Adjacent Codons Act in Concert to Modulate Translation Efficiency in Yeast.

Translation elongation efficiency is largely thought of as the sum of decoding efficiencies for individual codons. Here, we find that adjacent codon pairs modulate translation efficiency. Deploying an approach in Saccharomyces cerevisiae that scored the expression of over 35,000 GFP variants in which three adjacent codons were randomized, we have identified 17 pairs of adjacent codons associated with reduced expression. For many pairs, codon order is obligatory for inhibition, implying a more complex interaction than a simple additive effect. Inhibition mediated by adjacent codons occurs during translation itself as GFP expression is restored by increased tRNA levels or by non-native tRNAs with exact-matching anticodons. Inhibition operates in endogenous genes, based on analysis of ribosome profiling data. Our findings suggest translation efficiency is modulated by an interplay between tRNAs at adjacent sites in the ribosome and that this concerted effect needs to be considered in predicting the functional consequences of codon choice.

Identification of the determinants of tRNA function and susceptibility to rapid tRNA decay by high-throughput in vivo analysis.

Sequence variation in tRNA genes influences the structure, modification, and stability of tRNA; affects translation fidelity; impacts the activity of numerous isodecoders in metazoans; and leads to human diseases. To comprehensively define the effects of sequence variation on tRNA function, we developed a high-throughput in vivo screen to quantify the activity of a model tRNA, the nonsense suppressor SUP4oc of Saccharomyces cerevisiae. Using a highly sensitive fluorescent reporter gene with an ochre mutation, fluorescence-activated cell sorting of a library of SUP4oc mutant yeast strains, and deep sequencing, we scored 25,491 variants. Unexpectedly, SUP4oc tolerates numerous sequence variations, accommodates slippage in tertiary and secondary interactions, and exhibits genetic interactions that suggest an alternative functional tRNA conformation. Furthermore, we used this methodology to define tRNA variants subject to rapid tRNA decay (RTD). Even though RTD normally degrades tRNAs with exposed 5' ends, mutations that sensitize SUP4oc to RTD were found to be located throughout the sequence, including the anti-codon stem. Thus, the integrity of the entire tRNA molecule is under surveillance by cellular quality control machinery. This approach to assess activity at high throughput is widely applicable to many problems in tRNA biology.

RNA-ID, a highly sensitive and robust method to identify cis-regulatory sequences using superfolder GFP and a fluorescence-based assay.

We have developed a robust and sensitive method, called RNA-ID, to screen for cis-regulatory sequences in RNA using fluorescence-activated cell sorting (FACS) of yeast cells bearing a reporter in which expression of both superfolder green fluorescent protein (GFP) and yeast codon-optimized mCherry red fluorescent protein (RFP) is driven by the bidirectional GAL1,10 promoter. This method recapitulates previously reported progressive inhibition of translation mediated by increasing numbers of CGA codon pairs, and restoration of expression by introduction of a tRNA with an anticodon that base pairs exactly with the CGA codon. This method also reproduces effects of paromomycin and context on stop codon read-through. Five key features of this method contribute to its effectiveness as a selection for regulatory sequences: The system exhibits greater than a 250-fold dynamic range, a quantitative and dose-dependent response to known inhibitory sequences, exquisite resolution that allows nearly complete physical separation of distinct populations, and a reproducible signal between different cells transformed with the identical reporter, all of which are coupled with simple methods involving ligation-independent cloning, to create large libraries. Moreover, we provide evidence that there are sequences within a 9-nt library that cause reduced GFP fluorescence, suggesting that there are novel cis-regulatory sequences to be found even in this short sequence space. This method is widely applicable to the study of both RNA-mediated and codon-mediated effects on expression.

Susceptibility of two hymenopteran parasitoids of Agrilus planipennis (Coleoptera: Buprestidae) to the entomopathogenic fungus Beauveria bassiana (Ascomycota: Hypocreales).

Emerald ash borer (EAB), Agrilus planipennis Fairmaire, native to Asia, is killing ash trees (Fraxinus spp.) across 15 states and southeastern Canada. Integrated pest management using biological control is the only viable long-term approach for controlling the spread of EAB outside of host resistance. Three hymenopteran parasitoids, Spathius agrili Yang, Tetrastichus planipennisi Yang, and Oobius agrili Zhang and Huang were discovered attacking EAB in China and were approved for release in the United States in 2007. The objective of this study was to assess susceptibility of the larval parasitoid species S. agrili and T. planipennisi, relative to that of EAB, to Beauveria bassiana, an entomopathogenic fungus that infects and kills EAB adults when sprayed on ash bark or foliage. Adult EAB and parasitoids were exposed to B. bassiana inoculated ash twigs for 2 h and then monitored daily for death and signs of infection for up to 10 days. All EAB adults exposed to B. bassiana were fatally infected while mean survival for control EAB was 77%. Average survival in the treatment groups for T. planipennisi and S. agrili were 99% and 83%, respectively, indicating these parasitoids are relatively unaffected by exposure to B. bassiana. This research elucidates interactions between a fungal pathogen and two parasitoids of EAB, and provides data necessary to developing a successful multi-stage integrated management approach to control of EAB.

Control of translation efficiency in yeast by codon-anticodon interactions.

The choice of synonymous codons used to encode a polypeptide contributes to substantial differences in translation efficiency between genes. However, both the magnitude and the mechanisms of codon-mediated effects are unknown, as neither the effects of individual codons nor the parameters that modulate codon-mediated regulation are understood, particularly in eukaryotes. To explore this problem in Saccharomyces cerevisiae, we performed the first systematic analysis of codon effects on expression. We find that the arginine codon CGA is strongly inhibitory, resulting in progressively and sharply reduced expression with increased CGA codon dosage. CGA-mediated inhibition of expression is primarily due to wobble decoding of CGA, since it is nearly completely suppressed by coexpression of an exact match anticodon-mutated tRNA(Arg(UCG)), and is associated with generation of a smaller RNA fragment, likely due to endonucleolytic cleavage at a stalled ribosome. Moreover, CGA codon pairs are more effective inhibitors of expression than individual CGA codons. These results directly implicate decoding by the ribosome and interactions at neighboring sites within the ribosome as mediators of codon-specific translation efficiency.