Expanded CAG trinucleotide repeats in Huntington’s disease (HD) are causative for neurotoxicity. The mutant CAG repeat RNA encodes neurotoxic polyglutamine proteins and can lead to a toxic gain of function by aberrantly recruiting RNA-binding proteins. One of these is the MID1 protein, which induces aberrant Huntingtin (HTT) protein translation upon binding. Here we have identified a set of CAG repeat binder candidates by in silico methods. One of those, furamidine, reduces the level of binding of HTT mRNA to MID1 and other target proteins in vitro. Metadynamics calculations, fairly consistent with experimental data measured here, provide hints about the binding mode of the ligand. Importantly, furamidine also decreases the protein level of HTT in a HD cell line model. This shows that small molecules masking RNA–MID1 interactions may be active against mutant HTT protein in living cells.

Reducing Mutant Huntingtin Protein Expression in Living Cells by a Newly Identified RNA CAG Binder

Traditionally, studies on protein translation rely on systems, in which cells have been lysed prior determination of levels of the protein of interest. However, these assays do not reflect the protein synthesis in living cells in real time, but analyze protein levels after a given incubation time, leading to limitations in results based on experimental parameters. To overcome this problem, we have previously established a Fluorescence recovery after photobleaching (FRAP)-based technique to monitor protein translation in living cells. For this, the protein of interest fused to green fluorescent protein (GFP) is expressed in cell lines. After bleaching the entire cell, the fluorescent signal of the protein of interest is lost, allowing to capture the signal recovery of newly translated GFPtagged protein over time. Here we present two improved versions of this technique using different fluorescent dyes: tFRAP (translational FRAP). For the first improved version of tFRAP we have inserted a second fluorescent dye, red fluorescent protein (RFP), into the same expression vector that drives expression of the protein of interest fused to GFP driven by a second promoter. For the second improved version of tFRAP we have fused our protein of interest to a photo-switchable dye, Dendra2. Both improved versions allow to correct the fluorescence signal intensity of the protein of interest for different transfection rates of individual cells. These two advanced techniques are new powerful tools for quantifying translation rates in living cells and will be useful in future studies on mRNA translation.

Sybille Krau

Papers

tFRAP: A FRAP-Based Technique to Monitor Protein Translation in Living Cells