Piwi-interacting RNAs (piRNAs) and PIWI proteins are essential in germ cells to repress transposons and regulate mRNAs. InDrosophila, piRNAs bound to the PIWI protein Aubergine (Aub) are transferred maternally to the embryo and regulate maternal mRNA stability through two opposite roles. They target mRNAs by incomplete base pairing, leading to their destabilization in the soma and stabilization in the germ plasm. Here, we report a function of Aub in translation. Aub is required for translational activation ofnanosmRNA, a key determinant of the germ plasm. Aub physically interacts with the poly(A)-binding protein (PABP) and the translation initiation factor eIF3. Polysome gradient profiling reveals the role of Aub at the initiation step of translation. In the germ plasm, PABP and eIF3d assemble in foci that surround Aub-containing germ granules, and Aub acts with eIF3d to promotenanostranslation. These results identify translational activation as a new mode of mRNA regulation by Aub, highlighting the versatility of PIWI proteins in mRNA regulation.

Death domain-associated protein 6 (Daxx) is a multifunctional, ubiquitously expressed and highly conserved chaperone protein involved in numerous cellular processes, including apoptosis, transcriptional repression, and carcinogenesis. In 2015, we identified Daxx as an antiretroviral factor that interfered with HIV-1 replication by inhibiting the reverse transcription step. In the present study, we sought to unravel the molecular mechanism of Daxx-mediated restriction and, in particular, to identify the protein(s) that Daxx targets in order to achieve its antiviral activity. First, we show that the SUMO-interacting motif (SIM) located at the C-terminus of the protein is strictly required for Daxx to inhibit HIV-1 reverse transcription. By performing a quantitative proteomic screen combined with classical biochemical analyses, we found that Daxx associated with incoming HIV-1 cores through a SIM-dependent interaction with cyclophilin A (CypA) and capsid (CA). Daxx was found to reside within a multiprotein complex associated with viral capsids, also containing TNPO3, TRIM5α, and TRIM34. Given the well-known influence of these cellular factors on the stability of HIV-1 cores, we investigated the effect of Daxx on the cytoplasmic fate of incoming cores and found that Daxx prevented HIV-1 uncoating in a SIM-dependent manner. Altogether, our findings suggest that, by recruiting TNPO3, TRIM5α, and TRIM34 and possibly other proteins onto incoming HIV-1 cores through a SIM-dependent interaction with CA-bound CypA, Daxx increases their stability, thus preventing uncoating and reverse transcription. Our study uncovers a previously unknown function of Daxx in the early steps of HIV-1 infection and further illustrates how reverse transcription and uncoating are two tightly interdependent processes.

Exiguobacterium is a polyextremophile bacterial genus with a physiology that allows it to develop in different adverse environments. The Salar de Huasco is one of these environments due to its altitude, atmospheric pressure, solar radiation, temperature variations, pH, salinity, and the presence of toxic compounds such as arsenic. However, the physiological and/or molecular mechanisms that enable them to prosper in these environments have not yet been described. Our research group has isolated several strains of Exiguobacterium genus from different sites of Salar de Huasco, which show different resistance levels to As(III) and As(V). In this work, we compare the protein expression patterns of the three strains in response to arsenic by a proteomic approach; strains were grown in absence of the metalloid and in presence of As(III) and As(V) sublethal concentrations and the protein separation was carried out in 2D electrophoresis gels (2D-GE). In total, 999 spots were detected, between 77 and 173 of which showed significant changes for As(III) among the three strains, and between 90 and 143 for As(V), respectively, compared to the corresponding control condition. Twenty-seven of those were identified by mass spectrometry (MS). Among these identified proteins, the ArsA [ATPase from the As(III) efflux pump] was found to be up-regulated in response to both arsenic conditions in the three strains, as well as the Co-enzyme A disulfide reductase (Cdr) in the two more resistant strains. Interestingly, in this genus the gene that codifies for Cdr is found within the genic context of the ars operon. We suggest that this protein could be restoring antioxidants molecules, necessary for the As(V) reduction. Additionally, among the proteins that change their expression against As, we found several with functions relevant to stress response, e.g., Hpf, LuxS, GLpX, GlnE, and Fur. This study allowed us to shed light into the physiology necessary for these bacteria to be able to tolerate the toxicity and stress generated by the presence of arsenic in their niche.

