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...
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| 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 ∼ 1.5 Kb 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. |
| doi_str_mv | 10.1371/journal.pone.0085703 |
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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 ∼ 1.5 Kb 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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0085703</identifier><identifier>PMID: 24498264</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Baking yeast ; Biology ; Blotting, Northern ; Blotting, Western ; Chromatin ; Chromatin - genetics ; Chromatin - metabolism ; Chromatin - ultrastructure ; Cleavage ; Control systems ; Exonuclease ; Exoribonucleases - genetics ; Exoribonucleases - metabolism ; Hostages ; Immunology ; Kinetics ; Microscopy, Electron ; Modelling ; Mutation ; Quality control ; Ribonucleic acid ; Ribosomal subunits ; Ribosome Subunits, Large, Eukaryotic - genetics ; Ribosome Subunits, Large, Eukaryotic - metabolism ; Ribosomes - genetics ; Ribosomes - metabolism ; RNA ; RNA Precursors - genetics ; RNA Precursors - metabolism ; RNA processing ; RNA, Fungal - genetics ; RNA, Fungal - metabolism ; RNA, Ribosomal - genetics ; RNA, Ribosomal - metabolism ; RNA, Ribosomal, 5.8S - genetics ; RNA, Ribosomal, 5.8S - metabolism ; rRNA 18S ; rRNA 20S ; rRNA 25S ; rRNA 35S ; rRNA 5.8S ; Saccharomyces cerevisiae ; Saccharomyces cerevisiae - genetics ; Saccharomyces cerevisiae - metabolism ; Saccharomyces cerevisiae Proteins - genetics ; Saccharomyces cerevisiae Proteins - metabolism ; Synthesis ; Time Factors ; Transcription ; Transcription, Genetic ; Windows (intervals) ; Yeast</subject><ispartof>PloS one, 2014-02, Vol.9 (2), p.e85703-e85703</ispartof><rights>COPYRIGHT 2014 Public Library of Science</rights><rights>2014 Axt et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/legalcode (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 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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. 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We speculate that assembly of early-acting 60S synthesis factors is monitored as a quality control system prior to NTC.</description><subject>Baking yeast</subject><subject>Biology</subject><subject>Blotting, Northern</subject><subject>Blotting, Western</subject><subject>Chromatin</subject><subject>Chromatin - genetics</subject><subject>Chromatin - metabolism</subject><subject>Chromatin - ultrastructure</subject><subject>Cleavage</subject><subject>Control systems</subject><subject>Exonuclease</subject><subject>Exoribonucleases - genetics</subject><subject>Exoribonucleases - metabolism</subject><subject>Hostages</subject><subject>Immunology</subject><subject>Kinetics</subject><subject>Microscopy, Electron</subject><subject>Modelling</subject><subject>Mutation</subject><subject>Quality control</subject><subject>Ribonucleic acid</subject><subject>Ribosomal subunits</subject><subject>Ribosome Subunits, Large, Eukaryotic - genetics</subject><subject>Ribosome Subunits, Large, Eukaryotic - metabolism</subject><subject>Ribosomes - 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genetics</topic><topic>Chromatin - metabolism</topic><topic>Chromatin - ultrastructure</topic><topic>Cleavage</topic><topic>Control systems</topic><topic>Exonuclease</topic><topic>Exoribonucleases - genetics</topic><topic>Exoribonucleases - metabolism</topic><topic>Hostages</topic><topic>Immunology</topic><topic>Kinetics</topic><topic>Microscopy, Electron</topic><topic>Modelling</topic><topic>Mutation</topic><topic>Quality control</topic><topic>Ribonucleic acid</topic><topic>Ribosomal