Coming Together: RNAs and Proteins Assemble under the Single-Molecule Fluorescence Microscope

RNAs, across their numerous classes, often work in concert with proteins in RNA-protein complexes (RNPs) to execute critical cellular functions. Ensemble-averaging methods have been instrumental in revealing many important aspects of these RNA-protein interactions, yet are insufficiently sensitive to much of the dynamics at the heart of RNP function. Singlemolecule fluorescence microscopy (SMFM) offers complementary, versatile tools to probe RNP conformational and compositional changes in detail. In this review, we first outline the basic principles of SMFM as applied to RNPs, describing key considerations for labeling, imaging, and quantitative analysis. We then sample applications of in vitro and in vivo single-molecule visualization using the case studies of pre-messenger RNA (mRNA) splicing and RNA silencing, respectively. After discussing specific insights single-molecule fluorescence methods have yielded, we briefly review recent developments in the field and highlight areas of anticipated growth. Outline 1 Introduction 2 General considerations for applying singlemolecule fluorescence microscopy to RNA-protein interactions 3 Application example of in vitro singlemolecule fluorescence: The spliceosome 4 Deeper considerations for studying single RNPs in vitro 5 Single-RNP visualization in living cells 6 Application example of in vivo singlemolecule fluorescence: RNA silencing 7 Conclusions and outlook