In the final steps of yeast ribosome synthesis, immature translation-incompetent pre-40S particles that contain 20S pre-rRNA are converted to the mature translation-competent subunits containing the 18S rRNA. An assay for 20S pre-rRNA cleavage in purified pre-40S particles showed that cleavage by the PIN domain endonuclease Nob1 was strongly stimulated by the GTPase activity of Fun12, the yeast homolog of cytoplasmic translation initiation factor eIF5b. Cleavage of the 20S pre-rRNA was also inhibited in vivo and in vitro by blocking binding of Fun12 to the 25S rRNA through specific methylation of its binding site. Cleavage competent pre-40S particles stably associated with Fun12 and formed 80S complexes with 60S ribosomal subunits. We propose that recruitment of 60S subunits promotes GTP hydrolysis by Fun12, leading to structural rearrangements within the pre-40S particle that bring Nob1 and the pre-rRNA cleavage site together.
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This work was supported by the Wellcome Trust (S.L., C.S., S.G. and D.T., 077248; R.W.v.N. and N.J.W., WT089378MA), the Royal Society (C.S.), The Darwin Trust of Edinburgh (B.B.) and an European Molecular Biology Organisation Long-Term Fellowship (S.L.). The electron microscopy facility was supported by the Wellcome Trust and the Scottish Universities Life Sciences Alliance. Work in the Wellcome Trust Centre for Cell Biology is supported by Wellcome Trust core funding (092076). We thank E. Fayet-Lebaron for her technical support and critical reading of the manuscript. We thank members of the Edinburgh Protein Production Facility for protein purification. Use of the Edinburgh Protein Production Facility was supported by The Wellcome Trust, the Scottish Universities Life Sciences Alliance and the Biotechnology and Biological Sciences Research Council.
The authors declare no competing financial interests.
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Lebaron, S., Schneider, C., van Nues, R. et al. Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits. Nat Struct Mol Biol 19, 744–753 (2012). https://doi.org/10.1038/nsmb.2308
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