Volume 272, Number 40, Issue of October 3, 1997 pp. 25345-25352
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.

The Yeast Nucleolar Protein Nop4p Contains Four RNA Recognition Motifs Necessary for Ribosome Biogenesis

(Received for publication, February 13, 1997, and in revised form, May 14, 1997)

From the Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213

The Saccharomyces cerevisiae nucleolar protein Nop4p is necessary for processing of rRNA and assembly of 60 S ribosomal subunits. Nop4p is unusual in that it contains four RNA recognition motifs (RRMs) including one noncanonical RRM, as well as several auxiliary motifs, two acidic regions between the RRMs, and a carboxyl-terminal domain rich in lysines and arginines. To examine the functional importance of these motifs, we isolated random and site-directed mutations in NOP4 and assayed Nop4p function in vivo. Our results indicate that each RRM is essential for Nop4p function; mutations in conserved aromatic residues of Nop4p cause a temperature-sensitive lethal phenotype and diminished 60 S ribosomal subunit production. The carboxyl-terminal 68 amino acids are important but apparently not essential; carboxyl-terminal truncation of Nop4p causes slow growth, decreased ribosome production, and mislocalization of Nop4p. Deletion of both acidic motifs is lethal but replacement of most of the acidic residues with alanine has no apparent phenotype. These acidic residues may serve as spacers or tethers to separate the RRMs.

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