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Abstract
Analysis of general and specific protein synthesis provides important information, relevant to cellular physiology and function. However, existing methodologies, involving metabolic labelling by incorporation of radioactive amino acids into nascent polypeptides, cannot be applied to monitor protein synthesis in specific cells or tissues, in live specimens. We have developed a novel approach for monitoring protein synthesis in specific cells or tissues, in vivo. Fluorescent reporter proteins such as GFP are expressed in specific cells and tissues of interest or throughout animals using appropriate promoters. Protein synthesis rates are assessed by following fluorescence recovery after partial photobleaching of the fluorophore at targeted sites. We evaluate the method by examining protein synthesis rates in diverse cell types of live, wild type or mRNA translation-defective Caenorhabditis elegans animals. Because it is non-invasive, our approach allows monitoring of protein synthesis in single cells or tissues with intrinsically different protein synthesis rates. Furthermore, it can be readily implemented in other organisms or cell culture systems.
Highlights
Proper regulation of protein synthesis is critical for cell growth, cell proliferation and cell death
Overall protein synthesis activity can be assessed by polysomal profiling, which provides a relative estimate of mRNA loading onto actively translating polyribosomes
We used animals expressing GFP throughout somatic tissues under the control of the ife-2 gene promoter. ife-2 encodes one of the five nematode eIF4E isoforms that functions in somatic tissues [4,11]. eIF4E is a key mRNA translation initiation factor that binds the 7-methyl guanosine cap at the 59 end of all nuclear mRNAs and determines the rate of capdependent protein synthesis [12]
Summary
Proper regulation of protein synthesis is critical for cell growth, cell proliferation and cell death. Protein synthesis involves a complex series of protein-protein and protein-RNA interactions which result in the formation of peptide bonds between amino acids, as encoded by the mRNA being translated. One of the most widely used approaches for measuring general protein synthesis rate is metabolic labelling, typically in the form of radioactive amino acid incorporation into nascent polypeptides [5,6]. Overall protein synthesis activity can be assessed by polysomal profiling, which provides a relative estimate of mRNA loading onto actively translating polyribosomes. Polysomal profiling can be adapted to monitor translation of specific mRNAs [5]. These methodologies are useful for analyzing protein synthesis in cultured cells and in relatively homogenous, isolated tissues
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