Abstract
The spliceosome is a multimegadalton RNA-protein machine that removes noncoding sequences from nascent pre-mRNAs. Recruitment of the spliceosome to splice sites and subsequent splicing require a series of dynamic interactions among the spliceosome's component U snRNPs and many additional protein factors. These dynamics present several challenges for structural analyses, including purification of stable complexes to compositional homogeneity and assessment of conformational heterogeneity. We have isolated spliceosomes arrested before the second chemical step of splicing (C complex) in which U2, U5 and U6 snRNAs are stably associated. Using electron microscopy, we obtained images of C complex spliceosomes under cryogenic conditions and determined a three-dimensional structure of a core complex to a resolution of 30 Å. The structure reveals a particle of dimensions 27 × 22 × 24 nm with a relatively open arrangement of three primary domains.
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Acknowledgements
We thank T. Walz and M. Ohi for discussion and H. Stark for advice on sample preparation. N.G. is an assistant investigator and M.J.M is an associate investigator with the Howard Hughes Medical Institute; M.S.J. is a Paul Sigler/Agouron Institute fellow of the Helen Hay Whitney Foundation; D.R.S. is supported by a US National Science Foundation integrative graduate education and research training grant. This work was supported by US National Institutes of Health grant 1 P01 GM-62580 (N.G.) and GM 53007 (M.J.M.) and funding from the Keck Foundation.
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Jurica, M., Sousa, D., Moore, M. et al. Three-dimensional structure of C complex spliceosomes by electron microscopy. Nat Struct Mol Biol 11, 265–269 (2004). https://doi.org/10.1038/nsmb728
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DOI: https://doi.org/10.1038/nsmb728
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