Live Cell Imaging of Endogenous RNAs Using Pumilio Homology Domain Mutants: Principles and Applications

Abstract

Recently, dynamic changes in the location of RNA in space and time in living cells have become a target of interest in biology because of their essential roles in controlling physiological phenomena. To visualize RNA, methods for the fluorescent labeling of RNA in living cells have been developed. For RNA labeling, oligonucleotide-based RNA probes have mainly been used because of their high selectivity for target RNAs. By contrast, protein-based RNA probes have not been used widely because of their lack of design flexibility, although they have various potential advantages compared with nucleotide-based probes, such as controllability of intracellular localization, high detectability, and ease of introduction into cells and transgenic organisms in a cell type and tissue specific manner by genetic engineering techniques. This Perspective focuses on a possible approach to the development of protein-based RNA probes using Pumilio homology domain (PUM-HD) mutants. The PUM-HD is a domain of an RNA binding protein that allows custom-made modifications to recognize a given eight-base RNA sequence. PUM-HD-based RNA probes have been applied to visualize various RNAs in living cells. Here, the techniques and RNA imaging results obtained using the PUM-HD are introduced.

Import of Non-Coding RNAs into Human Mitochondria: A Critical Review and Emerging Approaches

“…Another group of methods is based on the detection of endogenous RNAs and does not need introduction of recombinant transcripts. In live cells, endogenous RNAs can be imaged with the use of designer sequence-specific Pumilio homology domain mutants [134]. Nowadays, the most popular and reliable technique for direct observation of endogenous RNAs is fluorescence in situ hybridization (FISH).…”

“…An original protein-based probe for imaging of endogenous RNAs has been developed on the basis of Pumilio homology domain (PUM-HD) mutants that have the ability to recognize single-stranded RNAs in a programmable and modular manner [134]. Ozawa et al constructed two mitochondrially targeted Pumilio proteins directed to two eight-base sequences in the mitochondrial MT-ND6 mRNA, which reconstitute a full-size fluorescent protein upon RNA binding [141].…”

Rhenium (I) Complexes as Probes for Prokaryotic and Fungal Cells by Fluorescence Microscopy: Do Ligands Matter?

“…Cell imaging has become a powerful tool to reveal particular biological structures and explore molecular mechanisms, unraveling dynamics, and functions of many different cellular processes (Rabuka et al, 2008; Hensle and Blum, 2013; Hananya et al, 2016; Majumder et al, 2016; Cui et al, 2017; Yoshimura, 2018). Accordingly, development of diverse transmitted light microscopy approaches, including fluorescence microscopy, is increasingly contributing to improve this technique (Roeffaers et al, 2008; Hauser et al, 2017).…”

Understanding spatiotemporal coupling of gene expression using single molecule RNA imaging technologies

“…Over the past three decades, numerous stem-loops have been optimized for aptamer-based mRNA imaging. They are derived either from bacteriophages (e.g., MS2 [ 107 ], PP7 [ 116 , 117 ], P22 [ 118 ]) or from RNA-protein binding pairs (e.g., U1A [ 119 , 120 ], Bgl bacterial anti-terminator [ 121 ], Pumilio recognition motif [ 122 ]). Association of stem-loop arrays with cognate RNA binding proteins (RBP) fused to an FP (e.g., GFP) or a fluorogenic tagging system (e.g., HALO) allows detecting individual mRNAs in living cells.…”