Opposing classes of prp8 alleles modulate the transition between the catalytic steps of pre-mRNA splicing

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Abstract

The spliceosome is thought to undergo a conformational change between the two catalytic steps of precursor messenger RNA splicing, although the specific events in this transition are poorly understood. We previously proposed a two-state model of splicing in which the conformations required for the first and second steps are in competition. Here, we identify and characterize a class of prp8 mutants that suppress first-step splicing defects and oppose the action of the previously described prp8 suppressors of second-step defects; these opposing effects parallel those of ribosomal 'ram' and 'restrictive' mutants, which alter fidelity of transfer RNA decoding. On the basis of genetic interactions, we propose that prp8-mediated substrate repositioning during the transition occurs between catalytic-center opening and closure mediated by the U6 small nuclear RNA and the DExH/D ATPase gene prp16. Modulation of these events alters splice-site selection and splicing fidelity.

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Figure 1: The prp8-R1753K allele suppresses first-step defects.
Figure 2: First-step prp8 alleles improve the first step (when limiting) and inhibit the second step.
Figure 3: Combination of first- and second-step suppressor mutations in the same Prp8 polypeptide restores a splicing phenotype similar to that of WT Prp8.
Figure 4: prp8 and U6-U57C alleles affect splicing at distinct steps.
Figure 5: prp8 and prp16 alleles affect splicing at distinct steps.
Figure 6: Proposed contributions of Prp8, U6 snRNA, Prp16 and Prp22 to distinct steps in spliceosomal transitions.

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Acknowledgements

We thank B. Schwer (Weill Cornell Medical College) for prp16 and prp22 mutant plasmids, C. Guthrie (University of California, San Francisco) for prp8-101 plasmid, D. McPheeters (Case Western Reserve University) for U6-U57 mutant plasmids, and D. Smith, J. Warner and members of our laboratories for helpful discussions and critical readings of the manuscript. This work was supported by US National Institutes of Health grants GM49044 to M.M.K. and GM57829 to C.C.Q., and by a Cancer Center Support (core) grant from the National Cancer Institute to Albert Einstein College of Medicine.

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Correspondence to Charles C Query or Maria M Konarska.

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Liu, L., Query, C. & Konarska, M. Opposing classes of prp8 alleles modulate the transition between the catalytic steps of pre-mRNA splicing. Nat Struct Mol Biol 14, 519–526 (2007) doi:10.1038/nsmb1240

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