Identification of proteins that form specific complexes with the highly conserved protein Translin in Schizosaccharomyces pombe.

Translin is a single-stranded DNA and RNA binding protein that has a high affinity for G-rich sequences. TRAX is a Translin paralog that associates with Translin. Both Translin and TRAX were highly conserved in eukaryotes. The nucleic acid binding form of Translin is a barrel-shaped homo-octamer. A Translin-TRAX hetero-octamer having a similar structure also binds nucleic acids. Previous reports suggested that Translin may be involved in chromosomal translocations, telomere metabolism and the control of mRNA transport and translation. More recent studies have indicated that Translin-TRAX hetero-octamers are involved in RNA silencing. To gain a further insight into the functions of Translin, we have undertaken to systematically search for proteins with which it forms specific complexes in living cells. Here we report the results of such a search conducted in the fission yeast Schizosaccharomyces pombe, a suitable model system. This search was carried out by affinity purification and immuno-precipitation techniques, combined with differential labeling of the intracellular proteins with the stable isotopes ¹5N and ¹4N. We identified for the first time two proteins containing an RNA Recognition Motif (RRM), which are specifically associated with the yeast Translin: (1) the pre-mRNA-splicing factor srp1 that belongs to the highly conserved SR family of proteins and (2) vip1, a protein conserved in fungi. Our data also support the presence of RNA in these intracellular complexes. Our experimental approach should be generally applicable to studies of weak intracellular protein-protein interactions and provides a clear distinction between false positive vs. truly interacting proteins.

Conformational transitions in human translin enable nucleic acid binding.

Translin is a highly conserved RNA- and DNA-binding protein that plays essential roles in eukaryotic cells. Human translin functions as an octamer, but in the octameric crystallographic structure, the residues responsible for nucleic acid binding are not accessible. Moreover, electron microscopy data reveal very different octameric configurations. Consequently, the functional assembly and the mechanism of nucleic acid binding by the protein remain unclear. Here, we present an integrative study combining small-angle X-ray scattering (SAXS), site-directed mutagenesis, biochemical analysis and computational techniques to address these questions. Our data indicate a significant conformational heterogeneity for translin in solution, formed by a lesser-populated compact octameric state resembling the previously solved X-ray structure, and a highly populated open octameric state that had not been previously identified. On the other hand, our SAXS data and computational analyses of translin in complex with the RNA oligonucleotide (GU)12 show that the internal cavity found in the octameric assemblies can accommodate different nucleic acid conformations. According to this model, the nucleic acid binding residues become accessible for binding, which facilitates the entrance of the nucleic acids into the cavity. Our data thus provide a structural basis for the functions that translin performs in RNA metabolism and transport.

Mapping of interaction sites of the Schizosaccharomyces pombe protein Translin with nucleic acids and proteins: a combined molecular genetics and bioinformatics study.

Translin is a single-stranded RNA- and DNA-binding protein, which has been highly conserved in eukaryotes, from man to Schizosaccharomyces pombe. TRAX is a Translin paralog associated with Translin, which has coevolved with it. We generated structural models of the S. pombe Translin (spTranslin), based on the solved 3D structure of the human ortholog. Using several bioinformatics computation tools, we identified in the equatorial part of the protein a putative nucleic acids interaction surface, which includes many polar and positively charged residues, mostly arginines, surrounding a shallow cavity. Experimental verification of the bioinformatics predictions was obtained by assays of nucleic acids binding to amino acid substitution variants made in this region. Bioinformatics combined with yeast two-hybrid assays and proteomic analyses of deletion variants, also identified at the top of the spTranslin structure a region required for interaction with spTRAX, and for spTranslin dimerization. In addition, bioinformatics predicted the presence of a second protein-protein interaction site at the bottom of the spTranslin structure. Similar nucleic acid and protein interaction sites were also predicted for the human Translin. Thus, our results appear to generally apply to the Translin family of proteins, and are expected to contribute to a further elucidation of their functions.

The oncolytic activity of Newcastle disease virus NDV-HUJ on chemoresistant primary melanoma cells is dependent on the proapoptotic activity of the inhibitor of apoptosis protein Livin.

Patients with advanced melanoma usually do not benefit from conventional chemotherapy treatment. There is therefore a true need for a new kind of therapy for melanoma. One factor responsible for the poor prognosis of melanoma is the inhibitor of apoptosis protein (IAP) family member Livin. In this study, we applied a novel approach for the treatment of melanoma, using a unique strain of the oncolytic Newcastle disease virus (NDV-HUJ). We found that, unlike chemotherapeutic drugs, NDV-HUJ, a one-cycle replicating virus, overcomes the resistance to apoptosis of melanoma primary cultures that over express the Livin protein. In contrast, melanoma tumor cells that do not express Livin are relatively resistant to NDV-HUJ treatment. Furthermore, we show that NDV-HUJ-induced oncolysis is attributed to the dual function of Livin: although Livin inhibits apoptosis through the inhibition of caspases, under the robust apoptotic stimulation of NDV-HUJ, caspases can cleave Livin to create a truncated protein with a paradoxical proapoptotic activity. Thus, NDV-HUJ is a potent inducer of apoptosis that can overcome the antiapoptotic effect of Livin and allow cleavage of Livin into the proapoptotic tLivin protein. Moreover, the results indicate that the interferon system, which is functional in melanoma, is not involved in NDV-induced oncolysis. Taken together, our data offer the possibility of a new viral oncolytic treatment for chemoresistant melanoma.

