To locate key RNA features in the structure of the spliceosome by EM, we fused a sequence-specific RNA binding protein to a protein with a distinct donut-shaped structure. We used this fusion to label spliceosomes assembled on a pre-mRNA that contained the target sequence in the exons. The label is clearly visible in EM images of the spliceosome, and subsequent image processing with averaging shows that the exons sit close to each other in the complex. This labeling strategy will serve as a general tool for analyzing the structures of RNA-containing macromolecular complexes.
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J. Ilagan provided support for EM data collection and image processing. M. O' Donnell (Rockefeller University, New York, USA) provided a plasmid encoding full-length DnaN and K. Collins (University of California, Berkeley, USA) gave us pET28ZZTPP7H including the PP7 sequence. We thank N. Grigorieff (Brandeis University, Waltham, Massachusetts, USA) and members of the Jurica laboratory for advice and discussion. This work was funded by US National Institutes of Health grant 5R01GM72649 to M.S.J., which included a Diversity Supplement for E.A.A.
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Alcid, E., Jurica, M. A protein-based EM label for RNA identifies the location of exons in spliceosomes. Nat Struct Mol Biol 15, 213–215 (2008). https://doi.org/10.1038/nsmb.1378
Development of a new protein labeling system to map subunits and domains of macromolecular complexes for electron microscopy
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