RESUMEN
The NSR1 gene product is involved in ribosomal RNA production and ribosome assembly in Saccharomyces cerevisiae. Yeast strains carrying a deletion of the NSR1 gene have a defect in rRNA processing, an aberrant ribosome profile and are sensitive to the drug paromomycin. This paper reports the isolation and characterization of spontaneous suppressors of the paromomycin sensitivity. Such suppressors could be isolated at very high frequency and do not exhibit straightforward single-gene inheritance patterns. The suppressors are not influenced by non-Mendelian factors such as psi or rho. Through a replacement of chromosomal rDNA with a plasmid rDNA system, I show that suppression of paromomycin sensitivity is mediated by rDNA. Swapping wild-type plasmid rDNA for chromosomal rDNA can reverse the suppression, but the effect does not appear to be due to amplification of rDNA or amplification of a pre-existing mutant rDNA copy.
Asunto(s)
Antibacterianos/farmacología , Proteínas Fúngicas/genética , Proteínas Nucleares , Paromomicina/farmacología , Proteínas de Unión al ARN , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Supresión Genética , ADN de Hongos/análisis , ADN Ribosómico/análisis , ADN Ribosómico/genética , Farmacorresistencia Microbiana , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Pruebas de Sensibilidad Microbiana , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/crecimiento & desarrolloRESUMEN
Several nucleolar proteins, such as nucleolin, NOP1/fibrillarin, SSB1, NSR1 and GAR1 share a common glycine and arginine rich structural motif called the GAR domain. To identify novel nucleolar proteins from fission yeast we screened Schizosaccharomyces pombe genomic DNA libraries with a probe encompassing the GAR structural motif. Here we report the identification and characterization of a S.pombe gene coding for a novel nucleolar protein, designated gar2. The structure of the fission yeast gar2 is reminiscent of that of nucleolin from vertebrates and NSR1 from Saccharomyces cerevisiae. In addition, like these proteins, gar2 has a nucleolar localisation. The disruption of the gar2+ gene affects normal cell growth, leads to an accumulation of 35S pre-rRNA and a decrease of mature 18S rRNA steady state levels. Moreover, ribosomal profiles of the mutant show an increase of free 60S ribosomal subunits and an absence of free 40S ribosomal subunits. gar2 is able to rescue a S.cerevisiae mutant lacking NSR1, thus establishing gar2 as a functional homolog of NSR1. We propose that gar2 helps the assembly of pre-ribosomal particles containing 18S rRNA.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas Nucleares/genética , ARN Ribosómico 18S/metabolismo , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Proteínas Fúngicas/aislamiento & purificación , Datos de Secuencia Molecular , Proteínas Nucleares/aislamiento & purificación , Proteínas Nucleares/metabolismo , Schizosaccharomyces/genéticaRESUMEN
NSR1 is a yeast nuclear localization sequence-binding protein showing striking similarity in its domain structure to nucleolin. Cells lacking NSR1 are viable but have a severe growth defect. We show here that NSR1, like nucleolin, is involved in ribosome biogenesis. The nsr1 mutant is deficient in pre-rRNA processing such that the initial 35S pre-rRNA processing is blocked and 20S pre-rRNA is nearly absent. The reduced amount of 20S pre-rRNA leads to a shortage of 18S rRNA and is reflected in a change in the distribution of 60S and 40S ribosomal subunits; there is no free pool of 40S subunits, and the free pool of 60S subunits is greatly increased in size. The lack of free 40S subunits or the improper assembly of these subunits causes the nsr1 mutant to show sensitivity to the antibiotic paromomycin, which affects protein translation, at concentrations that do not affect the growth of the wild-type strain. Our data support the idea that NSR1 is involved in the proper assembly of pre-rRNA particles, possibly by bringing rRNA and ribosomal proteins together by virtue of its nuclear localization sequence-binding domain and multiple RNA recognition motifs. Alternatively, NSR1 may also act to regulate the nuclear entry of ribosomal proteins required for proper assembly of pre-rRNA particles.