![]() ![]() Commonly used tags that are fused to a protein of interest for microscopy or biochemical applications range in size from 6 to 250 amino acids. Few dedicated techniques have been developed to advance the study of microproteins. Obstacles for systematically characterizing microproteins exist because the classic toolbox for studying protein function in vivo and vitro has historically been tailored to canonical proteins. (12) Finally, several research groups recently showed that the microprotein Myomixer/Minion/Myomerger is essential for skeletal muscle formation during embryogenesis. For example, MOTS-c, a signaling peptide encoded by the mitochondrial genome, has a systemic role in regulating insulin sensitivity and metabolic homeostasis, (11) whereas the primate-specific microprotein ORF0 is actively shaping our genomes by enhancing the mobility of LINE-1 retrotransposons. (6−10) A rapidly increasing number of microproteins are validated through detailed functional studies. Dedicated proteomic methods have provided further direct evidence for the existence of thousands of microproteins in mammalian cells. (4,5) It has become clear that sORF-encoded peptides (SEPs), also called microproteins or micropeptides, are abundantly produced in prokaryotic and eukaryotic cells. In the past decade, novel sequencing techniques, in particular RNA-sequencing and ribosome profiling, have provided a wealth of new insights into the coding potential of genomes. (1−3) Despite their protein-coding potential, sORFs remained relatively understudied in the absence of direct evidence of translation and function on the peptide level. ![]() sORFs can be present in mRNA molecules, translated alternatively to the main ORF, but also in otherwise noncoding transcripts, such as long noncoding RNAs, microRNAs, or antisense transcripts. The use of terminal residues for labeling provides a universally applicable and easily scalable strategy, which avoids alteration of the core sequence of the microprotein.Īcross genomes of all kingdoms of life, short open reading frames (sORFs) of 300 bp or less exceed canonical ORFs in number by orders of magnitude. Subsequent selective bioorthogonal reaction with a cell-permeable organic dye enables live cell imaging of microproteins with minimal perturbation of their native sequence. Efficient terminal noncanonical amino acid mutagenesis is achieved using a precursor tag that is tracelessly cleaved. ![]() Here, we develop single-residue terminal labeling (STELLA) tags that introduce a single noncanonical amino acid either at the N- or C-terminus of a protein or microprotein of interest for subsequent specific fluorescent labeling. The added molecular weight and structure of such fluorescent tag may thus significantly affect in vivo biophysical and biochemical properties of microproteins. While suitable for most proteins, common tags easily match and exceed the size of microproteins of 60 amino acids or less. Genetically encoded fluorescent tags for visualization of proteins in living cells add six to several hundred amino acids to the protein of interest. ![]()
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