The success of oral infection by viruses depends on their capacity to overcome the gut epithelial barrier of their host to crossing over apical, mucous extracellular matrices. As orally transmitted viruses, densoviruses, are also challenged by the complexity of the insect gut barriers, more specifically by the chitinous peritrophic matrix, that lines and protects the midgut epithelium; how capsids stick to and cross these barriers to reach their final cell destination where replication goes has been poorly studied in insects. Here, we analyzed the early interaction of the Junonia coenia densovirus (JcDV) with the midgut barriers of caterpillars from the pest Spodoptera frugiperda. Using combination of imaging, biochemical, proteomic and transcriptomic analyses, we examined in vitro, ex vivo and in vivo the early interaction of the capsids with the peritrophic matrix and the consequence of early oral infection on the overall gut function. We show that the JcDV particle rapidly adheres to the peritrophic matrix through interaction with different glycans including chitin and glycoproteins, and that these interactions are necessary for oral infection. Proteomic analyses of JcDV binding proteins of the peritrophic matrix revealed mucins and non-mucins proteins including enzymes already known to act as receptors for several insect pathogens. In addition, we show that JcDV early infection results in an arrest of N-Acetylglucosamine secretion and a disruption in the integrity of the peritrophic matrix, which may help viral particles to pass through. Finally, JcDV early infection induces changes in midgut genes expression favoring an increased metabolism including an increased translational activity. These dysregulations probably participate to the overall dysfunction of the gut barrier in the early steps of viral pathogenesis. A better understanding of early steps of densovirus infection process is crucial to build biocontrol strategies against major insect pests.

Biochemistry textbooks describe eukaryotic mRNAs as monocistronic. However, increasing evidence reveals the widespread presence and translation of upstream open reading frames preceding the “main” ORF. DNA and RNA viruses infecting eukaryotes often produce polycistronic mRNAs and viruses have evolved multiple ways of manipulating the host's translation machinery. Here, we introduce an experimental model to study gene expression regulation from virus‐like bicistronic mRNAs in human cells. The model consists of a short upstream ORF and a reporter downstream ORF encoding a fluorescent protein. We have engineered synonymous variants of the upstream ORF to explore large parameter space, including codon usage preferences, mRNA folding features, and splicing propensity. We show that human translation machinery can translate the downstream ORF from bicistronic mRNAs, albeit reporter protein levels are thousand times lower than those from the upstream ORF. Furthermore, synonymous recoding of the upstream ORF exclusively during elongation significantly influences its own translation efficiency, reveals cryptic splice signals, and modulates the probability of downstream ORF translation. Our results are consistent with a leaky scanning mechanism facilitating downstream ORF translation from bicistronic mRNAs in human cells, offering new insights into the role of upstream ORFs in translation regulation.

Redundancy in the genetic code allows for differences in transcription and/or translation efficiency between mRNA molecules carrying synonymous polymorphisms, with potential phenotypic impact at the molecular and at the organismal level. A combination of neutral and selective processes determines the global genome codon usage preferences, as well as local differences between genes within a genome and between positions along a single gene. The relative contribution of evolutionary forces at shaping codon usage bias in eukaryotes is a matter of debate, especially in mammals. The main riddle remains understanding the sharp contrast between the strong molecular impact of gene expression differences arising from codon usage preferences and the thin evidence for codon usage selection at the organismal level. Here we report a multiscale analysis of the consequences of alternative codon usage on heterologous gene expression in human cells. We generated synonymous versions of the shble antibiotic resistance gene, fused to a fluorescent reporter, and expressed independently them in human HEK293 cells. We analysed: i) mRNA-to-DNA and protein-to-mRNA ratios for each shble version; ii) cellular fluorescence, using flow cytometry, as a proxy for single cell-level construct expression; and iii) real-time cell proliferation in absence or presence of antibiotic, as a proxy for the cellular fitness. Our results show that differences in codon usage preferences in our focal gene strongly impacted the molecular and the cellular phenotype: i) they elicited large differences in mRNA and in protein levels, as well in mRNA-to-protein ratio; ii) they introduced splicing events not predicted by current algorithms; iii) they lead to reproducible phenotypic heterogeneity as different multimodal distributions of cellular fluorescence EGFP; iv) they resulted in a trade-off between burden of heterologous expression and antibiotic resistance. While certain codon usage-related variables monotonically correlated with protein expression, other variables (e.g. CpG content or mRNA folding energy) displayed a bell-like behaviour. We interpret that codon usage preferences strongly shape the molecular and cellular phenotype in human cells through a direct impact on gene expression.