Spliceosomal DEAH-Box ATPases Remodel Pre-mRNA to Activate Alternative Splice Sites
During pre-mRNA splicing, a central step in the expression and regulation of eukaryotic genes, the spliceosome selects splice sites for intron excision and exon ligation. In doing so, the spliceosome must distinguish optimal from suboptimal splice sites. At the catalytic stage of splicing, suboptimal splice sites are repressed by the DEAH-box ATPases Prp16 and Prp22. Here, using budding yeast, we show that these ATPases function further by enabling the spliceosome to search for and utilize alternative branch sites and 3′ splice sites. The ATPases facilitate this search by remodeling the splicing substrate to disengage candidate splice sites. Our data support a mechanism involving 3′ to 5′ translocation of the ATPases along substrate RNA and toward a candidate site, but, surprisingly, not across the site. Thus, our data implicate DEAH-box ATPases in acting at a distance by pulling substrate RNA from the catalytic core of the spliceosome.
Additional Information© 2016 Elsevier Under an Elsevier user license. Received 23 July 2014, Revised 8 January 2016, Accepted 15 January 2016, Available online 25 February 2016. We thank B. Schwer and C. Guthrie for plasmids and antibodies, J. Abelson and C. Guthrie for sharing unpublished data, the members of the J.P.S. and N.G.W. laboratories for helpful discussions, especially S. Fica, M. Kahlscheuer, and R. Toroney, who also provided experimental assistance, and C. Guthrie, A. Hoskins, and J. Piccirilli for comments on the manuscript. D.R.S. and M.R.B. were supported by a Genetics and Regulation Training Grant (T32GM07197) and a Cellular and Molecular Biology Training Grant (T32GM007315), respectively, both from the NIH. This work was funded by grants from the NIH (R01GM062264 and R01GM062264-08S1 to J.P.S.; R01GM098023 to N.G.W.). Author Contributions. D.R.S. and J.P.S. designed the experiments, interpreted the data, and wrote the paper with input from M.R.B. and N.G.W. D.R.S. generated reagents and performed all of the biochemistry experiments. D.R.S. and M.R.B. performed single-molecule FRET experiments. M.R.B. performed Gaussian fitting of histograms and statistical analysis of FRET trajectories.
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