Imaging of RNA-protein interactions in splicing complexes with dark-field STEM.

ABSTRACT

Splicing complexes were assembled in vitro on pre-messenger RNA. These complexes represent stages in the processing of pre-mRNA by snRNPs and accessory factors to remove the introns, thus forming messenger RNA. Complexes were purified using biotinylated oligonucleotide tags together with streptavidin-gold and simple centrifugation. The oligonucleotide tags with attached streptavidin-gold also serve as unambiguous labels of the splicing complexes for EM observation. The complexes were observed using dark-field scanning transmission electron microscopy (STEM). The high contrast and the high efficiency in detecting scattered electrons make it possible to visualize specimens at high resolution with low radiation dose. STEM visualization of the complexes allows a unique view of the events occurring in the splicing process. Three different classes of complex were identified. These complexes support the currently postulated steps in RNA splicing and demonstrate the dynamic nature of splicing complexes. This approach has broad application in high-resolution structural studies of nucleic acids and nucleic acid-protein interactions.