Sélection de promoteurs ARN reconnus par l'ARN polymérase de Escherichia coli

Les viroïdes sont de petits ARN circulaires simple brin qui infectent les plantes supérieures, causant ainsi d'importantes pertes économiques dans les domaines agro-alimentaire et horticole. Ces ARN ne sont pas encapsidés, ne possèdent pas de région codante et se répliquent de façon presque autonome par un mécanisme en cercle roulant. La seule étape du cycle de vie des viroïdes qui requiert la machinerie de l'hôte est la polymérisation de l'ARN. Par ailleurs, cette étape est certainement la moins bien connue, particulièrement chez le viroïde de la mosaïque latente du pêcher (PLMVd). Le but de ce travail est de découvrir des séquences importantes pour l'initiation de la réplication d'une molécule modèle d'ARN inspirée du PLMVd. Un protocole de sélection de promoteur en ARN a d'abord été mis au point. Dans ce type de protocole, des étapes pertinemment désignées s'enchaînent sous forme de cycles afin de sélectionner une molécule d'intérêt. Toutes les étapes constituant le complexe protocole ont été testées avec un témoin positif, basé sur une région minimale du PLMVd connue pour initier la réplication in vitro. Cette molécule a servi à la vérification et à l'optimisation individuelles des nombreuses étapes. Ce protocole amélioré a ensuite été utilisé pour sélectionner des molécules reconnues par l'ARNP d' E. coli à partir d'une population aléatoire d'ARN. Cette sélection a permis l'isolement de plusieurs variants qui ont rapidement montré des ressemblances en terme de séquence primaire au cours des cycles de sélection. En effet, sur trente positions aléatoires, une boite de six nucléotides (CAGACG) se retrouvant à divers endroits est apparue au quatrième cycle puis a été retrouvée presque intégralement dans toutes les séquences à partir du cinquième cycle. Une investigation sur cette boîte a mis en évidence une analogie avec une séquence retrouvée près du site d'initiation in vivo du PLMVd ainsi qu'avec des produits de sélection pour un promoteur ADN simple brin. Ceci semble pointer vers des éléments impliqués dans la réplication dépendante de l'ARN polymérase d'E. coli . L'effet de la boîte conservée CAGACG sur la réplication a été étudié. Le patron de réplication de molécules dont la boite a été modifiée (délétion ou modification) est distinct de celui de la molécule originale. Aussi, l'analyse de la séquence des petits ARN (pARN) produits lors de la réplication révèle l'implication des 6 nt pour fixer l'initiation de la polymérisation. En conclusion, ces résultats s'ajoutent aux évidences qui mèneront vers la découverte des séquences nécessaires à la réplication d'une molécule modèle d'ARN, autant in vitro (reconstitution d'un monde ARN) que in vivo (dans les viroïdes)

A Novel Ribozyme-Based Prophylaxis Inhibits Influenza A Virus Replication and Protects from Severe Disease

Influenza A virus seasonal outbreaks and occasional pandemics represent a global health threat. The high genetic instability of this virus permits rapid escape from the host immune system and emergence of resistance to antivirals. There is thus an urgent need to develop novel approaches for efficient treatment of newly emerging strains. Based on a sequence alignment of representatives from every subtype known to infect humans, we identified nucleic acid regions that are conserved amongst these influenza A populations. We then engineered SOFA-HDV-Ribozymes as therapeutic tools recognizing these conserved regions to catalytically cleave the corresponding viral mRNA targets. The most promising ribozymes were chosen based on an initial in silico screening, and their efficacy was assessed using in vitro cleavage assays. Further characterization of their antiviral effect in cell culture and in mice led to the gradual identification of prophylactic SOFA-HDV-Ribozyme combinations, providing proof-of-principle for the potential of this novel strategy to develop antivirals against genetically highly variable viruses

Direct Detection of Alternative Open Reading Frames Translation Products in Human Significantly Expands the Proteome

<div><p>A fully mature mRNA is usually associated to a reference open reading frame encoding a single protein. Yet, mature mRNAs contain unconventional alternative open reading frames (AltORFs) located in untranslated regions (UTRs) or overlapping the reference ORFs (RefORFs) in non-canonical +2 and +3 reading frames. Although recent ribosome profiling and footprinting approaches have suggested the significant use of unconventional translation initiation sites in mammals, direct evidence of large-scale alternative protein expression at the proteome level is still lacking. To determine the contribution of alternative proteins to the human proteome, we generated a database of predicted human AltORFs revealing a new proteome mainly composed of small proteins with a median length of 57 amino acids, compared to 344 amino acids for the reference proteome. We experimentally detected a total of 1,259 alternative proteins by mass spectrometry analyses of human cell lines, tissues and fluids. In plasma and serum, alternative proteins represent up to 55% of the proteome and may be a potential unsuspected new source for biomarkers. We observed constitutive co-expression of RefORFs and AltORFs from endogenous genes and from transfected cDNAs, including tumor suppressor p53, and provide evidence that out-of-frame clones representing AltORFs are mistakenly rejected as false positive in cDNAs screening assays. Functional importance of alternative proteins is strongly supported by significant evolutionary conservation in vertebrates, invertebrates, and yeast. Our results imply that coding of multiple proteins in a single gene by the use of AltORFs may be a common feature in eukaryotes, and confirm that translation of unconventional ORFs generates an as yet unexplored proteome.</p></div

Summary of LC-MS/MS analyses of human samples.

