Npa1p, a Component of Very Early Pre-60S Ribosomal Particles, Associates with a Subset of Small Nucleolar RNPs Required for Peptidyl Transferase Center Modification

Abstract

We have identified a novel essential nucleolar factor required for the synthesis of 5.8S and 25S rRNAs termed Npa1p. In the absence of Npa1p, the pre-rRNA processing pathway leading to 5.8S and 25S rRNA production is perturbed such that the C2 cleavage within internal transcribed spacer 2 occurs prematurely. Npa1p accumulates in the immediate vicinity of the dense fibrillar component of the nucleolus and is predominantly associated with the 27SA2 pre-rRNA, the RNA component of the earliest pre-60S ribosomal particles. By mass spectrometry, we have identified the protein partners of Npa1p, which include eight putative helicases as well as the novel Npa2p factor. Strikingly, we also show that Npa1p can associate with a subset of H/ACA and C/D small nucleolar RNPs (snoRNPs) involved in the chemical modification of residues in the vicinity of the peptidyl transferase center. Our results suggest that 27SA2-containing pre-60S ribosomal particles are located at the interface between the dense fibrillar and the granular components of the nucleolus and that these particles can contain a subset of snoRNPs.

We are very grateful to R. Capeyrou for experimental help. The gifts of plasmids pBS1479 from B. Séraphin (CNRS, Gif-sur-Yvette, France), pTL26 and pTL27 from D. Lafontaine (Université Libre de Bruxelles), of strain YDL402 from D. Lafontaine, and monoclonal anti-L3 antibody and polyclonal anti-S8 serum from J. Warner (Albert Einstein College of Medicine, Bronx, N.Y.) are gratefully acknowledged. We are thankful to members of the Ferrer lab for help and numerous discussions. We thank Y. de Préval for synthesis of oligonucleotides, D. Villa for art work, and A. Rivals for expert technical assistance. We are grateful to J. Rouquette and P.-E. Gleizes for advice. Detection of Npa1p-ZZ by immunoelectron microscopy was performed in the lab of N. Gas.

C.D. is a recipient of a postgraduate fellowship from the Association pour la Recherche contre le Cancer (ARC). This work was supported by the CNRS, the Université Paul Sabatier, and grants from La Ligue Nationale contre le Cancer (Equipe Labelisée) and the ACI program of the Ministère Délégué à la Recherche et aux Nouvelles Technologies to M.C.-F and from the Région Midi-Pyréneés and the Génopole Toulouse Midi-Pyrénées to B.M.