Neurons localize mRNAs near synapses where their translation can be regulated by synaptic demand and activity. Differences in the 3' UTRs of mRNAs can change their localization, stability, and translational regulation. Using 3' end RNA sequencing of microdissected rat brain slices, we discovered a huge diversity in mRNA 3' UTRs, with many transcripts showing enrichment for a particular 3' UTR isoform in either somata or the neuropil. The 3' UTR isoforms of localized transcripts are significantly longer than the 3' UTRs of non-localized transcripts and often code for proteins associated with axons, dendrites, and synapses. Surprisingly, long 3' UTRs add not only new, but also duplicate regulatory elements. The neuropil-enriched 3' UTR isoforms have significantly longer half-lives than somata-enriched isoforms. Finally, the 3' UTR isoforms can be significantly altered by enhanced activity. Most of the 3' UTR plasticity is transcription dependent, but intriguing examples of changes that are consistent with altered stability, trafficking between compartments, or local "remodeling" remain.
Protein synthesis is a dynamic process to tune the cellular proteome to internal and external demands. Metabolic labeling approaches identify the general proteomic response but missing is a tool to visualize within cells specific newly synthesized proteins. Here we describe a technique that couples non-canonical amino acid tagging or puromycylation with the proximity-ligation assay to visualize identified newly synthesized proteins and monitor their origin, redistribution and turnover in situ.
Monitoring newly synthesized proteins is becoming increasingly important to characterize proteome composition in regulatory networks. Puromycin is a peptidyl transfer inhibitor, widely used in cell biology for tagging newly synthesized proteins. Here, we report synthesis and application of an optimized puromycin carrying a photolabile protecting group as a powerful tool for tagging nascent proteins with high spatiotemporal resolution. The photocaged 7‐N,N‐(diethylaminocumarin‐4‐yl)‐methoxycarbonyl‐puromycin (DEACM‐puromycin) was synthesized and compared with the previously developed 6‐nitroveratryloxycarbonyl puromycin (NVOC‐puromycin). The photochemical behavior as well as the effectiveness in controlling puromycylation in living hippocampal neurons using two‐photon excitation is superior to the previously used NVOCpuromycin. We further report on the application of light‐controlled puromycylation to visualize new translated proteins in neurons.
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