iCodon customizes gene expression based on the codon composition.

Messenger RNA (mRNA) stability substantially impacts steady-state gene expression levels in a cell. mRNA stability is strongly affected by codon composition in a translation-dependent manner across species, through a mechanism termed codon optimality. We have developed iCodon ( www.iCodon.org ), an algorithm for customizing mRNA expression through the introduction of synonymous codon substitutions into the coding sequence. iCodon is optimized for four vertebrate transcriptomes: mouse, human, frog, and fish. Users can predict the mRNA stability of any coding sequence based on its codon composition and subsequently generate more stable (optimized) or unstable (deoptimized) variants encoding for the same protein. Further, we show that codon optimality predictions correlate with both mRNA stability using a massive reporter library and expression levels using fluorescent reporters and analysis of endogenous gene expression in zebrafish embryos and/or human cells. Therefore, iCodon will benefit basic biological research, as well as a wide range of applications for biotechnology and biomedicine.

Crosstalk between codon optimality and cis-regulatory elements dictates mRNA stability.

BACKGROUND: The regulation of messenger RNA (mRNA) stability has a profound impact on gene expression dynamics during embryogenesis. For example, in animals, maternally deposited mRNAs are degraded after fertilization to enable new developmental trajectories. Regulatory sequences in 3' untranslated regions (3'UTRs) have long been considered the central determinants of mRNA stability. However, recent work indicates that the coding sequence also possesses regulatory information. Specifically, translation in cis impacts mRNA stability in a codon-dependent manner. However, the strength of this mechanism during embryogenesis, as well as its relationship with other known regulatory elements, such as microRNA, remains unclear. RESULTS: Here, we show that codon composition is a major predictor of mRNA stability in the early embryo. We show that this mechanism works in combination with other cis-regulatory elements to dictate mRNA stability in zebrafish and Xenopus embryos as well as in mouse and human cells. Furthermore, we show that microRNA targeting efficacy can be affected by substantial enrichment of optimal (stabilizing) or non-optimal (destabilizing) codons. Lastly, we find that one microRNA, miR-430, antagonizes the stabilizing effect of optimal codons during early embryogenesis in zebrafish. CONCLUSIONS: By integrating the contributions of different regulatory mechanisms, our work provides a framework for understanding how combinatorial control of mRNA stability shapes the gene expression landscape.

Recombinant occlusion bodies of baculovirus as carriers of a non-structural protein of foot-and-mouth disease virus.

Here, we developed a diagnostic ELISA for foot-and-mouth disease using recombinant occlusion bodies (rOBs) of baculovirus. We fused Δ3AB1-3, a polypeptide derived from non-structural proteins of foot-and-mouth disease virus, to polyhedrin (POLH), the major constituent of OBs, under polh promoter. To further assess the most convenient strategy to improve yields, we designed two recombinant baculoviruses, vPOLH and vPOLHE44G. These carried the sequence of the fusion protein POLH-Δ3AB1-3 with an additional copy in cis of polh or polh E44G , respectively, under p10 promoter. Our results show that both viruses expressed POLH-Δ3AB1-3, which was detected by western blot in purified rOBs with anti-POLH and anti-3AB1 antibodies. We also found that vPOLHE44G produced larger polyhedra and a significant increase of antigen yield (p < 0.01). Furthermore, the chimeric protein POLH-Δ3AB1-3 was recognized by sera from experimentally infected animals, showing that translational fusion to POLH does not alter the antigenicity of Δ3AB1-3. Finally, the rOBs were successfully used in an ELISA test to differentiate infected from vaccinated animals. Taken together, these results demonstrate the great potential of rOBs to develop diagnostic schemes adaptable to animal infectious diseases.