BackgroundDue to the degeneracy of the genetic code, most amino acids can be encoded by multiple synonymous codons. Synonymous codons naturally occur with different frequencies in different organisms. The choice of codons may affect protein expression, structure, and function. Recombinant gene technologies commonly take advantage of the former effect by implementing a technique termed codon optimization, in which codons are replaced with synonymous ones in order to increase protein expression. This technique relies on the accurate knowledge of codon usage frequencies. Accurately quantifying codon usage bias for different organisms is useful not only for codon optimization, but also for evolutionary and translation studies: phylogenetic relations of organisms, and host-pathogen co-evolution relationships, may be explored through their codon usage similarities. Furthermore, codon usage has been shown to affect protein structure and function through interfering with translation kinetics, and cotranslational protein folding.ResultsDespite the obvious need for accurate codon usage tables, currently available resources are either limited in scope, encompassing only organisms from specific domains of life, or greatly outdated. Taking advantage of the exponential growth of GenBank and the creation of NCBI’s RefSeq database, we have developed a new database, the High-performance Integrated Virtual Environment-Codon Usage Tables (HIVE-CUTs), to present and analyse codon usage tables for every organism with publicly available sequencing data. Compared to existing databases, this new database is more comprehensive, addresses concerns that limited the accuracy of earlier databases, and provides several new functionalities, such as the ability to view and compare codon usage between individual organisms and across taxonomical clades, through graphical representation or through commonly used indices. In addition, it is being routinely updated to keep up with the continuous flow of new data in GenBank and RefSeq.ConclusionGiven the impact of codon usage bias on recombinant gene technologies, this database will facilitate effective development and review of recombinant drug products and will be instrumental in a wide area of biological research. The database is available at hive.biochemistry.gwu.edu/review/codon.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-017-1793-7) contains supplementary material, which is available to authorized users.

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Synonymous codons occur with different frequencies in different organisms, a phenomenon termed codon usage bias. Codon optimization, a common term for a variety of approaches used widely by the biopharmaceutical industry, involves synonymous substitutions to increase protein expression. It had long been presumed that synonymous variants, which, by definition, do not alter the primary amino acid sequence, have no effect on protein structure and function. However, a critical mass of reports suggests that synonymous codon variations may impact protein conformation. To investigate the impact of synonymous codons usage on protein expression and function, we designed an optimized coagulation factor IX (FIX) variant and used multiple methods to compare its properties to the wild-type FIX upon expression in HEK293T cells. We found that the two variants differ in their conformation, even when controlling for the difference in expression levels. Using ribosome profiling, we identified robust changes in the translational kinetics of the two variants and were able to identify a region in the gene that may have a role in altering the conformation of the protein. Our data have direct implications for codon optimization strategies, for production of recombinant proteins and gene therapies.

The liver is the primary source of a large number of plasma proteins and plays a critical role in multiple biological processes. Inadequate oxygen supply characterizing various clinical settings such as liver transplantation exposes the liver to hypoxic conditions. Studies assessing hypoxia-induced global translational changes in liver are lacking. Here, we employed a recently developed ribosome-profiling technique to assess global translational responses of human primary hepatocytes exposed to acute hypoxic stress (1% O2) for the short term. In parallel, transcriptome profiling was performed to assess mRNA expression changes. We found that translational responses appeared earlier and were predominant over transcriptional responses. A significant decrease in translational efficiency of several ribosome genes indicated translational inhibition of new ribosome protein synthesis in hypoxia. Pathway enrichment analysis highlighted altered translational regulation of MAPK signaling, drug metabolism, oxidative phosphorylation, and nonalcoholic fatty liver disease pathways. Gene Ontology enrichment analysis revealed terms related to translation, metabolism, angiogenesis, apoptosis, and response to stress. Transcriptional induction of genes encoding heat shock proteins was observed within 30 min of hypoxia. Induction of genes encoding stress response mediators, metabolism regulators, and proangiogenic proteins was observed at 240 min. Despite the liver being the primary source of coagulation proteins and the implicated role of hypoxia in thrombosis, limited differences were observed in genes encoding coagulation-associated proteins. Overall, our study demonstrates the predominance of translational regulation over transcription and highlights differentially regulated pathways or biological processes in short-term hypoxic stress responses of human primary hepatocytes.NEW & NOTEWORTHY The novelty of this study lies in applying parallel ribosome- and transcriptome-profiling analyses to human primary hepatocytes in hypoxia. To our knowledge, this is the first study to assess global translational responses using ribosome profiling in hypoxic hepatocytes. Our results demonstrate the predominance of translational responses over transcriptional responses in early hepatic hypoxic stress responses. Furthermore, our study reveals multiple pathways and specific genes showing altered regulation in hypoxic hepatocytes.