Biology subjects

Zinshteyn, B.

Publications and source records attributed to Zinshteyn, B..

3 recordsLinked to original sources

bioRxiv2020

Puromycin reactivity does not accurately localize translation at the subcellular level

Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays. More recently, in vivo puromycylation assays have become popular tools for localizing translating ribosomes in cells. These assays often use elongation inhibitors to purportedly inhibit the release of puromycin-labeled nascent peptides from ribosomes. Here, using in vitro and in vivo experiments, we demonstrate that, even in the presence of elongation inhibitors, puromycylated peptides are released and diffuse away from ribosomes. Puromycylation assays reveal subcellular sites, such as nuclei, where puromycylated peptides accumulate post-release and which do not necessarily coincide with sites of active translation. Our findings urge caution when interpreting puromycylation assays in the in vivo context.

biochemistry↗

bioRxiv2020

Direct analysis of ribosome targeting illuminates thousand-fold regulation of translation initiation

Translational control shapes the proteome in normal and pathophysiological conditions. Current high-throughput approaches reveal large differences in mRNA-specific translation activity but cannot identify the causative mRNA features. We developed direct analysis of ribosome targeting (DART) and used it to dissect regulatory elements within 5' untranslated regions that confer thousand-fold differences in ribosome recruitment in biochemically accessible cell lysates. Using DART, we identified novel translational enhancers and silencers, determined a functional role for most alternative 5' UTR isoforms expressed in yeast, and revealed a general mode of increased translation via direct binding to a core translation factor. DART enables systematic assessment of the translational regulatory potential of 5' UTR variants, whether native or disease-associated, and will facilitate engineering of mRNAs for optimized protein production in various systems. HighlightsO_LIDART illuminates thousand-fold differences in 5' UTR-specific translation activity C_LIO_LISNPs and alternative 5' UTR isoforms affect ribosome recruitment significantly C_LIO_LIInhibitory effects of RNA structures are highly dependent on 5' UTR context C_LIO_LI5' UTR motifs bind initiation factors directly, broadly stimulating translation C_LI

molecular biology↗

bioRxiv2020

Nuclease-mediated depletion biases in ribosome footprint profiling libraries

Ribosome footprint profiling is a high throughput sequencing based technique that provides detailed and global views of translation in living cells. An essential part of this technology is removal of unwanted, normally very abundant, ribosomal RNA sequences that dominate libraries and increase sequencing costs. The most effective commercial solution (Ribo-Zero) has been discontinued and a number of new, experimentally distinct commercial applications have emerged on the market. Here we evaluated several commercially available alternatives designed for RNA-seq of human samples and find them unsuitable for ribosome footprint profiling. We instead recommend the use of custom-designed biotinylated oligos, which were widely used in early ribosome profiling studies. Importantly, we warn that depletion solutions based on targeted nuclease cleavage significantly perturb the high-resolution information that can be derived from the data, and thus do not recommend their use for any applications that require precise determination of the ends of RNA fragments.

molecular biology↗