subunits</topic><topic>Ribosome Subunits, Large, Eukaryotic - genetics</topic><topic>Ribosome Subunits, Large, Eukaryotic - metabolism</topic><topic>Ribosomes - genetics</topic><topic>Ribosomes - metabolism</topic><topic>RNA</topic><topic>RNA Precursors - genetics</topic><topic>RNA Precursors - metabolism</topic><topic>RNA processing</topic><topic>RNA, Fungal - genetics</topic><topic>RNA, Fungal - metabolism</topic><topic>RNA, Ribosomal - genetics</topic><topic>RNA, Ribosomal - metabolism</topic><topic>RNA, Ribosomal, 5.8S - 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Academic</collection><collection>PubMed Central (Full Participant titles)</collection><collection>DOAJ Directory of Open Access Journals</collection><jtitle>PloS one</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Axt, Konstantin</au><au>French, Sarah L</au><au>Beyer, Ann L</au><au>Tollervey, David</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Kinetic analysis demonstrates a requirement for the Rat1 exonuclease in cotranscriptional pre-rRNA cleavage</atitle><jtitle>PloS one</jtitle><addtitle>PLoS One</addtitle><date>2014-02-03</date><risdate>2014</risdate><volume>9</volume><issue>2</issue><spage>e85703</spage><epage>e85703</epage><pages>e85703-e85703</pages><issn>1932-6203</issn><eissn>1932-6203</eissn><abstract>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 ∼ 1.5 Kb 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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>24498264</pmid><doi>10.1371/journal.pone.0085703</doi><tpages>e85703</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Baking yeast Biology Blotting, Northern Blotting, Western Chromatin Chromatin - genetics Chromatin - metabolism Chromatin - ultrastructure Cleavage Control systems Exonuclease Exoribonucleases - genetics Exoribonucleases - metabolism Hostages Immunology Kinetics Microscopy, Electron Modelling Mutation Quality control Ribonucleic acid Ribosomal subunits Ribosome Subunits, Large, Eukaryotic - genetics Ribosome Subunits, Large, Eukaryotic - metabolism Ribosomes - genetics Ribosomes - metabolism RNA RNA Precursors - genetics RNA Precursors - metabolism RNA processing RNA, Fungal - genetics RNA, Fungal - metabolism RNA, Ribosomal - genetics RNA, Ribosomal - metabolism RNA, Ribosomal, 5.8S - genetics RNA, Ribosomal, 5.8S - metabolism rRNA 18S rRNA 20S rRNA 25S rRNA 35S rRNA 5.8S Saccharomyces cerevisiae Saccharomyces cerevisiae - genetics Saccharomyces cerevisiae - metabolism Saccharomyces cerevisiae Proteins - genetics Saccharomyces cerevisiae Proteins - metabolism Synthesis Time Factors Transcription Transcription, Genetic Windows (intervals) Yeast |
| title | Kinetic analysis demonstrates a requirement for the Rat1 exonuclease in cotranscriptional pre-rRNA cleavage |
| url | https://sfx.bib-bvb.de/sfx_tum?ctx_ver=Z39.88-2004&ctx_enc=info:ofi/enc:UTF-8&ctx_tim=2025-02-08T20%3A23%3A38IST&url_ver=Z39.88-2004&url_ctx_fmt=infofi/fmt:kev:mtx:ctx&rfr_id=info:sid/primo.exlibrisgroup.com:primo3-Article-gale_plos_&rft_val_fmt=info:ofi/fmt:kev:mtx:journal&rft.genre=article&rft.atitle=Kinetic%20analysis%20demonstrates%20a%20requirement%20for%20the%20Rat1%20exonuclease%20in%20cotranscriptional%20pre-rRNA%20cleavage&rft.jtitle=PloS%20one&rft.au=Axt,%20Konstantin&rft.date=2014-02-03&rft.volume=9&rft.issue=2&rft.spage=e85703&rft.epage=e85703&rft.pages=e85703-e85703&rft.issn=1932-6203&rft.eissn=1932-6203&rft_id=info:doi/10.1371/journal.pone.0085703&rft_dat=%3Cgale_plos_%3EA478831940%3C/gale_plos_%3E%3Curl%3E%3C/url%3E&disable_directlink=true&sfx.directlink=off&sfx.report_link=0&rft_id=info:oai/&rft_pqid=1494055244&rft_id=info:pmid/24498264&rft_galeid=A478831940&rft_doaj_id=oai_doaj_org_article_3462cf860d8f4bd5a6029673144eb2c0&rfr_iscdi=true |