A single-molecule fluorescence imaging approach would complement these cellular and biochemical methods by providing precise spatiotemporal molecular information, including variance among mRNAs. Recently, translating RNA imaging by coat protein knock off (TRICK) ( 15) distinguished previously translated from untranslated mRNAs. Fluorescent protein–based translation assays ( 11– 14) require lengthy maturation of fluorophores. Cellular approaches, such as fluorescent noncanonical amino acid tagging (FUNCAT) ( 9) and puromycylation ( 10), measure overall protein synthesis but are not gene-specific. Pulse-labeling in cell culture quantifies newly synthesized proteins by means of mass spectrometry ( 1). Approaches that measure the association of ribosomes with mRNAs are not direct readouts of translation ( 8). Ensemble biochemical measurements such as ribosome profiling can provide a genome-wide measurement of translation ( 6, 7). However, the translation of localized mRNA in living cells remains poorly understood because unlike transcription, a single-molecule method to directly image the process is lacking ( 5). Numerous studies have concentrated on RNA localization and its underlying mechanism ( 4). Translational regulation allows cells to respond rapidly to environmental cues and synthesize proteins with precise timing and at specific subcellular locations ( 2, 3). Genome-wide studies have shown that protein abundance is predominantly controlled at the level of translation ( 1).
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