality control system prior to NTC. |
format | Online Article Text |
id | pubmed-3911906 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-39119062014-02-04 Kinetic Analysis Demonstrates a Requirement for the Rat1 Exonuclease in Cotranscriptional Pre-rRNA Cleavage Axt, Konstantin French, Sarah L. Beyer, Ann L. Tollervey, David PLoS One Research Article During yeast ribosome synthesis, three early cleavages generate the 20S precursor to the 18S rRNA component of the 40S subunits. These cleavages can occur either on the nascent transcript (nascent transcript cleavage; NTC) or on the 35S pre-rRNA that has been fully transcribed and released from the rDNA (released transcript cleavage; RTC). These alternative pathways cannot be assessed by conventional RNA analyses, since the pre-rRNA products of NTC and RTC are identical. They can, however, be distinguished kinetically by metabolic labeling and quantified by modeling of the kinetic data. The aim of this work was to use these approaches as a practical tool to identify factors that mediate the decision between utilization of NTC and RTC. The maturation pathways of the 40S and 60S ribosomal subunits are largely distinct. However, depletion of some early-acting 60S synthesis factors, including the 5′-exonuclease Rat1, leads to accumulation of the 35S pre-rRNA and delayed 20S pre-rRNA synthesis. We speculated that this might reflect the loss of NTC. Rat1 acts catalytically in 5.8S and 25S rRNA processing but binds to the pre-rRNA prior to these activities. Kinetic data for strains depleted of Rat1 match well with the modeled effects of strongly reduced NTC. This was confirmed by EM visualization of “Miller” chromatin spreads of nascent pre-rRNA transcripts. Modeling further indicates that NTC takes place in a limited time window, when the polymerase has transcribed ∼1.5Kb past the A2 cleavage site. We speculate that assembly of early-acting 60S synthesis factors is monitored as a quality control system prior to NTC. Public Library of Science 2014-02-03 /pmc/articles/PMC3911906/ /pubmed/24498264 http://dx.doi.org/10.1371/journal.pone.0085703 Text en © 2014 Axt et al http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited. |
spellingShingle | Research Article Axt, Konstantin French, Sarah L. Beyer, Ann L. Tollervey, David Kinetic Analysis Demonstrates a Requirement for the Rat1 Exonuclease in Cotranscriptional Pre-rRNA Cleavage |
title | Kinetic Analysis Demonstrates a Requirement for the Rat1 Exonuclease in Cotranscriptional Pre-rRNA Cleavage |
title_full | Kinetic Analysis Demonstrates a Requirement for the Rat1 Exonuclease in Cotranscriptional Pre-rRNA Cleavage |
title_fullStr | Kinetic Analysis Demonstrates a Requirement for the Rat1 Exonuclease in Cotranscriptional Pre-rRNA Cleavage |
title_full_unstemmed | Kinetic Analysis Demonstrates a Requirement for the Rat1 Exonuclease in Cotranscriptional Pre-rRNA Cleavage |
title_short | Kinetic Analysis Demonstrates a Requirement for the Rat1 Exonuclease in Cotranscriptional Pre-rRNA Cleavage |
title_sort | kinetic analysis demonstrates a requirement for the rat1 exonuclease in cotranscriptional pre-rrna cleavage |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3911906/ https://www.ncbi.nlm.nih.gov/pubmed/24498264 http://dx.doi.org/10.1371/journal.pone.0085703 |
work_keys_str_mv | AT axtkonstantin kineticanalysisdemonstratesarequirementfortherat1exonucleaseincotranscriptionalprerrnacleavage AT frenchsarahl kineticanalysisdemonstratesarequirementfortherat1exonucleaseincotranscriptionalprerrnacleavage AT beyerannl kineticanalysisdemonstratesarequirementfortherat1exonucleaseincotranscriptionalprerrnacleavage AT tollerveydavid kineticanalysisdemonstratesarequirementfortherat1exonucleaseincotranscriptionalprerrnacleavage |