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

Methods : a Companion to Methods in Enzymology
Clarisse van der Feltz, Aaron A Hoskins

Abstract

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.

Citations

Aug 21, 2019·Wiley Interdisciplinary Reviews. RNA·Donny D LicatalosiEckhard Jankowsky
Jun 17, 2020·Wiley Interdisciplinary Reviews. RNA·Fatu Badiane MarkeyMona Batish
Apr 4, 2021·Journal of Molecular Biology·Leonard Schärfen, Karla M Neugebauer

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