Pri peptides temporally coordinate transcriptional programs during epidermal differentiation.

To achieve a highly differentiated state, cells undergo multiple transcriptional processes whose coordination and timing are not well understood. In Drosophila embryonic epidermal cells, polished-rice (Pri) smORF peptides act as temporal mediators of ecdysone to activate a transcriptional program leading to cell shape remodeling. Here, we show that the ecdysone/Pri axis concomitantly represses the transcription of a large subset of cuticle genes to ensure proper differentiation of the insect exoskeleton. The repression relies on the transcription factor Ken and persists for several days throughout early larval stages, during which a soft cuticle allows larval crawling. The onset of these cuticle genes normally awaits the end of larval stages when the rigid pupal case assembles, and their premature expression triggers abnormal sclerotization of the larval cuticle. These results uncovered a temporal switch to set up distinct structures of cuticles adapted to the animal lifestyle and which might be involved in the evolutionary history of insects.

In Depth Exploration of the Alternative Proteome of Drosophila melanogaster.

Recent studies have shown that hundreds of small proteins were occulted when protein-coding genes were annotated. These proteins, called alternative proteins, have failed to be annotated notably due to the short length of their open reading frame (less than 100 codons) or the enforced rule establishing that messenger RNAs (mRNAs) are monocistronic. Several alternative proteins were shown to be biologically active molecules and seem to be involved in a wide range of biological functions. However, genome-wide exploration of the alternative proteome is still limited to a few species. In the present article, we describe a deep peptidomics workflow which enabled the identification of 401 alternative proteins in Drosophila melanogaster. Subcellular localization, protein domains, and short linear motifs were predicted for 235 of the alternative proteins identified and point toward specific functions of these small proteins. Several alternative proteins had approximated abundances higher than their canonical counterparts, suggesting that these alternative proteins are actually the main products of their corresponding genes. Finally, we observed 14 alternative proteins with developmentally regulated expression patterns and 10 induced upon the heat-shock treatment of embryos, demonstrating stage or stress-specific production of alternative proteins.

Small ORFs as New Regulators of Pri-miRNAs and miRNAs Expression in Human and Drosophila.

MicroRNAs (miRNAs) are small regulatory non-coding RNAs, resulting from the cleavage of long primary transcripts (pri-miRNAs) in the nucleus by the Microprocessor complex generating precursors (pre-miRNAs) that are then exported to the cytoplasm and processed into mature miRNAs. Some miRNAs are hosted in pri-miRNAs annotated as long non-coding RNAs (lncRNAs) and defined as MIRHGs (for miRNA Host Genes). However, several lnc pri-miRNAs contain translatable small open reading frames (smORFs). If smORFs present within lncRNAs can encode functional small peptides, they can also constitute cis-regulatory elements involved in lncRNA decay. Here, we investigated the possible involvement of smORFs in the regulation of lnc pri-miRNAs in Human and Drosophila, focusing on pri-miRNAs previously shown to contain translatable smORFs. We show that smORFs regulate the expression levels of human pri-miR-155 and pri-miR-497, and Drosophila pri-miR-8 and pri-miR-14, and also affect the expression and activity of their associated miRNAs. This smORF-dependent regulation is independent of the nucleotidic and amino acidic sequences of the smORFs and is sensitive to the ribosome-stalling drug cycloheximide, suggesting the involvement of translational events. This study identifies smORFs as new cis-acting elements involved in the regulation of pri-miRNAs and miRNAs expression, in both Human and Drosophila melanogaster.

Drosophila primary microRNA-8 encodes a microRNA-encoded peptide acting in parallel of miR-8.

BACKGROUND: Recent genome-wide studies of many species reveal the existence of a myriad of RNAs differing in size, coding potential and function. Among these are the long non-coding RNAs, some of them producing functional small peptides via the translation of short ORFs. It now appears that any kind of RNA presumably has a potential to encode small peptides. Accordingly, our team recently discovered that plant primary transcripts of microRNAs (pri-miRs) produce small regulatory peptides (miPEPs) involved in auto-regulatory feedback loops enhancing their cognate microRNA expression which in turn controls plant development. Here we investigate whether this regulatory feedback loop is present in Drosophila melanogaster. RESULTS: We perform a survey of ribosome profiling data and reveal that many pri-miRNAs exhibit ribosome translation marks. Focusing on miR-8, we show that pri-miR-8 can produce a miPEP-8. Functional assays performed in Drosophila reveal that miPEP-8 affects development when overexpressed or knocked down. Combining genetic and molecular approaches as well as genome-wide transcriptomic analyses, we show that miR-8 expression is independent of miPEP-8 activity and that miPEP-8 acts in parallel to miR-8 to regulate the expression of hundreds of genes. CONCLUSION: Taken together, these results reveal that several Drosophila pri-miRs exhibit translation potential. Contrasting with the mechanism described in plants, these data shed light on the function of yet undescribed primary-microRNA-encoded peptides in Drosophila and their regulatory potential on genome expression.

