Fanconi anemia A protein participates in nucleolar homeostasis maintenance and ribosome biogenesis.

Fanconi anemia (FA), the most common inherited bone marrow failure and leukemia predisposition syndrome, is generally attributed to alterations in DNA damage responses due to the loss of function of the DNA repair and replication rescue activities of the FANC pathway. Here, we report that FANCA deficiency, whose inactivation has been identified in two-thirds of FA patients, is associated with nucleolar homeostasis loss, mislocalization of key nucleolar proteins, including nucleolin (NCL) and nucleophosmin 1 (NPM1), as well as alterations in ribosome biogenesis and protein synthesis. FANCA coimmunoprecipitates with NCL and NPM1 in a FANCcore complex-independent manner and, unique among the FANCcore complex proteins, associates with ribosomal subunits, influencing the stoichiometry of the translational machineries. In conclusion, we have identified unexpected nucleolar and translational consequences specifically associated with FANCA deficiency that appears to be involved in both DNA damage and nucleolar stress responses, challenging current hypothesis on FA physiopathology.

HIV-1 DNA Flap formation promotes uncoating of the pre-integration complex at the nuclear pore.

The HIV-1 central DNA Flap acts as a cis-acting determinant of HIV-1 genome nuclear import. Indeed, DNA-Flap re-insertion within lentiviral-derived gene transfer vectors strongly stimulates gene transfer efficiencies. In this study, we sought to understand the mechanisms by which the central DNA Flap mediates HIV-1 nuclear import. Here, we show that reverse transcription (RT degrees) occurs within an intact capsid (CA) shell, independently of the routing process towards the nuclear membrane, and that uncoating is not an immediate post-fusion event, but rather occurs at the nuclear pore upon RT degrees completion. We provide the first observation with ultrastructural resolution of intact intracellular HIV-1 CA shells by scanning electron microscopy. In the absence of central DNA Flap formation, uncoating is impaired and linear DNA remains trapped within an integral CA shell precluding translocation through the nuclear pore. These data show that DNA Flap formation, the very last event of HIV-1 RT degrees, acts as a viral promoting element for the uncoating of HIV-1 at the nuclear pore.

Wild-type and central DNA flap defective HIV-1 lentiviral vector genomes: intracellular visualization at ultrastructural resolution levels.

HIV-1 and other lentiviruses have the unique ability among retroviruses to efficiently replicate in non-dividing cells as a result of the active nuclear import of their DNA genome across an interphasic nuclear membrane. Previous work has shown that a three-stranded DNA structure synthesized during HIV-1 reverse transcription, called the central DNA flap, acts as a cis-determinant of HIV-1 genome nuclear import. Concordantly, DNA Flap re-insertion in lentiviral-derived gene therapy vectors stimulates gene transfer efficiencies and complements the level of nuclear import to wild-type levels quantitatively indistinguishable from wild-type virus in all cell types and tissues examined so far. In order to define the precise nature of the replicative defect of DNA flap mutant viruses, we carried out in situ DNA hybridization experiments with electron microscopy to determine the subcellular localization of DNA flap mutant and wild-type HIV-1 genomes. We found that Flap defective DNA genomes accumulate at the cytoplasmic face of the nuclear membrane with no overlap across the nuclear membrane, whereas wild-type genomes localize throughout the nuclear compartment. These data provide an unequivocal confirmation of the role of the DNA flap in HIV-1 nuclear import and further establish that the DNA flap controls a step that immediately precedes translocation through the nuclear pore. Further, the widespread distribution of wild-type genomes within the open chromatin confirms the recent genome-wide mapping of HIV-1 cDNA integration sites and points to an as-yet poorly understood step of intranuclear transport of HIV-1 pre-integration complexes.

The relationship between BCL6 bodies and nuclear sites of normal and halogenated DNA and RNA synthesis.

BCL6 is a POZ/BTB and zinc finger transcription factor that self-interacts and accumulates into discrete nuclear "bodies" of unknown function. We recently reported that BCL6 bodies associate with bromodeoxyuridine (BrdU)-substituted DNA, suggesting their implication in replication. To examine this possibility, we examine here by electron and confocal microscopy the relation between BCL6 bodies and replication foci (RF) using incorporation of various halogenated nucleotides (BrdU, chlorodeoxyuridine, CldU, and iododeoxyuridine, IdU) or PCNA (proliferating cell nuclear antigen) staining. We show that BCL6 bodies are found associated with RF, as revealed by PCNA staining. However, such association is markedly prolonged upon BrdU or CldU incorporation, but less, or not at all, upon IdU incorporation. Pulse-chase and double-labeling experiments indicate that IdU-substituted DNA leaves BCL6 bodies after a few tenths of minutes while BrdU- or CldU-substituted DNA stalls in their vicinity for several hours, thereby giving the characteristic "crowns" of DNA entirely surrounding BCL6 bodies. In all cases, however, the halogenated DNA ends up undergoing a movement from BCL6 bodies toward nucleoplasm and nuclear periphery to reach euchromatin and heterochromatin, respectively. We propose that replicating DNA is prone to be bound by BCL6, while BrdU/CldU incorporation increases this propensity possibly because these two events have synergistic effects on the structure and chromatinisation of the newly synthesized DNA. Finally, despite the known proximity between nuclear sites of transcription and replication, we show via several approaches that BCL6 bodies do not appear to be involved either in RNA synthesis or storage.

Adenovirus infection targets the cellular protein kinase CK2 and RNA-activated protein kinase (PKR) into viral inclusions of the cell nucleus.

The effects of the adenovirus infection on the distribution of the cellular protein kinase CK2 and double-stranded RNA-activated protein kinase (PKR) were examined at the ultrastructural level. Immunogold labeling revealed the redistribution of CK2 subunits and PKR to morphologically distinct structures of the cell nucleus. The electron-clear amorphous structures, designated pIX nuclear bodies in our previous work (Rosa-Calatrava et al., 2001), contained CK2 alpha and PKR. The protein crystals, which result from the regular assembly of hexon, penton base, and fiber proteins [Boulanger et al. (1970) J Gen Virol 6:329-332], contained CK2 beta and PKR. Both viral structures were devoid of viral RNA, including the PKR-inhibitor VA1 RNA generated by the RNA polymerase III. Instead, VA1 RNA accumulated in PKR-free viral compact rings in which the viral RNA generated by the RNA polymerase II was excluded.