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Kinetic Analysis Demonstrates a Requirement for the Rat1 Exonuclease in Cotranscriptional Pre-rRNA Cleavage

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  • Konstantin Axt
  • Sarah L French
  • Ann L Beyer
  • David Tollervey

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 ∼1.5Kb 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.

Suggested Citation

  • Konstantin Axt & Sarah L French & Ann L Beyer & David Tollervey, 2014. "Kinetic Analysis Demonstrates a Requirement for the Rat1 Exonuclease in Cotranscriptional Pre-rRNA Cleavage," PLOS ONE, Public Library of Science, vol. 9(2), pages 1-11, February.
  • Handle: RePEc:plo:pone00:0085703
    DOI: 10.1371/journal.pone.0085703
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    References listed on IDEAS

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    1. Song Xiang & Amalene Cooper-Morgan & Xinfu Jiao & Megerditch Kiledjian & James L. Manley & Liang Tong, 2009. "Structure and function of the 5′→3′ exoribonuclease Rat1 and its activating partner Rai1," Nature, Nature, vol. 458(7239), pages 784-788, April.
    2. François Dragon & Jennifer E. G. Gallagher & Patricia A. Compagnone-Post & Brianna M. Mitchell & Kara A. Porwancher & Karen A. Wehner & Steven Wormsley & Robert E. Settlage & Jeffrey Shabanowitz & Yvo, 2002. "A large nucleolar U3 ribonucleoprotein required for 18S ribosomal RNA biogenesis," Nature, Nature, vol. 417(6892), pages 967-970, June.
    3. Minkyu Kim & Nevan J. Krogan & Lidia Vasiljeva & Oliver J. Rando & Eduard Nedea & Jack F. Greenblatt & Stephen Buratowski, 2004. "The yeast Rat1 exonuclease promotes transcription termination by RNA polymerase II," Nature, Nature, vol. 432(7016), pages 517-522, November.
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