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International audience; The integration of the retroviral genome into the chromatin of the infected cell is catalysed by the integrase (IN)•viral DNA complex (intasome). This process requires functional association between the integration complex and the nucleosomes. Direct intasome/histone contacts have been reported to modulate the interaction between the integration complex and the target DNA (tDNA). Both prototype foamy virus (PFV) and HIV-1 integrases can directly bind histone amino-terminal tails. We have further investigated this final association by studying the effect of isolated histone tails on HIV-1 integration. We show here that the binding of HIV-1 IN to a peptide derived from the H4 tail strongly stimulates integration catalysis in vitro. This stimulation was not observed with peptide tails from other variants or with alpha-retroviral (RAV) and spuma-retroviral PFV integrases. Biochemical analyses show that the peptide tail induces both an increase in the IN oligomerization state and affinity for the target DNA, which are associated with substantial structural rearrangements in the IN carboxy-terminal domain (CTD) observed by NMR. Our data indicate that the H4 peptide tail promotes the formation of active strand transfer complexes (STCs) and support an activation step of the incoming intasome at the contact of the histone tail.

Retroviral integrase (IN) proteins catalyze the permanent integration of the viral genome into host DNA. They can productively recruit cellular proteins, and the human Bromodomain and Extra-Terminal domain (hBET) proteins have been shown to be co-factors for integration of gamma-retroviruses such as Murine Leukemia Virus (MLV) into human cells. By using two-hybrid, co-immunoprecipitation and in vitro interaction assays, we showed that IN of the gamma- Porcine Endogenous Retrovirus-A/C (PERV IN) interacts through its C-terminal domain (CTD) with hBET proteins. We observed that PERV IN interacts with the BRD2, BRD3 and BRD4 proteins in vitro and that the BRD2 protein specifically binds and co-localizes with PERV IN protein in the nucleus of cells. We further mapped the interaction sites to the conserved Extra-Terminal (ET) domain of the hBET proteins and to several amino acids of the of the C-terminal tail of the PERV IN CTD. Finally, we determined the first experimental structure of an IN CTD - BET ET complex from small-angle X-ray scattering data (SAXS). We showed that the two factors assemble as two distinct modules linked by a short loop which confers partial flexibility. The SAXS-restrained model is structurally compatible with the binding of the PERV intasome to BRD2. Altogether, these data confirm the important role of host BET proteins in the gamma-retroviruses' targeting site and efficiency of integration.

International audience; The RNA helicase Upf1 is a multifaceted eukaryotic enzyme involved in DNA replication, telomere metabolism and several mRNA degradation pathways. Upf1 plays a central role in nonsense-mediated mRNA decay (NMD), a surveillance process in which it links premature translation termination to mRNA degradation with its conserved partners Upf2 and Upf3. In human, both the ATP-dependent RNA helicase activity and the phosphorylation of Upf1 are essential for NMD. Upf1 activation occurs when Upf2 binds its N-terminal domain, switching the enzyme to the active form. Here, we uncovered that the C-terminal domain of Upf1, conserved in higher eukaryotes and containing several essential phosphorylation sites, also inhibits the flanking helicase domain. With different biochemical approaches we show that this domain, named SQ, directly interacts with the helicase domain to impede ATP hydrolysis and RNA unwinding. The phosphorylation sites in the distal half of the SQ domain are not directly involved in this inhibition. Therefore, in the absence of multiple binding partners, Upf1 is securely maintained in an inactive state by two intramolecular inhibition mechanisms. This study underlines the tight and intricate regulation pathways required to activate multifunctional RNA helicases like Upf1.

The exon-junction complex (EJC) functionally links splicing to subsequent mRNA localization, translation and stability. Sequence-independent binding of the EJC core to RNA is ensured by the DEAD-box helicase eIF4AIII. Here, we identified the splicing factor CWC22 as a new eIF4AIII partner in flies and humans. CWC22 coexists with eIF4AIII in large protein complexes distinct from EJCs. Recombinant CWC22 directly contacts eIF4AIII and prevents it from binding RNA. In vitro splicing assays revealed that CWC22 introduces eIF4AIII to spliceosomes before remodeling to facilitate eIF4AIII incorporation into the EJC. Finally, using knockdowns in vivo, we showed that CWC22 is essential for EJC assembly. We elucidated the initial step of EJC assembly and the duality of CWC22 function that hinders eIF4AIII from nonspecifically binding RNA and escorts it to the splicing machinery to promote EJC assembly on mature mRNAs.

Degradation of eukaryotic mRNAs harboring a premature translation termination codon is ensured by the process of nonsense-mediated mRNA decay (NMD). The main effector of this quality-control pathway is the conserved RNA helicase UPF1 that forms a surveillance complex with the proteins UPF2 and UPF3. In all the organisms tested, the ATPase activity of UPF1 is essential for NMD. Here, we describe the expression of active recombinant UPF proteins and the reconstitution of the surveillance complex in vitro. To understand how UPF1 is regulated during NMD, we developed different biochemical approaches. We describe methods to monitor UPF1 binding to RNA, ATP hydrolysis and RNA unwinding in the presence of its binding partner UPF2. This functional analysis is an important complement for structural studies of protein complexes containing RNA helicases.

Summary Upf1 is a crucial factor in nonsense-mediated mRNA decay, the eukaryotic surveillance pathway that degrades mRNAs containing premature stop codons. The essential RNA-dependent ATPase activity of Upf1 is triggered by the formation of the surveillance complex with Upf2-Upf3. We report crystal structures of Upf1 in the presence and absence of the CH domain, captured in the transition state with ADP:AlF 4 − and RNA. In isolation, Upf1 clamps onto the RNA, enclosing it in a channel formed by both the catalytic and regulatory domains. Upon binding to Upf2, the regulatory CH domain of Upf1 undergoes a large conformational change, causing the catalytic helicase domain to bind RNA less extensively and triggering its helicase activity. Formation of the surveillance complex thus modifies the RNA binding properties and the catalytic activity of Upf1, causing it to switch from an RNA-clamping mode to an RNA-unwinding mode.