A protein-based EM label for RNA identifies the location of exons in spliceosomes

Abstract

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.

Figure 1

Beta-PP7 label design, RNA binding and imaging. (a) Schematic of Beta-PP7 modeled from the separate X-ray crystallographic structures of the coliphage coat protein PP7 and the βsubunit of E. coli DNA polymerase III, dnaN[ED: OK] ,. dnaN dimerizes to form a ‘donut’ structure (shaded darker gray), and two copies of PP7 (lighter-gray crescents) serve to bind RNA. (b) Western analysis of Beta-PP7 pull-downs. Using a fusion of the coliphage coat protein MS2 and maltose binding protein (MS2-MBP) pull-downs of Beta PP7 were carried out with MS2-tagged pre-mRNA substrates [ED: Changed as requested] either possessing a PP7 site (+) or lacking one (−). The antibody was specific to the 6×His tag on Beta-PP7. (c) EM micrograph of the + PP7 elution stained with 2% (w/v) uranyl acetate. Arrows indicate the Beta-PP7 rings. (d) Schematic of the pre-mRNA substrate for spliceosome purification and labeling. A single PP7 site (large black hairpin) is inserted at indicated sites of a pre-mRNA splicing substrate containing 3 MS2 sites (small gray hairpins) used for affinity purification of C-complex spliceosomes.

Figure 2

Difference maps of two-dimensional averages of labeled and unlabeled C complexes. (a) Filtered single image of C complex labeled at the 5′ exon and the corresponding two-dimensional average. (b) As in a, with the C complex labeled at the 3′ exon instead of the 5′ exon. (c) As in a, with unlabeled C complex. (d) From left to right: the difference map between the two-dimensional averages of 5′ exon – labeled C complex and unlabeled C complex; the statistically significant region of the difference map at the 99% confidence level and the difference map (in white) superimposed on a two-dimensional average of unlabeled C complex. (e) As in d, with the C complex labeled at the 3′ exon instead of the 5′ exon.