Fluorescent Probes for Imaging Endogenous β-Actin mRNA in Living Cells Using Fluorescent Protein-Tagged Pumilio

Abstract

Subcellular localization and dynamics of mRNAs control various physiological functions in living cells. A novel technique for visualizing endogenous mRNAs in living cells is necessary for investigation of the spatiotemporal movement of mRNAs. A pumilio homology domain of human pumilio 1 (PUM-HD) is a useful RNA binding protein as a tool for mRNA recognition because the domain can be modified to bind a specific 8-base sequence of target mRNA. In this study, we designed PUM-HD to match the sequence of β-actin mRNA and developed an mRNA probe consisting of two PUM-HD mutants flanking full-length enhanced green fluorescent protein (EGFP). Fluorescence microscopy with the probe in living cells revealed that the probe was labeled precisely with the β-actin mRNA in cytosol. Fluorescent spots from the probe were colocalized with microtubules and moved directionally in living cells. The PUM-HD mutants conjugated with full-length EGFP can enable visualization of β-actin mRNA localization and dynamics in living cells.

Programmable RNA-binding protein composed of repeats of a single modular unit

“…This assay is sometimes seen as qualitative, because it does not indicate actual binding affinities, but it has proven useful in the study of RNA-binding proteins such as Pumilio because it allows for such interactions to be measured at a functional level in living cells (23)(24)(25)(26). In particular, split fluorescent protein reconstitution was used to test on-target binding of three different Pum variants to NRE variants, and also previously used to visualize binding of PumHD variants to the mRNAs for human β-actin and NADH dehydrogenase subunit 6 (23)(24)(25)(26). Based upon earlier literature,…”

“…Previous works had demonstrated, using proteins that bind to specific RNA sequences, the measurement of mRNA expression level (23,24), imaging of mRNA dynamics (23)(24)(25)(26)32), and enhancement and suppression of mRNA translation (6,30,31,33) with variants of natural RNA-binding proteins. We demonstrated that our Pumby architecture, which uses a single repeated module to support protein generation (analogous to the TALE design), enables performance equivalent to the original Pumilio protein.…”

Pumilio-Based RNA In Vivo Imaging

“…Pumilio-based RNA imaging has enabled subcellular detection of the mRNAs of mitochondrial NADH dehydrogenase and -actin, as well as retroviral RNA in mammalian cultured cells (20)(21)(22)(23), and of the genomes of Tobacco mosaic virus, Potato virus X and Turnip mosaic virus in live plant tissue (24)(25)(26)(27). Of particular relevance to the topic of this volume, Pumilio-based RNA imaging has recently shown that Potato virus X is present in membrane structures at the entrances of plasmodesmata that also contain the viral replicase (27).…”

“…However, a fusion with the FP inserted between the PUMHDs (NLS-PUMHD-FP-PUMHD) was recently used in mammalian cells (23) and may also work in plants.…”

“…PUM-BiFC imaging can be used in two different modes: PUMHDs can be modified to bind to the native RNA of interest (20,21,(23)(24)(25), or the RNA can be tagged with recognition motifs of previously characterised PUMHD variants (22,24,26,27,33) (Table 1). As described in the introduction, leaving the RNA unmodified has the advantage of avoiding potential RNA processing and localisation artefacts that could be caused by a tag, and by the expression of the tagged construct from an artificial genomic context.…”