Effects of group housing on reproductive performance, lameness, injuries and saliva cortisol in gestating sows.

In many countries sows are kept in individual stalls from insemination up to just few days prior to farrowing. The overall objective of this study was to examine group housing management system for sows during gestation as an alternative for individual confinement stalls, and the possible effects on their welfare, production and reproduction performances. Accordingly, the study included three specific objectives: (1) to compare parameters of production, reproduction, and welfare of sows housed in groups (either 30 or 7 sows/group; Large Group: LG, Small Group: SG, respectively) during gestation as compared to individual confinement stalls (IS); (2) to compare saliva cortisol of pregnant sows throughout gestation, when housed in groups of three different sizes (either 7, 15 or 30 sows per pen group); and (3) to compare sows' production and reproduction performances at the herd level, before, during and after practically transforming from a management of individual confinement stalls to a group housing system, in a large commercial swine farm over a six-year period. Mean cycle length (weaning-to-weaning) was shorter in group housing management as compared to individual stalls (P = 0.0110), but gestation length did not differ among the three groups. Overall farrowing rate (sows farrowed out of those inseminated) was higher (P ≤ 0.0134) for sows housed in groups (either SG or LG). Furthermore, there was a tendency towards a higher number of total born (P = 0.1033), and born alive piglets (P = 0.0862), in group housing system as compared to individual housing management; however, it did not differ between the LG and SG groups. Injuries and lameness index (ILI) of sows improved significantly over the gestation period in group housing management. Group saliva cortisol during gestation did not differ significantly among groups of 7, 15, or 30 sows, except on the first saliva sampling, just after sows were mixed into groups, where cortisol level was significantly higher in sows housed in a pen of 30 sows. Production and reproduction performances at the herd level, over a 6-years period- before, during and after transforming to a group housing system, improved significantly: shortened cycle length, increased farrowing rate, and increased number of total born and born alive piglets. In conclusion, group housing management during gestation was associated with better reproduction, productivity and welfare of sows, as compared to individual stalls. A welfare friendly housing system can be beneficial and effective for both the farmers and the animals.

A novel open-barrel structure of octameric translin reveals a potential RNA entryway.

The single-stranded DNA (ssDNA)/RNA binding protein translin was suggested to be involved in chromosomal translocations, telomere metabolism, and mRNA transport and translation. Oligonucleotide binding surfaces map within a closed cavity of translin octameric barrels, raising the question as to how DNA/RNA gain access to this inner cavity, particularly given that, to date, none of the barrel structures reported hint to an entryway. Here, we argue against a mechanism by which translin octamers may "dissociate and reassemble" upon RNA binding and report a novel "open"-barrel structure of human translin revealing a feasible DNA/RNA entryway into the cavity. Additionally, we report that translin not only is confined to binding of ssDNA oligonucleotides, or single-stranded extensions of double-stranded DNA (dsDNA), but also can bind single-stranded sequences internally embedded in dsDNA molecules.

A Search for Ribonucleic Antiterminator Sites in Bacterial Genomes: Not Only Antitermination?

BglG/LicT-like proteins are transcriptional antiterminators that prevent termination of transcription at intrinsic terminators by binding to ribonucleic antiterminator (RAT) sites and stabilizing an RNA conformation which is mutually exclusive with the terminator structure. The known RAT sites, which are located in intergenic regions of sugar utilization operons, show low sequence conservation but significant structural analogy. To assess the prevalence of RATs in bacterial genomes, we employed bioinformatic tools that describe RNA motifs based on both sequence and structural constraints. Using descriptors with different stringency, we searched the genomes of Escherichiacoli K12, uropathogenic E. coli and Bacillus subtilis for putative RATs. Our search identified all known RATs and additional putative RAT elements. Surprisingly, most putative RATs do not overlap an intrinsic terminator and many reside within open reading frames (ORFs). The ability of one of the putative RATs, which is located within an antiterminator-encoding ORF and does not overlap a terminator, to bind to its cognate antiterminator protein in vitro and in vivo was confirmed experimentally. Our results suggest that the capacity of RAT elements has been exploited during evolution to mediate activities other than antitermination, for example control of transcription elongation or of RNA stability.