Although it has been overlooked for a long time, translation, and more precisely the initiation step is the target of many regulatory mechanisms. In particular, it enables the cell to store mRNA in a repressed state that can be swiftly mobilised to react to stimuli. Among a galore of recently discovered examples we could cite the cases of maternal mRNA in oocytes, of the interferon mediated response to viral infection, or of post synaptic events in neurones. A great amount of structural and biochemical information on translation initiation has been accumulated in recent years, most notably, the cryo-electronic microscopy images and the crystal derived structures of ribosomal subunits complexed or not to a classical mRNA or Internal Ribosome Entry Sites (IRES). Our work aims at understanding the molecular mechanisms ruling translation initiation in eukaryotes, and more specifically the influence of the mRNA structure on this process. To this end we study viral translation, and our main model is HIV genomic RNA translation. We aim at defining the specificity of viral translation to identify new therapeutic targets.
Translation initiation in few words:
Translation initiation in eukaryotes begins by the attachment to the “cap” at the 5’ end of the mRNA of a complex composed by the small ribosomal subunit (40S) and at least ten protein factors known as the eukaryotic Initiation Factors (eIF). This complex then progresses along the 5’ UnTranslated Region (5’UTR) searching for the initiation codon during a process known as “scanning”. Each eIF has a precise function allowing the assembly of a translation-competent ribosome on the bona fide initiation codon. This step is highly regulated.
Canonical “cap-dependant” initiation
However in numerous exceptions, the 5’ UTR can recruit the small ribosomal subunit at, or close to the initiation codon. The 5’ UTR is then qualified of Internal Ribosome Entry Site (IRES). In most cases, this property is due to its 3D structure which is able to specifically interact with the ribosome and/or one of the initiation factors. In those cases, initiation only requires a subset of the initiation factors, the role of the missing eIF being fulfilled by the RNA itself. This allows specific genes to be expressed while general translation is shut down due to a physiological (general translation regulation) or pathological (stress) situation. Interestingly, such property enables viruses to hijack the host translation machinery. For instance, the virus produces a protease that degrades one or several of the initiation factors essential to mRNA translation, and while the cellular translation is shut down, the viral genome is translated through an IRES mechanism which does not require all the initiation factors.
The different types of IRESes and the initiation factors required
HIV genomic RNA translation: Most of our work focuses on the mechanisms leading to the translation of HIV Gag polyprotein. We have recently discovered an IRES located within the coding region of gag of the HIV (Herbreteau et al, 2005).
Internal ribosome entry and cap-dependance of Gag translation
HIV genomic RNA can recruit ribosomal complexes using a unique mechanism which does not require the 5’ UTR, and we have shown that it is dependant of a secondary structure adopted by the Gag coding region of all primates lentiviruses (Weill et al, 2009). This structure allows the viral RNA to directly bind the small ribosomal subunit as well as the pivotal initiation factor eIF3 (Locker et al, 2011).
Secondary structure of the gag open reading frame of primate lentiviruses
Actually, HIV genomic RNA appears to be translated through at least three mechanisms: the canonical cap-dependant, and two IRES-driven mechanisms which depend either on the 5’UTR (specifically activated during the G2/M phase of the cellular cycle), or on the coding region (Balvay et al, 2007; Chamond et al, 2010; de Breyne et al, 2012; Vallejos et al, 2012; Vallejos et al, 2011). This could reflect a regulation of Gag production throughout the viral cycle.
Model for Internal ribosome entry site on HIV genomic RNA
Defining the specific features of viral translation as therapeutic targets:
Our goal is to better understand HIV and the general mechanism of ribosome recruitment on a start codon, with a particular focus on the role of mRNA structures. To this end we have acquire several tools including a semi-automated RNA secondary structure determination method ( SHAPE , other chemical and enzymatic probes). This allows us to probe the structure of RNAs of interest in many conditions, to monitor the influence of mutations, and to determine protein binding sites on RNA. For instance, we recently identified new determinants for HIV genome dimerization (Deforges et al, 2012), we defined G2/M specific protein footprint on HIV 5’UTR (Vallejos et al, 2011) and modelled FIV 5’UTR secondary structure (James & Sargueil, 2008). To characterize in details the cascade of events leading to initiation on viral RNA, we reconstitute this process entirely in vitro form purified components (10 eIF, 2 ribosomal subunits, tRNAMet, and accessory proteins). Viruses constitute a baffling example of information compaction, their RNAs being endowed with both the coding information and structural properties conferring them additional properties. Our goal is to define the specificity of the structures involved in translation to validate them as therapeutic target. As a first step we will screen large libraries of small molecules to isolate compounds that specifically inhibit viral translation.
Balvay L, Lastra ML, Sargueil B, Darlix JL, Ohlmann T (2007) Translational control of retroviruses. Nat Rev Microbiol 5 : 128-140
Chamond N, Locker N, Sargueil B (2010) The different pathways of HIV genomic RNA translation. Biochem Soc Trans 38 : 1548-1552
de Breyne S, Chamond N, Decimo D, Trabaud MA, Andre P, Sargueil B, Ohlmann T (2012) In vitro studies reveal that different modes of initiation on HIV-1 mRNA have different levels of requirement for eIF4F. The FEBS journal 279 : 3098-3111
Deforges J, Chamond N, Sargueil B (2012) Structural investigation of HIV-1 genomic RNA dimerization process reveals a role for the Major Splice-site stem loop. Biochimie 94 : 1481-1489
Herbreteau CH, Weill L, Decimo D, Prevot D, Darlix JL, Sargueil B, Ohlmann T (2005) HIV-2 genomic RNA contains a novel type of IRES located downstream of its initiation codon. Nat Struct Mol Biol 12 : 1001-1007
James L, Sargueil B (2008) RNA secondary structure of the feline immunodeficiency virus 5’UTR and Gag coding region. Nucleic Acids Res 36 : 4653-4666
Locker N, Chamond N, Sargueil B (2011) A conserved structure within the HIV gag open reading frame that controls translation initiation directly recruits the 40S subunit and eIF3. Nucleic Acids Res 39 : 2367-2377
Vallejos M, Carvajal F, Pino K, Navarrete C, Ferres M, Huidobro-Toro JP, Sargueil B, Lopez-Lastra M (2012) Functional and structural analysis of the internal ribosome entry site present in the mRNA of natural variants of the HIV-1. PLoS One 7 : e35031
Vallejos M, Deforges J, Plank TD, Letelier A, Ramdohr P, Abraham CG, Valiente-Echeverria F, Kieft JS, Sargueil B, Lopez-Lastra M (2011) Activity of the human immunodeficiency virus type 1 cell cycle-dependent internal ribosomal entry site is modulated by IRES trans-acting factors. Nucleic Acids Res 39 : 6186-6200
Weill L, James L, Ulryck N, Chamond N, Herbreteau CH, Ohlmann T, Sargueil B (2010) A new type of IRES within gag coding region recruits three initiation complexes on HIV-2 genomic RNA. Nucleic Acids Res 38 : 1367-1381