Griesbach, E. | Search bioRxiv

bioRxiv2025

Quantitative comparison of methodologies for translation site imaging in living cells

Single-molecule imaging of translation sites in living cells has enabled the dynamics of protein synthesis to be investigated with high spatial and temporal resolution. These methodologies utilize the interaction between a multimerized epitope tag and its cognate fluorescent nanobody to detect nascent polypeptides as they emerge from the ribosome. Here, we present a systematic comparison of current methodologies and determine that the ALFA-tag reduces perturbations of mRNA expression and increases the fluorescent signal of translation sites.

biophysics↗

bioRxiv2021

Single-molecule imaging reveals the coupling of translation and mRNA decay

The relationship between mRNA translation and decay is incompletely understood, with conflicting reports suggesting that translation can either promote decay or stabilize mRNAs. The effect of translation on mRNA decay has mainly been studied using ensemble measurements and global inhibitors of transcription and translation, which can mask the underlying mechanisms. We developed a single-molecule imaging approach to control the translation of a specific transcript that enabled simultaneous measurement of translation and mRNA decay. Our results demonstrate that mRNAs undergoing translation are degraded faster than non-translating ones, although with slower kinetics than translation-coupled degradation of transcripts targeted by NMD. Furthermore, our results indicate that miRNAs mediate efficient degradation of both translating and non-translating target mRNAs. Single-molecule measurements of translation and decay reveal a predominant role of mRNA decay in miRNA-mediated regulation. Simultaneous visualization of translation and decay on single mRNAs provides a framework to study how these processes are interconnected in cells.

molecular biology↗