Summary Severe sepsis remains a poorly understood systemic inflammatory condition with high mortality rates and limited therapeutic options in addition to organ support measures. Here we show that the clinically approved group of anthracyclines acts therapeutically at a low dose regimen to confer robust protection against severe sepsis in mice. This salutary effect is strictly dependent on the activation of DNA damage response and autophagy pathways in the lung, as demonstrated by deletion of the ataxia telangiectasia mutated (Atm) or the autophagy-related protein 7 (Atg7) specifically in this organ. The protective effect of anthracyclines occurs irrespectively of pathogen burden, conferring disease tolerance to severe sepsis. These findings demonstrate that DNA damage responses, including the ATM and Fancony Anemia pathways, are important modulators of immune responses and might be exploited to confer protection to inflammation-driven conditions, including severe sepsis.

U2AF65 is an essential splicing factor that promotes binding of U2 small nuclear (sn)RNP at the pre-mRNA branchpoint. Here we describe a novel monoclonal antibody that reacts specifically with U2AF65. Using this antibody, we show that U2AF65 is diffusely distributed in the nucleoplasm with additional concentration in nuclear speckles, which represent subnuclear compartments enriched in splicing snRNPs and other splicing factors. Furthermore, transient expression assays using epitope-tagged deletion mutants of U2AF65 indicate that targeting of the protein to nuclear speckles is not affected by removing either the RNA binding domain, the RS domain, or the region required for interaction with U2AF35. The association of U2AF65 with speckles persists during mitosis, when transcription and splicing are downregulated. Moreover, U2AF65 is localized to nuclear speckles in early G1 cells that were treated with transcription inhibitors during mitosis, suggesting that the localization of U2AF65 in speckles is independent of the presence of pre-mRNA in the nucleus, which is consistent with the idea that speckles represent storage sites for inactive splicing factors. After adenovirus infection, U2AF65 redistributes from the speckles and is prefferentially detected at sites of viral transcription. By combining adenoviral infection with transient expression of deletion mutants, we show a specific requirement of the RS domain for recruitment of U2AF65 to sites of active splicing in the nucleus. This suggests that interactions involving the RS region of U2AF65 may play an important role in targeting this protein to spliceosomes in vivo.

The U2 small nuclear ribonucleoprotein auxiliary factor (U2AF) is a heterodimeric splicing factor composed of 65-kDa (U2AF 65 ) and 35-kDa (U2AF 35) subunits. The large subunit of U2AF recognizes the intronic polypyrimidine tract, a sequence located adjacent to the 3 splice site that serves as an important signal for both constitutive and regulated pre-mRNA splicing. The small subunit U2AF35 interacts with the 3 splice site dinucleotide AG and is essential for regulated splicing. Like several other proteins involved in constitutive and regulated splicing, both U2AF 65 and U2AF 35 contain an arginine/ serine-rich (RS) domain. In the present study we determined the role of RS domains in the subcellular localization of U2AF. Both U2AF 65 and U2AF 35 are shown to shuttle continuously between the nucleus and the cytoplasm by a mechanism that involves carrier receptors and is independent from binding to mRNA. The RS domain on either U2AF 65 or U2AF 35 acts as a nuclear localization signal and is sufficient to target a heterologous protein to the nuclear speckles. Furthermore, the results suggest that the presence of an RS domain in either U2AF subunit is sufficient to trigger the nucleocytoplasmic import of the heterodimeric complex. Shuttling of U2AF between nucleus and cytoplasm possibly represents a means to control the availability of this factor to initiate spliceosome assembly and therefore contribute to regulate splicing.Pre-mRNA splicing is an essential step in eukaryotic gene expression that is often regulated in a cell type-specific or developmental manner. The splicing reaction takes place in the spliceosome, a multicomponent RNA-protein complex containing five uracil-rich small nuclear ribonucleoproteins (snRNP) 1 and 50 -100 non-snRNP protein splicing factors (1, 2). To ensure the precise excision of introns, components of the spliceosome recognize weakly conserved sequences in the pre-mRNA (3). The decision to splice a given pre-mRNA and the selection of splice sites takes place during the first stages of spliceosome assembly. These early steps are therefore crucial for splicing regulation (3-5). The stable association of U2 snRNP with the 3Ј region of the intron (3, 6, 7) requires an auxiliary factor, U2AF (8). U2AF consists of two subunits that form a stable heterodimer (9). U2AF 65 binds directly to the intronic polypyrimidine tract and is essential for splicing (10). U2AF35 recognizes the 3Ј splice site dinucleotide AG (11-13) and is required for the regulated splicing of a subset of pre-mRNAs (14).Regulatory mechanisms underlying the selection of alternative exons remain poorly defined (5, 15). A significant advance in understanding splice site recognition was provided by the observation that exon sequences also play a critical role in splicing. Constitutive as well as regulated exon splicing enhancers have been identified (16,17). These sequences contain binding sites for SR proteins (18, 19), a family of essential splicing factors with a modular structure consisting of one or two RRMs and a C-term...

The spatial separation of mRNA synthesis from translation, while providing eukaryotes with the possibility to achieve higher complexity through a more elaborate regulation of gene expression, has set the need for transport mechanisms through the nuclear envelope. In a simplistic view of nucleocytoplasmic transport, nuclear proteins are imported into the nucleus while RNAs are exported to the cytoplasm. The reality is, however, that transport of either proteins or RNAs across the nuclear envelope can be bi-directional. During the past years, an increasing number of proteins have been identified that shuttle continuously back and forth between the nucleus and the cytoplasm. The emerging picture is that shuttling proteins are key factors in conveying information on nuclear and cytoplasmic activities within the cell. ß