<p>Summary of LC-MS/MS analyses of human samples.</p

Co-expression of alternative and reference proteins in cDNA transfection experiments is common.

<p>(<i>A</i>) Distribution of the number of predicted RefORFs-contained AltORFs per gene in the human genome. Top, schematic representation of a mRNA with a RefORF (grey)-containing AltORF (green). By definition, RefORFs are present in the +1 reading frame and AltORFs are present in the non-canonical +2 and +3 reading frames. (<i>B</i>) Strategy to detect the co-expression of reference and alternative proteins in cDNA transfection experiments. HA and GFP tags permit the detection of reference and alternative proteins, respectively. Top, graphical representation of a mRNA with a RefORF-contained AltORF. Middle, typical cDNA construct used in transfection experiments. Bottom, representation of constructs used in (<i>C</i>). (<i>C</i>) Western blot analyses of HA-tagged LGALS3BP (Lectin galactoside-binding soluble 3 binding protein), VEGFC (vascular endothelium growth factor), p53 (cellular tumor antigen p53), CDC42 (cell division cycle 42), BDKRB2 (bradykinin receptor), and SRSF1 (serine/arginine-rich splicing factor 1), and their respective GFP-tagged alternative proteins using anti-HA and anti-GFP antibodies (top panels). Bottom panels show the cellular distribution of alternative proteins by confocal fluorescence microscopy (differential interference contrast and Hoechst, left panels; GFP, right panels). Scale bar: 10 μm.</p

Transfection of tagged constructs validate the expression and translation initiation site prediction of alternative proteins detected by LC-MS/MS.

<p>Top diagrams represent the constructs used to detect the co-expression of HA-tagged reference and GFP-tagged alternative proteins by western blot analyses of HeLa cell lysates. GFP is inserted before the alternative stop codon in frame with the AltORF. The black line represents a specific region of the endogenous mRNA. For AltORFs located in 5′UTRs of <i>ZNF83</i> and <i>SLC35A4</i>, the constructs do not contain the RefORF since the insertion of GFP may prevent the expression of the downstream RefORF. For AltORFs overlapping the 5′UTR and the RefORF (<i>IDH3B</i>), and for AltORFs overlapping the RefORF (<i>BDH2</i>, <i>NIPA1</i>, <i>SCARB2</i>), the HA tag was introduced before the GFP tag in frame with the RefORFs. Western blots show the co-expression of reference and corresponding alternative proteins in cell lysates with anti-HA and anti-GFP antibodies, respectively. The left and right lanes are cell lysates from cells expressing a construct with a normal alternative initiation AUG codon or with an inactivated alternative initiation AAG codon, respectively. NPTII, encoded in the expression plasmid, was used as a transfection control. Molecular weight markers in kDa are indicated on the right. Bottom panels show confocal/DIC images with the various cellular distributions of GFP-tagged alternative proteins. Nuclei were stained with Hoechst. Scale bar: 10 μm. * The reference protein was not detected due to the small size (<3 kDa) of the truncated HA-tagged reference IDH3B protein.</p

A database to predict AltORFs in human mRNAs.

<p>(<i>A</i>) A canonical mRNA and its possible AltORFs. The RefORF is the main protein coding ORF annotated in current nucleotide databases. An AltORF is a nucleotide region comprised between an AUG codon and a stop codon distinct from the RefORF and is predicted to encode an alternative protein. AltORFs may be localized in 5′ UTRs, overlapping the 5′UTR and the RefORF, overlapping the RefORF, overlapping the RefORF and the 3′UTR, or in the 3′UTR. (<i>B</i>) Representation of the database generation process. Distinct AltORFs number indicates the total number of predicted AltORFs that encode alternative proteins with unique amino acid sequences. Since an AltORF may be present in several transcripts, the total number of AltORFs in the transcriptome exceeds the number of distinct AltORFs. (<i>C</i>) Distribution in % of AltORFs. (<i>D,E</i>) Distribution of the number of predicted AltORFs per mRNA in (<i>D</i>) and the size distribution of AltORFs (empty bars, left and bottom scale) compared to RefORFs (grey bars, right and top scale) in (<i>E</i>). Boxes and arrows indicate the median.</p

Endogenous expression of alternative proteins in cultured cells.

<p>(<i>A</i>) Alternative proteins expression was analyzed by LC-MS/MS in HeLa cells, and a schematic distribution of AltORFs in absolute numbers is shown. There were a total of 129 identified alternative proteins (indicated in the center). (<i>B</i>) Average peptide intensity plot of both the reference and alternative proteins that were co-expressed from 42 genes. (<i>C</i>) Same as (<i>A</i>) with colon cell lines. (<i>D</i>) Venn diagram showing the number of alternative proteins identified in HeLa cells and colon cell lines. The overlap identifies a common list of 14 alternative proteins.</p