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.HighlightsDART illuminates thousand-fold differences in 5′ UTR-specific translation activitySNPs and alternative 5′ UTR isoforms affect ribosome recruitment significantlyInhibitory effects of RNA structures are highly dependent on 5′ UTR context5′ UTR motifs bind initiation factors directly, broadly stimulating translation

Second Generation Drosophila Chemical Tags: Sensitivity, Versatility and Speed

Labeling and visualizing cells and sub-cellular structures within thick tissues, whole organs and even intact animals is key to studying biological processes. This is particularly true for studies of neural circuits where neurons form sub-micron synapses but have arbors that may span millimeters in length. Traditionally labeling is achieved by immunofluorescence; however diffusion of antibody molecules (>100 kDa) is slow and often results in uneven labeling with very poor penetration into the centre of thick specimens; these limitations can be partially addressed by extending staining protocols to over a week (Drosophila brain) and months (mice). Recently we developed an alternative approach using genetically encoded chemical tags CLIP, SNAP, Halo and TMP for tissue labeling; this resulted in >100 fold increase in labeling speed in both mice and Drosophila, at the expense of a considerable drop in absolute sensitivity when compared to optimized immunofluorescence staining. We now present a second generation of UAS and LexA responsive CLIP, SNAPf and Halo chemical labeling reagents for flies. These multimerized tags with translational enhancers display up to 64 fold increase in sensitivity over first generation reagents. In addition we developed a suite of conditional reporters (4xSNAPf tag and CLIP-SNAP-Halo) that are activated by the DNA recombinase Bxb1. Our new reporters can be used with weak and strong GAL4 and LexA drivers and enable stochastic, intersectional and multicolor Brainbow labeling. These improvements in sensitivity and experimental versatility, while still retaining the substantial speed advantage that is a signature of chemical labeling, should significantly increase the scope of this technology.

Tombusvirus Y-Shaped Translational Enhancer Forms a Complex with eIF4F and Can Be Functionally Replaced by Heterologous Translational Enhancers

ABSTRACTCertain plus-strand RNA plant viruses that are uncapped and nonpolyadenylated rely on RNA elements in their 3′ untranslated region, termed 3′-cap-independent translational enhancers (3′CITEs), for efficient translation of their proteins. Here, we have investigated the properties of the Y-shaped class of 3′CITE present in the tombusvirusCarnation Italian ringspot virus(CIRV). While some types of 3′CITE have been found to function through recruitment of translation initiation factors to the viral genome, notrans-acting translation-related factors have yet been identified for the Y-shaped 3′CITE. Our results indicate that the CIRV 3′CITE complexes with eIF4F and eIFiso4F, with the former mediating translation more efficiently than the latter. In nature, some classes of 3′CITE are present in several different viral genera, suggesting that these elements hold a high degree of modularity. Here, we test this concept by engineering chimeric viruses containing heterologous 3′CITEs and show that the Y-shaped class of 3′CITE in CIRV can be replaced by two alternative types of 3′CITE, i.e., aPanicum mosaic virus-like 3′CITE or an I-shaped 3′CITE, without any major loss inin vitrotranslation or replication efficiency in protoplasts. The heterologous 3′CITEs also mediated whole-plant infections ofNicotiana benthamiana, where distinct symptoms were observed for each of the alternative 3′CITEs and 3′CITE evolution occurred during serial passaging. Our results supply new information on Y-shaped 3′CITE function and provide insights into 3′CITE virus-host compatibilities.