New insights into the spliceosome by single molecule fluorescence microscopy

Splicing is an essential eukaryotic process in which introns are excised from precursors to messenger RNAs and exons ligated together. This reaction is catalyzed by a multi-MegaDalton machine called the spliceosome, composed of 5 small nuclear RNAs (snRNAs) and a core set of ∼100 proteins minimally required for activity. Because of the spliceosome's size, its low abundance in cellular extracts, and its highly dynamic assembly pathway, analysis of the kinetics of splicing and the conformational rearrangements occurring during spliceosome assembly and disassembly has proven extraordinarily challenging. Here, we review recent progress in combining chemical biology methodologies with single molecule fluorescence techniques to provide a window into splicing in real time. These methods complement ensemble measurements of splicing in vivo and in vitro to facilitate kinetic dissection of pre-mRNA splicing.

► Pre-mRNA splicing is carried out by a MegaDalton machine called the spliceosome.
► Single molecule fluorescence combined with chemical biology techniques has recently enabled real time visualization of splicing and spliceosome assembly.
► Single molecule FRET measurements reveal pre-mRNA dynamics during splicing.
► Monitoring the assembly of spliceosomes on single pre-mRNAs exposes both ordering and reversibility of key steps.