MMP14 is required for delamination of chick neural crest cells independently of its catalytic activity.

Matrix metalloproteinases have a broad spectrum of substrates ranging from extracellular matrix components and adhesion molecules to chemokines and growth factors. Despite being mostly secreted, MMPs have been detected in the cytosol, the mitochondria or the nucleus. Although most of the attention is focused on their role in matrix remodeling, the diversity of their substrates and their complex trafficking open the possibility for non-canonical functions. Yet in vivo examples and experimental demonstration of the physiological relevance of such activities are rare. Here, we have used chick neural crest (NC) cells, a highly migratory stem cell population likened to invasive cancer cells, as a model for physiological epithelial-mesenchymal transition (EMT). We demonstrate that MMP14 is required for NC delamination. Interestingly, this role is independent of its cytoplasmic tail and of its catalytic activity. Our in vivo data indicate that, in addition to being a late pro-invasive factor, MMP14 is also likely to be an early player, owing to its role in EMT.

Calcium Delivery by Electroporation Induces In Vitro Cell Death through Mitochondrial Dysfunction without DNA Damages.

Adolescent cancer survivors present increased risks of developing secondary malignancies due to cancer therapy. Electrochemotherapy is a promising anti-cancer approach that potentiates the cytotoxic effect of drugs by application of external electric field pulses. Clinicians proposed to associate electroporation and calcium. The current study aims to unravel the toxic mechanisms of calcium electroporation, in particular if calcium presents a genotoxic profile and if its cytotoxicity comes from the ion itself or from osmotic stress. Human dermal fibroblasts and colorectal HCT-116 cell line were treated by electrochemotherapy using bleomycin, cisplatin, calcium, or magnesium. Genotoxicity, cytotoxicity, mitochondrial membrane potential, ATP content, and caspases activities were assessed in cells grown on monolayers and tumor growth was assayed in tumor spheroids. Results in monolayers show that unlike cisplatin and bleomycin, calcium electroporation induces cell death without genotoxicity induction. Its cytotoxicity correlates with a dramatic fall in mitochondrial membrane potential and ATP depletion. Opposite of magnesium, over seven days of calcium electroporation led to spheroid tumor growth regression. As non-genotoxic, calcium has a better safety profile than conventional anticancer drugs. Calcium is already authorized by different health authorities worldwide. Therefore, calcium electroporation should be a cancer treatment of choice due to the reduced potential of secondary malignancies.

Nanosecond electric pulse effects on gene expression.

Gene electrotransfection using micro- or millisecond electric pulses is a well-established method for safe gene transfer. For efficient transfection, plasmid DNA has to reach the nucleus. Shorter, high-intensity nanosecond electric pulses (nsEPs) affect internal cell membranes and may contribute to an increased uptake of plasmid by the nucleus. In our study, nsEPs were applied to Chinese hamster ovary (CHO) cells after classical gene electrotransfer, using micro- or millisecond pulses with a plasmid coding the green fluorescent protein (GFP). Time gaps between classical gene electrotransfer and nsEPs were varied (0.5, 2, 6 and 24 h) and three different nsEP parameters were used: 18 ns-10 kV/cm, 10 ns-40 kV/cm and 15 ns-60 kV/cm. Results analyzed by either fluorescence microscopy or flow cytometry showed that neither the percentage of electrotransfected cells nor the amount of GFP expressed was increased by nsEP. All nsEP parameters also had no effects on GFP fluorescence intensity of human colorectal tumor cells (HCT-116) with constitutive expression of GFP. We thus conclude that nsEPs have no major contribution to gene electrotransfer in CHO cells and no effect on constitutive GFP expression in HCT-116 cells.