A new type of IRES within gag coding region recruits three initiation complexes on HIV-2 genomic RNA

Genomic RNA of primate lentiviruses serves both as an mRNA that encodes Gag and Gag-Pol polyproteins and as a propagated genome. Translation of this RNA is initiated by standard cap dependant mechanism or by internal entry of the ribosome. Two regions of the genomic RNA are able to attract initiation complexes, the 5′ untranslated region and the gag coding region itself. Relying on probing data and a phylogenetic study, we have modelled the secondary structure of HIV-1, HIV-2 and SIVMac coding region. This approach brings to light conserved secondary-structure elements that were shown by mutations to be required for internal entry of the ribosome. No structural homologies with other described viral or cellular IRES can be identified and lentiviral IRESes show many peculiar properties. Most notably, the IRES present in HIV-2 gag coding region is endowed with the unique ability to recruit up to three initiation complexes on a single RNA molecule. The structural and functional properties of gag coding sequence define a new type of IRES. Although its precise role is unknown, the conservation of the IRES among fast evolving lentiviruses suggests an important physiological role

HIV-1 gRNA, a biological substrate, uncovers the potency of DDX3X biochemical activity

International audienceDEAD-box helicases play central roles in the metabolism of many RNAs and ribonucleoproteins by assisting their synthesis, folding, function and even their degradation or disassembly. They have been implicated in various phenomena, and it is often difficult to rationalize their molecular roles from in vivo studies. Once purified in vitro, most of them only exhibit a marginal activity and poor specificity. The current model is that they gain specificity and activity through interaction of their intrinsically disordered domains with specific RNA or proteins. DDX3 is a DEAD-box cellular helicase that has been involved in several steps of the HIV viral cycle, including transcription, RNA export to the cytoplasm and translation. In this study, we investigated DDX3 biochemical properties in the context of a biological substrate. DDX3 was overexpressed, purified and its enzymatic activities as well as its RNA binding properties were characterized using both model substrates and a biological substrate, HIV-1 gRNA. Biochemical characterization of DDX3 in the context of a biological substrate identifies HIV-1 gRNA as a rare example of specific substrate and unravels the extent of DDX3 ATPase activity. Analysis of DDX3 binding capacity indicates an unexpected dissociation between its binding capacity and its biochemical activity. We further demonstrate that interaction of DDX3 with HIV-1 gRNA relies both on specific RNA determinants and on the disordered N-and C-terminal regions of the protein. These findings shed a new light regarding the potentiality of DDX3 biochemical activity supporting its multiple cellular functions

Two ribosome recruitment sites direct multiple translation events within HIV1 Gag open reading frame

International audienceIn the late phase of the HIV virus cycle, the full length unspliced genomic RNA is exported to the cytoplasm and serves as mRNA to translate the Gag and Gag-pol polyproteins. Three different translation initiation mechanisms responsible for Gag production have been described. However a rationale for the involvement of as many translation pathways in gRNA translation is yet to be defined. The Gag-IRES has the singularity to be located within the Gag open reading frame and to directly recruit the 40S ribosomal subunit. To further characterize this interaction, we first probed the Gag-IRES RNA structure. We then developed an innovative integrative modelling approach and propose a novel secondary structure model for the Gag-IRES. The minimal 40S ribosomal subunit binding site was further mapped using different assays. To our surprise, we found that at least two regions within Gag-IRES can independently recruit the ribosome. Next, we validated that these two regions influence Gag translation both in vitro and in cellulo. These binding sites are mostly unstructured and highly A-rich, such sequences have previously been shown to be sufficient to recruit the ribosome and to support an IRES function. A combination of biochemical and functional data give insight into the Gag-IRES molecular mechanism and provide compelling evidences for its importance. Hypothesis about its physiological role reflecting its conservation amongst primate lentiviruses are proposed

Pby1 is a direct partner of the Dcp2 decapping enzyme

International audienceMost eukaryotic mRNAs harbor a characteristic 5 m 7 GpppN cap that promotes pre-mRNA splicing, mRNA nucleocytoplasmic transport and translation while also protecting mRNAs from exonucleolytic attacks. mRNA caps are eliminated by Dcp2 during mRNA decay, allowing 5-3 exonucleases to degrade mRNA bodies. However, the Dcp2 decapping enzyme is poorly active on its own and requires binding to stable or transient protein partners to sever the cap of target mRNAs. Here, we analyse the role of one of these partners, the yeast Pby1 factor, which is known to co-localize into P-bodies together with decapping factors. We report that Pby1 uses its C-terminal domain to directly bind to the decapping enzyme. We solved the structure of this Pby1 domain alone and bound to the Dcp1-Dcp2-Edc3 de-capping complex. Structure-based mutant analyses reveal that Pby1 binding to the decapping enzyme is required for its recruitment into P-bodies. Moreover, Pby1 binding to the decapping enzyme stimulates growth in conditions in which decapping activation is compromised. Our results point towards a direct connection of Pby1 with decapping and P-body formation , both stemming from its interaction with the Dcp1-Dcp2 holoenzyme

Stoichiometry of the MexA-OprM binding, as investigated by blue native gel electrophoresis

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Evolutionary insights into Trm112-methyltransferase holoenzymes involved in translation between archaea and eukaryotes

International audienceProtein synthesis is a complex and highly coordinated process requiring many different protein factors as well as various types of nucleic acids. All translation machinery components require multiple maturation events to be functional. These include post-transcriptional and post-translational modification steps and methylations are the most frequent among these events. In eukaryotes, Trm112, a small protein (COG2835) conserved in all three domains of life, interacts and activates four methyltransferases (Bud23, Trm9, Trm11 and Mtq2) that target different components of the translation machinery (rRNA, tRNAs, release factors). To clarify the function of Trm112 in archaea, we have characterized functionally and structurally its interaction network using Haloferax volcanii as model system. This led us to unravel that methyltransferases are also privileged Trm112 partners in archaea and that this Trm112 network is much more complex than anticipated from eukaryotic studies. Interestingly, among the identified enzymes, some are functionally orthologous to eukaryotic Trm112 partners, emphasizing again the similarity between eukaryotic and archaeal translation machineries. Other partners display some similarities with bacterial methyltransferases, suggesting that Trm112 is a general partner for methyltransferases in all living organisms

The human 18S rRNA m6A methyltransferase METTL5 is stabilized by TRMT112

International audienceN6-methyladenosine (m6A) has recently been found abundantly on messenger RNA and shown to regulate most steps of mRNA metabolism. Several important m6A methyltransferases have been described functionally and structurally, but the enzymes responsible for installing one m6A residue on each subunit of human ribosomes at functionally important sites have eluded identification for over 30 years. Here, we identify METTL5 as the enzyme responsible for 18S rRNA m6A modification and confirm ZCCHC4 as the 28S rRNA modification enzyme. We show that METTL5 must form a heterodimeric complex with TRMT112, a known methyltransferase activator, to gain metabolic stability in cells. We provide the first atomic resolution structure of METTL5–TRMT112, supporting that its RNA-binding mode differs distinctly from that of other m6A RNA methyltransferases. On the basis of similarities with a DNA methyltransferase, we propose that METTL5–TRMT112 acts by extruding the adenosine to be modified from a double-stranded nucleic acid