Npa1p is an essential trans-acting factor required for an early step in the assembly of 60S ribosomal subunits in Saccharomyces cerevisiae

Abstract

Ribosome biogenesis requires >100 nonribosomal proteins, which are associated with different preribosomal particles. The substrates, the interacting partners, and the timing of action of most of these proteins are largely unknown. To elucidate the functional environment of the putative ATP-dependent RNA helicase Dbp6p from Saccharomyces cerevisiae, which is required for 60S ribosomal subunit assembly, we have previously performed a synthetic lethal screen and thereby revealed a genetic interaction network between Dbp6p, Rpl3p, Nop8p, and the novel Rsa3p. In this report, we extended the characterization of this functional network by performing a synthetic lethal screen with the rsa3 null allele. This screen identified the so far uncharacterized Npa1p (YKL014C). Polysome profile analysis indicates that there is a deficit of 60S ribosomal subunits and an accumulation of half-mer polysomes in the slowly growing npa1-1 mutant. Northern blotting and primer extension analysis shows that the npa1-1 mutation negatively affects processing of all 27S pre-rRNAs and the normal accumulation of both mature 25S and 5.8S rRNAs. In addition, 27SA 2 pre-rRNA is prematurely cleaved at site C 2 . Moreover, GFP-tagged Npa1p localizes predominantly to the nucleolus and sediments with large complexes in sucrose gradients, which most likely correspond to pre-60S ribosomal particles. We conclude that Npa1p is required for ribosome biogenesis and operates in the same functional environment of Rsa3p and Dbp6p during early maturation of 60S ribosomal subunits.

Rrp15p, a novel component of pre-ribosomal particles required for 60S ribosome subunit maturation

“…6E). This phenotype has been already described for other components of early pre-60S particles (Fatica et al , 2003Dez et al 2004;Rosado and de La Cruz 2004). Decrease levels of 5.8S and 5S mature rRNAs were also observed (Fig.…”

“…Coprecipitation of the 27SA 2 , 27SA 3 , and 27SB, with lower efficiency of 7S pre-rRNAs, but not of the mature 5.8S and 25S rRNAs indicate that Rrp15p remains associated in pre-ribosomal particles during all the processing steps of the 27SA 2 , dissociating at C 2 cleavage. Indeed, as reported for other pre-60S components (Fatica et al , 2003Dez et al 2004;Rosado and de La Cruz 2004), the presence of Rrp15p is required to block premature cleavage of 27SA 2 at site C 2 . This leads to the conclusion that the still unidentified endonuclease responsible for this cleavage event can act even on a not properly assembled preribosome and that the function of some pre-60S components is to provide quality control systems that guarantee the right timing of the 27SA 2 processing events.…”

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

“…Here, we provide evidence that the predominantly nucleolar Npa2p protein is an additional member of the aforementioned network of proteins. Our results indicate that Npa2p is required for 27S pre-rRNA processing and suggest that Npa2p has an early role during 60S r-subunit assembly similar to that described previously for Dbp6p (56), Dbp7p (12), Dbp9p (11), Nop8p (108), Npa1p (19,70,78), or Rsa3p (16,78). Recently, some of us have reported the composition of very early pre-60S r-particles purified using Npa1p-TAP as bait (19).…”

“…Most yeast strains used in this study (see Table S1 in the supplemental material) are derivatives of strain W303 (MATa/MAT␣ ade2-1/ade2-1 his3- 11,15/his3-11,15 leu2-3,112/leu2-3,112 trp1-1/ trp1-1 ura3-1/ura3-1 can1-100/can1-100). The isolation of synthetic lethal (sl) mutants has been previously described (78). Strain IVY317 [YCplac33-NPA2] was obtained after four consecutive backcrosses of W303-1A with a meiotic npa2::KanMX4 segregant of Y26839 (Euroscarf collection) containing the YCplac33-NPA2 plasmid.…”

“…We are interested in the identification of partners of the putative RNA helicase Dbp6p, which is required for 60S r-subunit assembly and has been proposed to act at an early step during this process (56). Our initial genetic and functional analyses have revealed specific interactions between Dbp6p, different 60S r-subunit assembly factors (Dbp7p, Dbp9p, Nop8p, Npa1p/Urb1p, and Rsa3p), and the 60S r-protein Rpl3p (16,78). Here, we provide evidence that the predominantly nucleolar Npa2p protein is an additional member of the aforementioned network of proteins.…”