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Published June 1, 2010 | Published + Supplemental Material
Journal Article Open

Spliceosome discards intermediates via the DEAH box ATPase Prp43p


To promote fidelity in nuclear pre-mRNA splicing, the spliceosome rejects and discards suboptimal substrates that have engaged the spliceosome. Whereas DExD/H box ATPases have been implicated in rejecting suboptimal substrates, the mechanism for discarding suboptimal substrates has remained obscure. Corroborating evidence that suboptimal, mutated lariat intermediates can be exported to the cytoplasm for turnover, we have found that the ribosome can translate mutated lariat intermediates. By glycerol gradient analysis, we have found that the spliceosome can dissociate mutated lariat intermediates in vivo in a manner that requires the DEAH box ATPase Prp43p. Through an in vitro assay, we demonstrate that Prp43p promotes the discard of suboptimal and optimal 5′ exon and lariat intermediates indiscriminately. Finally, we demonstrate a requirement for Prp43p in repressing splicing at a cryptic splice site. We propose a model for the fidelity of exon ligation in which the DEAH box ATPase Prp22p slows the flow of suboptimal intermediates through exon ligation and Prp43p generally promotes discard of intermediates, thereby establishing a pathway for turnover of stalled intermediates. Because Prp43p also promotes spliceosome disassembly after exon ligation, this work establishes a parallel between the discard of suboptimal intermediates and the dissociation of a genuine excised intron product.

Additional Information

© 2010 National Academy of Sciences. Edited by Joan A. Steitz, Howard Hughes Medical Institute, New Haven, CT, and approved April 6, 2010 (received for review June 24, 2009). We thank Joe Piccirilli for comments on the manuscript, Peter Sarnow for strain H2545, Eliza Small and Angela Hilliker for plasmids, John Abelson for the UBC4 transcription template, John Abelson and Soo-Chen Cheng for sharing results before publication, and the Staley lab for helpful discussions. R.M.M. was supported by a Ford Foundation Predoctoral Diversity Fellowship and H.M. by a Yamada Foundation Postdoctoral Fellowship. This work was supported by American Cancer Society Illinois Division Stephen F. Sener, M.D.—Research Scholar Award 06-099-01-GMC (to J.P.S.) and Grant GM62264 (to J.P.S.) from the National Institutes of Health. Materials and Methods. Strains (Table S1), plasmids (Table S2), and further experimental details are provided in SI Text. R.M.M., H.M., and D.R.S. contributed equally to this work. Author contributions: R.M.M., H.M., D.R.S., and J.P.S. designed research; R.M.M., H.M., and D.R.S. performed research; R.M.M., H.M., and D.R.S. contributed new reagents/analytic tools; R.M.M., H.M., D.R.S., and J.P.S. analyzed data; and R.M.M., H.M., D.R.S., and J.P.S. wrote the paper. The authors declare no conflict of interest. This Direct Submission article had a prearranged editor. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.0906022107/-/DCSupplemental.

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Published - 10020.full.pdf

Supplemental Material - pnas.0906022107_SI.pdf


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August 22, 2023
March 5, 2024