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Proofreading of pre-40S ribosome maturation by a translation initiation factor and 60S subunits

Abstract

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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Figure 1: In vitro RNA cleavage by Nob1 is not affected by sequences 3′ to site A2.
Figure 2: In vitro cleavage in pre-40S particles is stimulated by ATP or GTP addition.
Figure 3: Fun12 is associated with pre-rRNA.
Figure 4: Fun12 is required for efficient pre-20S pre-rRNA processing.
Figure 5: Fun12 is responsible for GTP-mediated stimulation of in vitro cleavage.
Figure 6: Fun12 binding to 25S rRNA is required for efficient 20S processing.
Figure 7: Pre-40S particles stably associate with Fun12 and mature 60S particles before final 40S particle maturation.
Figure 8: Model for the role of Fun12 in pre-40S processing.

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Acknowledgements

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.

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S.L., C.S., R.W.v.N., A.S., S.G., N.J.W. and D.T. designed experiments; S.L., C.S., R.W.v.N., A.S., D.W., B.B. and S.G. performed experiments; S.L., C.S., R.W.v.N., A.S., B.B., S.G., N.J.W. and D.T. analyzed data; and S.L., C.S., R.W.v.N., A.S., S.G., N.J.W. and D.T. wrote the paper.

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Correspondence to David Tollervey.

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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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