A protein-based EM label for RNA identifies the location of exons in spliceosomes.
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.
Alcid
Eric A
EA
Department of Molecular, Cell and Developmental Biology and Center for Molecular Biology of RNA, University of California, 1156 High Street, Santa Cruz, California 95064, USA.
Jurica
Melissa S
MS
D005091:Exons; D006367:HeLa Cells; D006801:Humans; D007091:Image Processing, Computer-Assisted; D046529:Microscopy, Electron, Transmission; D011485:Protein Binding; D012322:RNA Precursors / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D017381:Spliceosomes / Q000737:chemistry / Q000648:ultrastructure*; D013194:Staining and Labeling / Q000379:methods*
D012322:RNA Precursors; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins