Characterization of Saccharomyces cerevisiae Npa2p (Urb2p) reveals a low-molecular-mass complex containing Dbp6p, Npa1p (Urb1p), Nop8p, and Rsa3p involved in early steps of 60S ribosomal subunit biogenesis.
We report the characterization of the yeast Npa2p (Urb2p) protein, which is essential for 60S ribosomal subunit biogenesis. We identified this protein in a synthetic lethal screening with the rsa3 null allele. Rsa3p is a genetic partner of the putative RNA helicase Dbp6p. Mutation or depletion of Npa2p leads to a net deficit in 60S subunits and a decrease in the levels all 27S pre-rRNAs and mature 25S and 5.8S rRNAs. This is likely due to instability of early pre-60S particles. Consistent with a role of Npa2p in 60S subunit biogenesis, green fluorescent protein-tagged Npa2p localizes predominantly to the nucleolus and TAP-tagged Npa2p sediments with large complexes in sucrose gradients and is associated mainly with 27SA(2) pre-rRNA-containing preribosomal particles. In addition, we reveal a genetic synthetic interaction between Npa2p, several factors required for early steps of 60S subunit biogenesis (Dbp6p, Dbp7p, Dbp9p, Npa1p, Nop8p, and Rsa3p), and the 60S protein Rpl3p. Furthermore, coimmunoprecipitation and gel filtration analyses demonstrated that at least Npa2p, Dbp6p, Npa1p, Nop8p, and Rsa3p are present together in a subcomplex of low molecular mass whose integrity is independent of RNA. Our results support the idea that these five factors work in concert during the early steps of 60S subunit biogenesis.
Rosado
Iván V
IV
Departamento de Genética, Facultad de Biología, Universidad de Sevilla, Avenida Reina Mercedes 6, E-41012 Seville, Spain.
Dez
Christophe
C
Lebaron
Simon
S
Caizergues-Ferrer
Michèle
M
Henry
Yves
Y
de la Cruz
Jesús
J
D000483:Alleles; D002466:Cell Nucleolus / Q000378:metabolism; D053487:DEAD-box RNA Helicases; D008970:Molecular Weight; D046912:Multiprotein Complexes / Q000378:metabolism; D050505:Mutant Proteins / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000172:deficiency / Q000378:metabolism*; D010641:Phenotype; D011485:Protein Binding; D021381:Protein Transport; D012316:RNA Nucleotidyltransferases / Q000378:metabolism; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D012335:RNA, Ribosomal / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D012269:Ribosomal Proteins / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015534:Trans-Activators / Q000378:metabolism
D046912:Multiprotein Complexes; D050505:Mutant Proteins; C494530:Nop8 protein, S cerevisiae; D009687:Nuclear Proteins; D012322:RNA Precursors; D012335:RNA, Ribosomal; D016601:RNA-Binding Proteins; C494529:RSA3 protein, S cerevisiae; D012269:Ribosomal Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; C488428:URB1 protein, S cerevisiae; C517809:URB2 protein, S cerevisiae; D012316:RNA Nucleotidyltransferases; C490617:DBP6 protein, S cerevisiae; D053487:DEAD-box RNA Helicases