Methodologies for studying the spliceosome's RNA dynamics with single-molecule FRET

- Single-molecule FRET is useful for studying spliceosome RNA dynamics.
- This review summarizes steps involved in a typical single-molecule FRET experiment.
- These methods have been used to study snRNA and pre-mRNA dynamics during splicing.

The spliceosome is an extraordinarily dynamic molecular machine in which significant changes in composition as well as protein and RNA conformation are required for carrying out pre-mRNA splicing. Single-molecule fluorescence resonance energy transfer (smFRET) can be used to elucidate these dynamics both in well-characterized model systems and in entire spliceosomes. These types of single-molecule data provide novel information about spliceosome components and can be used to identify sub-populations of molecules with unique behaviors. When smFRET is combined with single-molecule fluorescence colocalization, conformational dynamics can be further linked to the presence or absence of a given spliceosome component. Here, we provide a description of experimental considerations, approaches, and workflows for smFRET with an emphasis on applications for the splicing machinery.