Diazaborine Treatment of Yeast Cells Inhibits Maturation of the 60S Ribosomal Subunit

Abstract

Diazaborine treatment of yeast cells was shown previously to cause accumulation of aberrant, 3-elongated mRNAs. Here we demonstrate that the drug inhibits maturation of rRNAs for the large ribosomal subunit. Pulse-chase analyses showed that the processing of the 27S pre-rRNA to consecutive species was blocked in the drug-treated wild-type strain. The steady-state level of the 7S pre-rRNA was clearly reduced after short-term treatment with the inhibitor. At the same time an increase of the 35S pre-rRNA was observed. Longer incubation with the inhibitor resulted in a decrease of the 27S precursor. Primer extension assays showed that an early step in 27S pre-rRNA processing is inhibited, which results in an accumulation of the 27SA2 pre-rRNA and a strong decrease of the 27SA3, 27SB1L, and 27SB1S precursors. The rRNA processing pattern observed after diazaborine treatment resembles that reported after depletion of the RNA binding protein Nop4p/ Nop77p. This protein is essential for correct pre-27S rRNA processing. Using a green fluorescent protein-Nop4 fusion, we found that diazaborine treatment causes, within minutes, a rapid redistribution of the protein from the nucleolus to the periphery of the nucleus, which provides a possible explanation for the effect of diazaborine on rRNA processing.Translation of mRNA in the cytoplasm relies on the ribosome. While the principal steps in initiation and elongation of protein synthesis were uncovered in the middle of the last century, details of the biogenesis of the ribosome itself remained elusive. However, the development of novel techniques in affinity purification and refined techniques of protein identification by mass spectrometry within the last several years provided novel insights into the steps of assembly of the ribosomal subunits and how they are coordinated with the processing of the rRNA precursors (for recent reviews see references 15 and 47). Much of this detailed knowledge was worked out in the baker's yeast, Saccharomyces cerevisiae. This organism constitutes a simple model for the reaction sequence occurring in more complex eukaryotic cells.While the 5S rRNA is transcribed by RNA polymerase III, the 18S, 25S, and 5.8S rRNAs are transcribed as a single precursor molecule by RNA polymerase I. The primary transcript is cotranscriptionally processed at its 3Ј external transcribed spacer (ETS) end into the 35S pre-rRNA (26). The rRNAs for both the large and the small subunits are then generated from the 35S pre-rRNA by several subsequent processing events. In the first two processing steps for the 35S rRNA, the 5Ј ETS is successively removed by endonucleolytic cleavages at positions A0 and A1. The following step, which separates the rRNA precursors for the two ribosomal subunits, is a cleavage reaction at position A2 in internal transcribed spacer 1 (ITS1) located between the 18S and 5.8S rRNAs (23,27). This cleavage reaction results in the release of the 20S pre-rRNA, which matures in the cytoplasm to the 18S rRNA present in the small subunit of the ribosome ...

Cytoplasmic Recycling of 60S Preribosomal Factors Depends on the AAA Protein Drg1

“…Recently we demonstrated that the 60S ribosomal subunit formation defects caused by the drug diazaborine are compensated for by allelic forms of Drg1 (24). To test whether Drg1 plays a role in ribosome biogenesis, we obtained polysome profiles from the temperature-sensitive drg1-ts strain, the intragenic suppressor strain (drg1-sup), and the isogenic wild-type strain (Fig.…”

“…Cells were suspended in 5 M guanidinium-HCl buffer and broken by vigorous vortexing with glass beads. RNA was extracted and transferred onto nylon membranes as described previously (24). After UV cross-linking, the radioactivity on the membrane was detected by phosphorimaging using a tritium screen (Kodak).…”

“…Other allelic forms of DRG1 confer resistance to the drug diazaborine in yeast (35). Diazaborine inhibits maturation of the 60S ribosomal subunit by blocking 27SA2 pre-rRNA processing (24). This processing step is dependent on the nucleolar protein Nop4 (3,30), which is relocalized from the nucleolus to the nuclear periphery upon diazaborine treatment.…”

“…This processing step is dependent on the nucleolar protein Nop4 (3,30), which is relocalized from the nucleolus to the nuclear periphery upon diazaborine treatment. In the diazaborine-resistant DRG1-1 mutant, no inhibition of 60S biogenesis and no relocalization of Nop4 were observed (24).…”

The Drug Diazaborine Blocks Ribosome Biogenesis by Inhibiting the AAA-ATPase Drg1

“…We showed previously that the drug diazaborine blocks ribosome biogenesis in yeast (10). Diazaborine is a heterocyclic boron-containing compound with strong antimicrobial activity (11).…”