The Splicing ATPase Prp43p Is a Component of Multiple Preribosomal Particles

Abstract

Prp43p is a putative helicase of the DEAH family which is required for the release of the lariat intron from the spliceosome. Prp43p could also play a role in ribosome synthesis, since it accumulates in the nucleolus. Consistent with this hypothesis, we find that depletion of Prp43p leads to accumulation of 35S pre-rRNA and strongly reduces levels of all downstream pre-rRNA processing intermediates. As a result, the steady-state levels of mature rRNAs are greatly diminished following Prp43p depletion. We present data arguing that such effects are unlikely to be solely due to splicing defects. Moreover, we demonstrate by a combination of a comprehensive two-hybrid screen, tandem-affinity purification followed by mass spectrometry, and Northern analyses that Prp43p is associated with 90S, pre-60S, and pre-40S ribosomal particles. Prp43p seems preferentially associated with Pfa1p, a novel specific component of pre-40S ribosomal particles. In addition, Prp43p interacts with components of the RNA polymerase I (Pol I) transcription machinery and with mature 18S and 25S rRNAs. Hence, Prp43p might be delivered to nascent 90S ribosomal particles during pre-rRNA transcription and remain associated with preribosomal particles until their final maturation steps in the cytoplasm. Our data also suggest that the ATPase activity of Prp43p is required for early steps of pre-rRNA processing and normal accumulation of mature rRNAs.

ACKNOWLEDGMENTS

We thank J. Staley (University of Chicago) and S. Stevens (University of Texas at Austin) for communicating results prior to publication. The gifts of plasmid pBS1539 from B. Séraphin (CNRS, Gif-sur-Yvette) are gratefully acknowledged. We are thankful to members of the Ferrer laboratory 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.

S.L. is a recipient of a postgraduate fellowship from the Ministère Délégué à l'Enseignement Supérieur età la Recherche. This work was supported by the CNRS, the Université Paul Sabatier, and grants from La Ligue Nationale contre le Cancer (“Equipe Labelisée”) to M.C.-F. and from the Région Midi-Pyrénées and the Génopole Toulouse Midi-Pyrénées to B.M.