NMD3 Encodes an Essential Cytoplasmic Protein Required for Stable 60S Ribosomal Subunits in Saccharomyces cerevisiae

Abstract

A mutation in NMD3 was found to be lethal in the absence of XRN1, which encodes the major cytoplasmic exoribonuclease responsible for mRNA turnover. Molecular genetic analysis of NMD3 revealed that it is an essential gene required for stable 60S ribosomal subunits. Cells bearing a temperature-sensitive allele of NMD3 had decreased levels of 60S subunits at the nonpermissive temperature which resulted in the formation of half-mer polysomes. Pulse-chase analysis of rRNA biogenesis indicated that 25S rRNA was made and processed with kinetics similar to wild-type kinetics. However, the mature RNA was rapidly degraded, with a half-life of 4 min. Nmd3p fractionated as a cytoplasmic protein and sedimented in the position of free 60S subunits in sucrose gradients. These results suggest that Nmd3p is a cytoplasmic factor required for a late cytoplasmic assembly step of the 60S subunit but is not a ribosomal protein. Putative orthologs of Nmd3p exist in Drosophila, in nematodes, and in archaebacteria but not in eubacteria. The Nmd3 protein sequence does not contain readily recognizable motifs of known function. However, these proteins all have an amino-terminal domain containing four repeats of Cx₂C, reminiscent of zinc-binding proteins, implicated in nucleic acid binding or protein oligomerization.

ACKNOWLEDGMENTS

We thank Alan Sachs for providing strain YAS398, Janet Lindsley and Jim Wang for providing anti-Topo II antibodies, Jonathan Warner for anti-L3 antibodies, and Roy Parker for plasmid pRP485. We are grateful to Xianmei Yang for the spb2Δ cross with xrn1Δ and Justin Brown for c-Myc-NMD3 construct pAJ153. We are especially appreciative of John Woolford for critical reading of the manuscript and of Allan Jacobson for sharing results before publication. DNA sequence analysis was done by the Core Facility at the Institute for Cellular and Molecular Biology, University of Texas at Austin.

This work was supported by NIH grant GM056355 to A. W. Johnson.

ADDENDUM IN PROOF

Because we found that the c-Myc signal of epitope-tagged Nmd3p was weak, even when expressed from a galactose-inducible promoter, we have since tagged the genomic NMD3 with 13 tandem copies of c-Myc to verify the cytoplasmic localization. The multiply tagged protein was functional and by indirect immunofluorescence techniques it was found in the cytoplasm and excluded from the nucleus.