Individual influenza A virus mRNAs show differential dependence on cellular NXF1/TAP for their nuclear export

The influenza A virus RNA-dependent RNA polymerase produces capped and polyadenylated mRNAs in the nucleus of infected cells that resemble mature cellular mRNAs, but are made by very different mechanisms. Furthermore, only two of the 10 viral protein-coding mRNAs are spliced: most are intronless, while two contain unremoved introns. The mechanism(s) by which any of these mRNAs are exported from the nucleus is uncertain. To probe the involvement of the primary cellular mRNA export pathway, we treated cells with siRNAs against NXF1, Aly or UAP56, or with the drug 5,6-dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB), an inhibitor of RNA polymerase II phosphorylation previously shown to inhibit nuclear export of cellular mRNA as well as influenza virus segment 7 mRNAs. Depletion of NXF1 or DRB treatment had similar effects, inhibiting the nuclear export of several of the viral mRNAs. However, differing degrees of sensitivity were seen, depending on the particular segment examined. Intronless HA mRNA and spliced M2 or unspliced M1 transcripts (all encoding late proteins) showed a strong requirement for NXF1, while intronless early gene mRNAs, especially NP mRNA, showed the least dependency. Depletion of Aly had little effect on viral mRNA export, but reduction of UAP56 levels strongly inhibited trafficking and/or translation of the M1, M2 and NS1 mRNAs. Synthesis of NS2 from the spliced segment 8 transcript was, however, resistant. We conclude that influenza A virus co-opts the main cellular mRNA export pathway for a subset of its mRNAs, including most but not all late gene transcripts

Histone H3 Serine 57 and Lysine 56 Interplay in Transcription Elongation and Recovery from S-Phase Stress

Contains fulltext : 84400.pdf (publisher's version ) (Open Access

Histone H3K56 Acetylation, CAF1, and Rtt106 Coordinate Nucleosome Assembly and Stability of Advancing Replication Forks

Chromatin assembly mutants accumulate recombinogenic DNA damage and are sensitive to genotoxic agents. Here we have analyzed why impairment of the H3K56 acetylation-dependent CAF1 and Rtt106 chromatin assembly pathways, which have redundant roles in H3/H4 deposition during DNA replication, leads to genetic instability. We show that the absence of H3K56 acetylation or the simultaneous knock out of CAF1 and Rtt106 increases homologous recombination by affecting the integrity of advancing replication forks, while they have a minor effect on stalled replication fork stability in response to the replication inhibitor hydroxyurea. This defect in replication fork integrity is not due to defective checkpoints. In contrast, H3K56 acetylation protects against replicative DNA damaging agents by DNA repair/tolerance mechanisms that do not require CAF1/Rtt106 and are likely subsequent to the process of replication-coupled nucleosome deposition. We propose that the tight connection between DNA synthesis and histone deposition during DNA replication mediated by H3K56ac/CAF1/Rtt106 provides a mechanism for the stabilization of advancing replication forks and the maintenance of genome integrity, while H3K56 acetylation has an additional, CAF1/Rtt106-independent function in the response to replicative DNA damage

The Ccr4-Not Complex Interacts with the mRNA Export Machinery

The Ccr4-Not complex is a key eukaryotic regulator of gene transcription and cytoplasmic mRNA degradation. Whether this complex also affects aspects of post-transcriptional gene regulation, such as mRNA export, remains largely unexplored. Human Caf1 (hCaf1), a Ccr4-Not complex member, interacts with and regulates the arginine methyltransferase PRMT1, whose targets include RNA binding proteins involved in mRNA export. However, the functional significance of this regulation is poorly understood.Here we demonstrate using co-immunoprecipitation approaches that Ccr4-Not subunits interact with Hmt1, the budding yeast ortholog of PRMT1. Furthermore, using genetic and biochemical approaches, we demonstrate that Ccr4-Not physically and functionally interacts with the heterogenous nuclear ribonucleoproteins (hnRNPs) Nab2 and Hrp1, and that the physical association depends on Hmt1 methyltransferase activity. Using mass spectrometry, co-immunoprecipitation and genetic approaches, we also uncover physical and functional interactions between Ccr4-Not subunits and components of the nuclear pore complex (NPC) and we provide evidence that these interactions impact mRNA export.Taken together, our findings suggest that Ccr4-Not has previously unrealized functional connections to the mRNA processing/export pathway that are likely important for its role in gene expression. These results shed further insight into the biological functions of Ccr4-Not and suggest that this complex is involved in all aspects of mRNA biogenesis, from the regulation of transcription to mRNA export and turnover

HIV infection of non-dividing cells: a divisive problem

Understanding how lentiviruses can infect terminally differentiated, non-dividing cells has proven a very complex and controversial problem. It is, however, a problem worth investigating, for it is central to HIV-1 transmission and AIDS pathogenesis. Here I shall attempt to summarise what is our current understanding for HIV-1 infection of non-dividing cells. In some cases I shall also attempt to make sense of controversies in the field and advance one or two modest proposals

Homolog of BRCA2-interacting Dss1p and Uap56p link Mlo3p and Rae1p for mRNA export in fission yeast

The breast cancer tumor suppressor BRCA2-interacting protein, DSS1, and its homologs are critical for DNA recombination in eukaryotic cells. We found that Dss1p, along with Mlo3p and Uap56p, Schizosaccharomyces pombe homologs of two messenger RNA (mRNA) export factors of the NXF–NXT pathway, is required for mRNA export in S. pombe. Previously, we showed that the nuclear pore-associated Rae1p is an essential mRNA export factor in S. pombe. Here, we show that Dss1p and Uap56p function by linking mRNA adapter Mlo3p to Rae1p for targeting mRNA–protein complex (mRNP) to the proteins of the nuclear pore complex (NPC). Dss1p preferentially recruits to genes in vivo and interacts with –FG (phenylalanine glycine) nucleoporins in vivo and in vitro. Thus, Dss1p may function at multiple steps of mRNA export, from mRNP biogenesis to their targeting and translocation through the NPC

A Kaposi's sarcoma virus RNA element that increases the nuclear abundance of intronless transcripts

The Kaposi's sarcoma-associated herpesvirus produces a 1077 nucleotide noncoding, polyadenylated, exclusively nuclear RNA called PAN that is highly expressed in lytically infected cells. We report that PAN contains a novel post-transcriptional element essential for its abundant accumulation. The element, PAN–ENE (PAN RNA expression and nuclear retention element), increases the efficiency of 3′-end formation in vivo and is sufficient to enhance RNA abundance from an otherwise inefficiently expressed intronless β-globin construct. The PAN–ENE does not concomitantly increase the production of encoded protein. Rather, it retains the unspliced β-globin mRNA in the nucleus. Tethering of export factors can override the nuclear retention of the PAN–ENE, supporting a mechanism whereby the PAN–ENE blocks assembly of an export-competent mRNP. The activities of the PAN–ENE are specific to intronless constructs, since inserting the PAN–ENE into a spliced β-globin construct has no effect on mRNA abundance and does not affect localization. This is the first characterization of a cis-acting element that increases RNA abundance of intronless transcripts but inhibits assembly of an export-competent mRNP