FlAsH Forward

Small fluorescent labels such as the fluorescein arsenical hairpin binder (FlAsH) make popular alternatives to bulky fluorescent proteins. FlAsH, along with its color variant ReAsH (red arsenical hairpin binder) interacts with a tetra-cysteine motif whose small size minimizes disruption to protein function. In addition (because it doesn't require a flexible linker), the fluorophore is locked into a more easily definable position, a feature that is appealing for Förster resonance energy transfer (FRET) experiments. In previous FRET studies, FlAsH has been used in combination with cyan fluorescent protein (CFP), so the potential limitations associated with fluorescent proteins remain a concern. A more effective system would be to use FlAsH and ReAsH as FRET partners. Although a sequential labeling strategy for these fluorophores has been described in purified proteins, the method didn't address selective labeling of protein sites in intact cells. In Bioconjugate Chemistry, Zürn et al. fill this gap by showing selective labeling of the membrane-bound parathyroid hormone (PTH) receptor and its cytosolic binding partner ß-arrestin2. The strategy rests on placing a 12–amino acid high-affinity FlAsH/ReAsH binding motif in one protein, and a lower-affinity binding motif (of 6 amino acids) in the other. After incubation with ReAsH, the cells are washed with dimercaptopropanol, a competitive inhibitor of FlAsH/ReAsH binding. The dimercaptopropanol concentration is set so that the fluorophore's binding to the lower-affinity sequence is nearly fully outcompeted, while its interaction with the high-affinity motif is essentially unaffected. When the cells are subsequently incubated with FlAsH, the lower-affinity motifs become labeled, but the high-affinity motifs remain bound to ReAsH. This simple approach delivers selective labeling with minimal background, the proteins can be tracked by confocal microscopy, and the fluorescent tags do not interfere with normal behavior of the proteins in response to ligand binding to the PTH receptor. Measurements of the emission ratios of FlAsH and ReAsH showed that FRET occurred when colocalization was triggered. The system worked equally well when the binding motifs were swapped between the two proteins, suggesting the domains perform equivalently in different structural contexts. Ongoing development of brighter biarsenical fluorophore tags of various colors should open new opportunities for cell labeling and tracking.

Sequential labeling strategy for selective binding of ReAsH and FlAsH to distinct proteins. (Click to enlarge)

Zürn et al. Site-specific, orthogonal labeling of proteins in intact cells with two small biarsenical fluorophores. Bioconjug Chem. [Epub ahead of print, April 29, 2010; doi: 10.1021/bc900394j].

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The importance of the 3' untranslated region (UTR) to post-transcriptional regulation of eukaryotic gene expression is increasingly appreciated but hard to study in isolation. A central difficulty is that stimuli that act on UTRs often also affect promoter activity. For instance, AU-rich mRNA destabilizing elements (AREs) respond to cytokines such as tumor necrosis factor a (TNF-a) and interleukin 1-a(IL-1a), both of which induce the cytomegalovirus (CMV) promoter. In an article in RNA, Hitti et al. introduce a new reporter that permits selective examination of the regulatory effect of the 3' end of the transcript. The authors designed a green fluorescent protein (GFP) reporter construct where expression is driven by an optimized promoter from the ribosomal protein S30 (RPS30), which shows that this modified promoter is impervious to various stress inducers known to act through ARE-mediated pathways. When AREs from IL-8 or TNF-a were placed within the reporter, fluorescence was significantly more reduced (reflecting ARE-mediated transcript destabilization) than in a CMV-driven construct, where the effect was likely obscured by promoter effects. In further studies, the authors explain how comparing RNA level (by quantitative reverse transcription PCR) and protein amount (by GFP fluorescence) helps coax apart transcript stability and translational blockage. The system is also suitable for studying regulation by RNA-binding proteins, and gives more meaningful results than traditional reporter vectors. For example, cotransfection with a plasmid expressing the ARE-binding protein HuR increases signal from a CMV-driven reporter even in the absence of an ARE. In contrast, the modulation of the RPS30-based reporter is ARE-dependent. A helpful feature of the reporter is that transcriptional response elements can easily be appended, permitting study of the combined effects of transcriptional and post-transcriptional modulation. The authors also describe a Tet-Off variant of the modified RPS30 promoter for mRNA half-life determinations without the need for agents such as actinomycin D, which can have nonspecific toxicity. These features make the new reporter a flexible tool for analyzing mRNA regulation, all in the context of a robust promoter and modular structure that simplifies the study of functional elements, particularly in the 3' end of the transcript.

Hitti et al. A versatile ribosomal protein promoter-based reporter system for selective assessment of RNA stability and post-transcriptional control. RNA. [Epub ahead of print, April 23, 2010; doi: 10.1261/rna.2026310].