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

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

doi:10.1371/journal.pone.0085703

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.

Highlights

During the yeast ribosome synthesis pathway, the 18S, 5.8S and 25S rRNAs are cotranscribed as a single precursor that undergoes a multi-step processing pathway to generate the mature rRNAs (Figure 1)
Following the electron microscopy (EM) analyses, a Chisquared test was used to determine if the proportion of genes with no nascent transcript cleavage (NTC) was significantly altered by the experimental conditions
The unlabeled pre-rRNAs in the cells are at steady-state, and the metabolic labeling reveals the kinetics of the approach to this steady state over time [7]

Summary

Introduction

During the yeast ribosome synthesis pathway, the 18S, 5.8S and 25S rRNAs are cotranscribed as a single precursor that undergoes a multi-step processing pathway to generate the mature rRNAs (Figure 1). The NTC:RTC ratio can be determined by fast kinetic analyses of the appearance of labeled, newly-synthesized pre-rRNA [7]. This relies on the fact that newly labeled 20S region of the pre-rRNA can only be observed as a discrete species when cleavage at sites A1 and A2 has occurred. NTC allows cleavage and 20S appearance before the polymerase has reached the end of the transcription unit This time difference can be detected by metabolic labeling, following the incorporation of [3H] uracil into pre-rRNA species [7].

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Conclusion