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Genetic evidence for 18S rRNA binding and an Rps19p assembly function of yeast nucleolar protein Nep1p

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Abstract

The nucleolar protein Nep1 and its human homologue were previously shown to be involved in the maturation of 18S rRNA and to interfere directly or indirectly with a methylation reaction. Here, we report that the loss-of-function mutation Δsnr57 and multicopy expression of the ribosomal 40S subunit protein 19 (Rps19p) can partially suppress the Saccharomyces cerevisiae Δnep1 growth defect. SnR57 mediates 2′-O-ribose-methylation of G1570 in the 18S rRNA. By performing a three-hybrid screen, we isolated several short RNA sequences with strong binding affinity to Nep1p. All isolated RNAs shared a six-nucleotide consensus motif C/UUCAAC. Furthermore, one of the isolated RNAs exactly corresponded to nucleotides 1553–1577 of the 18S rRNA, which includes G1570, the site of snR57-dependent 18S rRNA methylation. From protein–protein crosslink data and the cryo-EM map of the S. cerevisiae small ribosomal subunit, we suggest that Rps19p is localized in close vicinity to the Nep1p 18S rRNA binding site. Our results suggest that Nep1p binds adjacent to helix 47 of the 18S rRNA and possibly supports the association of Rps19p to pre-ribosomal particles.

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Acknowledgments

This work was supported by the Deutsche Forschungsgemeinschaft, SFB 579, Project B3. We thank M. Wickens and S. Fields for yeast three-hybrid reagents and the hybrid RNA expression library and M. Snyder for the mTn3-lacZ/LEU2 library and plasmid pRSQ2-URA3.

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Authors and Affiliations

  1. Institute of Molecular Biosciences, Johann Wolfgang Goethe-University, Marie-Curie-Str. 9, 60439, Frankfurt/Main, Germany

    Markus Buchhaupt, Britta Meyer, Peter Kötter & Karl-Dieter Entian

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  1. Markus Buchhaupt
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  2. Britta Meyer
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  3. Peter Kötter
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  4. Karl-Dieter Entian
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Correspondence to Karl-Dieter Entian.

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Communicated by S. Hohmann

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Buchhaupt, M., Meyer, B., Kötter, P. et al. Genetic evidence for 18S rRNA binding and an Rps19p assembly function of yeast nucleolar protein Nep1p. Mol Genet Genomics 276, 273–284 (2006). https://doi.org/10.1007/s00438-006-0132-x

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  • DOI: https://doi.org/10.1007/s00438-006-0132-x

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