C3P3-G1: first generation of a eukaryotic artificial cytoplasmic expression system

Jaïs, Philippe H; Decroly, Etienne; Jacquet, Eric; Le Boulch, Marine; Jaïs, Aurélien; Jean-Jean, Olivier; Eaton, Heather; Ponien, Prishila; Verdier, Fréderique; Canard, Bruno; Goncalves, Sergio; Chiron, Stéphane; Le Gall, Maude; Mayeux, Patrick; Shmulevitz, Maya

modifications performed by the current generation of the C3P3 system might be incomplete. Our semi-quantitative assay ( Figure 4) suggests that the majority of mRNAs are capped by C3P3-G1. Nevertheless, even minor amounts of uncapped 5'-triphosphate RNA might induce retinoic acidinducible protein I (RIG-I)-mediated interferon-␣ response and host-cell translation shut-down (70, 71) . Interferon-␣ response can be also induced by the absence of cap-1 at the 5 -ends of transcripts, which is not accommodated by C3P3-G1 (72) . Strikingly, no frank modifications of ribosome distribution patterns were brought into focus by polysome profiling analysis. This finding does not support the hypothesis of global inhibition of host-cell translation as expected in case of strong interferon-response, but closer investigation of content of the polysomal fractions. The length of the polyadenylation tail is also known be critical for mRNA translation and should be increased by further generations of the C3P3 system. Secondly, other post-transcriptional modifications which are not performed by C3P3-G1 might be critical for mRNA translational efficiency. For instance, the reversible N6,2'-O-dimethyladenosine methylation at the first encoded nucleotide adjacent to the cap was recently found to influence cellular mRNA fate (73) . Alternatively, the absence of other modifications might induce the shut-down of host-cell translation, via interferon pathway or others (70) . For instance, internal bases can be subjected to modifications such as pseudouridination or methylation at uridine or cytosine residues, which decrease RNA-dependent protein kinase activation in response to exogenous RNA, then phosphorylates translation initiation factor 2-␣ (eIF2-␣) and inhibits translation (74, 75) . Thirdly, host-cell proteins, eventually deposited onto pre- messenger RNA or mRNA, might participate in translation. For instance, the exon junction complex, which is assembled onto pre-messenger RNA at the nuclear stage, contributes to both mRNA translational efficiency and decay (76, 77) . Fourthly, C3P3 transcripts might be inappropriately positioned in the cytoplasmic compartment for their translation by the host-cell machinery. For comparison, large cytoplasmic replicating DNA viruses, such as Poxvirus or Asfarvirus, form viral factories that sequester transcription factors, ribosomes, and chaperonins in circumscribed cytoplasmic sub-compartments. Altogether, these hypotheses will be systemically explored by deeper transcriptional and translational analysis for further improvements of the C3P3 system.