Yeast Pre-rRNA Processing and Modification Occur Cotranscriptionally

Martin Koš,David Tollervey

doi:10.1016/j.molcel.2010.02.024

Martin Koš, David Tollervey

Open Access

https://doi.org/10.1016/j.molcel.2010.02.024

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Abstract

SummaryTo better understand yeast ribosome synthesis, we developed techniques for the rapid harvesting and analysis of metabolically labeled cultures. Modeling of the resulting kinetic data allowed predicted lifetimes and processing patterns to be compared with the experimental data. This supported a transcription time for the 35S primary transcripts of ∼170 s at 30°C (∼40 nt s−1), with a high fraction (∼70%) of nascent transcripts cleaved at the early processing sites that generate the 20S precursor to the 18S rRNA. This level of nascent transcript cleavage apparently conflicted with previous reports that modification of yeast pre-rRNA exclusively occurred on released transcripts. A second round of high-resolution kinetic labeling showed that 20S pre-rRNA predominately undergoes methylation as nascent transcripts, whereas the 27S precursor to the 25S/5.8S rRNAs was partially methylated on the nascent transcript. The results demonstrate that quantitative analyses of pre-rRNA processing can yield important biological insights.

Highlights

The synthesis of eukaryotic ribosomes is a remarkably complex system, with multiple intermediate steps in maturation of the rRNAs that involve a vast number of trans-acting factors
To better understand yeast ribosome synthesis, we developed techniques for the rapid harvesting and analysis of metabolically labeled cultures
Modeling of the resulting kinetic data allowed predicted lifetimes and processing patterns to be compared with the experimental data. This supported a transcription time for the 35S primary transcripts of $170 s at 30C ($40 nt sÀ1), with a high fraction ($70%) of nascent transcripts cleaved at the early processing sites that generate the 20S precursor to the 18S rRNA

Summary

SUMMARY

To better understand yeast ribosome synthesis, we developed techniques for the rapid harvesting and analysis of metabolically labeled cultures. Modeling of the resulting kinetic data allowed predicted lifetimes and processing patterns to be compared with the experimental data This supported a transcription time for the 35S primary transcripts of $170 s at 30C ($40 nt sÀ1), with a high fraction ($70%) of nascent transcripts cleaved at the early processing sites that generate the 20S precursor to the 18S rRNA. This level of nascent transcript cleavage apparently conflicted with previous reports that modification of yeast pre-rRNA exclusively occurred on released transcripts. The results demonstrate that quantitative analyses of pre-rRNA processing can yield important biological insights

INTRODUCTION

RESULTS

DISCUSSION

EXPERIMENTAL PROCEDURES