Synonymous codon choice can have dramatic effects on ribosome speed and protein expression. Ribosome profiling experiments have underscored that ribosomes do not move uniformly along mRNAs. We modeled this variation in translation elongation using a feedforward neural network to predict the ribosome density at each codon as a function of its sequence neighborhood. Our approach revealed sequence features affecting translation elongation and characterized large technical biases in ribosome profiling. We applied our model to design synonymous variants of a fluorescent protein spanning the range of translation speeds predicted with our model. Levels of the fluorescent protein in budding yeast closely tracked the predicted translation speeds across their full range. We therefore demonstrate that our model captures information determining translation dynamics in vivo , that we can harness this information to design coding sequences, and that control of translation elongation alone is sufficient to produce large, quantitative differences in protein output.

Mosquito-borne flaviviruses (MBFVs) including dengue, West Nile, yellow fever, and Zika viruses have an RNA genome encoding one open reading frame flanked by 5′ and 3′ untranslated regions (UTRs). The 3′ UTRs of MBFVs contain regions of high sequence conservation in structured RNA elements known as dumbbells (DBs). DBs regulate translation and replication of the viral RNA genome, functions proposed to depend on the formation of an RNA pseudoknot. To understand how DB structure provides this function, we solved the x-ray crystal structure of the Donggang virus DB to 2.1Å resolution and used structural modeling to reveal the details of its three-dimensional fold. The structure confirmed the predicted pseudoknot and molecular modeling revealed how conserved sequences form a four-way junction that appears to stabilize the pseudoknot. Single-molecule FRET suggests that the DB pseudoknot is a stable element that can regulate the switch between translation and replication during the viral lifecycle by modulating long-range RNA conformational changes.

All flaviviruses contain conserved RNA structures in the 3′ untranslated region (3′ UTR) that are important for flavivirus RNA replication, translation, and pathogenesis. Flaviviruses like Zika virus (ZIKV) contain multiple conserved RNA structures in the viral 3′ UTR, including the structure known as dumbbell-1 (DB-1). Previous research has shown that the DB-1 structure is important for flavivirus positive-strand genome replication, but the functional role of the flavivirus DB-1 structure and the mechanism by which it contributes to viral pathogenesis are not known. Using the recently solved flavivirus DB RNA structural data, we designed two DB-1 mutant ZIKV infectious clones, termed ZIKV-TL.PK and ZIKV-p.2.5′, which disrupt DB-1 tertiary folding. We found that viral positive-strand genome replication of both ZIKV DB-1 mutant clones is similar to wild-type (WT) ZIKV, but ZIKV DB-1 mutants exhibit significantly decreased cytopathic effect due to reduced caspase-3 activation. We next show that ZIKV DB-1 mutants exhibit decreased levels of sfRNA species compared to ZIKV-WT during infection. However, ZIKV DB-1 mutant 3′ UTRs exhibit unchanged sfRNA biogenesis following XRN1 degradation in vitro . We also found that ZIKV DB-1 mutant virus (ZIKV-p.2.5′) exhibited enhanced sensitivity to type I interferon treatment, and both ZIKV-DB-1 mutants exhibit reduced morbidity and mortality due to tissue-specific attenuated viral replication in brain tissue of interferon type I/II receptor knockout mice. We propose that the flavivirus DB-1 RNA structure maintains sfRNA levels during infection despite maintained sfRNA biogenesis, and these results indicate that ZIKV DB-dependent maintenance of sfRNA levels support caspase-3-dependent, cytopathic effect, type I interferon resistance, and viral pathogenesis in mammalian cells and in a ZIKV murine model of disease. IMPORTANCE The group of viruses termed flaviviruses cause important disease throughout the world and include dengue virus, Zika virus, Japanese encephalitis virus, and many more. All of these flaviviruses have highly conserved RNA structures in the untranslated regions of the virus genome. One of the shared RNA structures, termed the dumbbell region, is not well studied, but mutations in this region are important for vaccine development. In this study, we made structure-informed targeted mutations in the Zika virus dumbbell region and studied the effect on the virus. We found that Zika virus dumbbell mutants are significantly weakened or attenuated due to a decreased ability to produce non-coding RNA that is needed to support infection, support virus-induced cell death, and support escape from the host immune system. These data show that targeted mutations in the flavivirus dumbbell RNA structure may be an important approach to develop future vaccine candidates.

*contributed equallySynonymous codon choice can have dramatic effects on ribosome speed, RNA stability, and protein expression. Ribosome profiling experiments have underscored that ribosomes do not move uniformly along mRNAs, exposing a need for models of coding sequences that capture the full range of empirically observed variation. We present a method, Iχnos, that models this variation in translation elongation using a feedforward neural network to predict the translation elongation rate at each codon as a function of its sequence neighborhood. Our approach revealed sequence features affecting translation elongation and quantified the impact of large technical biases in ribosome profiling. We applied our model to design synonymous variants of a fluorescent protein spanning the range of possible translation speeds predicted with our model. We found that levels of the fluorescent protein in yeast closely tracked the predicted translation speeds across their full range. We therefore demonstrate that our model captures information determining translation dynamics in vivo , and that control of translation elongation alone is sufficient to produce large, quantitative differences in protein output.As the ribosome moves along a transcript, it encounters diverse codons, tRNAs, and amino acids. This diversity affects translation elongation and, ultimately, gene expression. For instance, exogenous gene expression can be seriously hampered by a mismatch between the choice of synonymous codons and the availability of tRNAs. The consequences of endogenous variation in codon use have been more elusive, but new methods have revealed that changes in translation speed due to synonymous coding mutations, upregulation of tRNAs, or mutations within tRNAs can have dramatic effects on protein expression, folding, or stability [1][2][3] . However, translation initiation has been considered the rate-limiting step in translation, implying that changes in elongation speed should have limited effects 4 . Recent work has suggested a relationship between codon use and RNA stability; slower translation may destabilize mRNAs . CC-BY 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/201517 doi: bioRxiv preprint first posted online Oct. 11, 2017; and thus decrease protein expression 5,6 . These opposing viewpoints have yet to be fully reconciled, leaving us with an incomplete understanding of what defines a favorable sequence for translation.With the advent of high-throughput methods to measure translation elongation in vivo , we can understand the functional implications of codon usage. Ribosome profiling measures translation transcriptome-wide by capturing and sequencing the regions of mRNA protected within ribosomes, called ribosome footprints 7 . Each footprint reflects the position of an individual ribosome on a transcript, and we can reliably infer the A site codon -the site of tRNA decoding -in each footprint (F...