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Exon ligation is proofread by the DExD/H-box ATPase Prp22p

Nature Structural & Molecular Biology volume 13, pages 482–490 (2006)Cite this article

Abstract

To produce messenger RNA, the spliceosome excises introns from precursor (pre)-mRNA and splices the flanking exons. To establish fidelity, the spliceosome discriminates against aberrant introns, but current understanding of such fidelity mechanisms is limited. Here we show that an ATP-dependent activity represses formation of mRNA from aberrant intermediates having mutations in any of the intronic consensus sequences. This proofreading activity is disabled by mutations that impair the ATPase or RNA unwindase activity of Prp22p, a conserved spliceosomal DExD/H-box ATPase. Further, cold-sensitive prp22 mutants permit aberrant mRNA formation from a mutated 3′ splice-site intermediate in vivo. We conclude that Prp22p generally represses splicing of aberrant intermediates, in addition to its known ATP-dependent role in promoting release of genuine mRNA. This dual function for Prp22p validates a general model in which fidelity can be enhanced by a DExD/H-box ATPase.

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Figure 1: An ATP-dependent mechanism represses exon ligation at near-consensus 3′ splice sites.
Figure 2: An ATP-dependent mechanism represses exon ligation at a mutated, but not a wild-type, 3′ splice site.
Figure 3: Repression of aberrant exon ligation at mutated 3′ splice sites is compromised in vitro and in vivo by ATPase- and RNA unwindase–deficient variants of the DEAH-box ATPase Prp22p.
Figure 4: An ATP- and Prp22p-dependent mechanism also represses aberrant exon ligation of intermediates having mutations of the branch site or 5′ splice site.
Figure 5: A dual role for Prp22p in fidelity and mRNA release suggests a proofreading mechanism7,8,9 for exon ligation.

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Acknowledgements

We thank S.-C. Cheng (Academia Sinica) for the gift of antibodies to Ntc20; D. Bishop, L. Cochella, B. Glick, R. Green, J. Piccirilli, H. Singh, E. Sontheimer and members of the Staley laboratory for critical reading of the manuscript; and C. Jordan, V. Shaw and M. Norman for technical assistance. This research was supported by a predoctoral fellowship from the Ford Foundation to R.M.M. and by grants from the US National Institutes of Health and the Packard Foundation to J.P.S.

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  1. Hiroshi Maita

    Present address: Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-ku, Sapporo, 060-0812, Japan

Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, The University of Chicago, Cummings Life Science Center 817, 920 E. 58th Street, Chicago, 60637, Illinois, USA

    Rabiah M Mayas

  2. Department of Molecular Genetics and Cell Biology, The University of Chicago, Cummings Life Science Center 817, 920 E. 58th Street, Chicago, 60637, Illinois, USA

    Hiroshi Maita & Jonathan P Staley

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Supplementary information

Supplementary Fig. 1

A prp43 mutant does not permit exon ligation at an aberrant 3′ splice site (PDF 721 kb)

Supplementary Methods

Oligonucleotides, plasmids, strains and RT-PCR (PDF 106 kb)

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Mayas, R., Maita, H. & Staley, J. Exon ligation is proofread by the DExD/H-box ATPase Prp22p. Nat Struct Mol Biol 13, 482–490 (2006). https://doi.org/10.1038/nsmb1093

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