Background The yeast yCCR4 factor belongs to the CCR4-NOT transcriptional regulatory complex, in which it interacts, through its leucine-rich repeat (LRR) motif with yPOP2. Recently, yCCR4 was shown to be a component of the major cytoplasmic mRNA deadenylase complex, and to contain a fold related to the Mg2+-dependent endonuclease core. Results Here, we report the identification of nineteen yCCR4-related proteins in eukaryotes (including yeast, plants and animals), which all contain the yCCR4 endonuclease-like fold, with highly conserved CCR4-specific residues. Phylogenetic and genomic analyses show that they form four distinct families, one of which contains the yCCR4 orthologs. The orthologs in animals possess a leucine-rich repeat domain. We show, using two-hybrid and far-Western assays, that the human member binds to the human yPOP2 homologs, i.e. hCAF1 and hPOP2, in a LRR-dependent manner. Conclusions We have identified the mammalian orthologs of yCCR4 and have shown that the human member binds to the human yPOP2 homologs, thus strongly suggesting conservation of the CCR4-NOT complex from yeast to human. All members of the four identified yCCR4-related protein families show stricking conservation of the endonuclease-like catalytic motifs of the yCCR4 C-terminal domain and therefore constitute a new family of potential deadenylases in mammals.

Background Soluble guanylyl cyclase (sGC) is the main receptor for nitric oxide (NO) when the latter is produced at low concentrations. This enzyme exists mainly as a heterodimer consisting of one α and one β subunit and converts GTP to the second intracellular messenger cGMP. In turn, cGMP plays a key role in regulating several physiological processes in the nervous system. The aim of the present study was to explore the effects of a NO donor on sGC activity and its protein and subunit mRNA levels in a neural cell model. Results Continuous exposure of bovine adrenal chromaffin cells in culture to the nitric oxide donor, diethylenetriamine NONOate (DETA/NO), resulted in a lower capacity of the cells to synthesize cGMP in response to a subsequent NO stimulus. This effect was not prevented by an increase of intracellular reduced glutathione level. DETA/NO treatment decreased sGC subunit mRNA and β1 subunit protein levels. Both sGC activity and β1 subunit levels decreased more rapidly in chromaffin cells exposed to NO than in cells exposed to the protein synthesis inhibitor, cycloheximide, suggesting that NO decreases β1 subunit stability. The presence of cGMP-dependent protein kinase (PKG) inhibitors effectively prevented the DETA/NO-induced down regulation of sGC subunit mRNA and partially inhibited the reduction in β1 subunits. Conclusions These results suggest that activation of PKG mediates the drop in sGC subunit mRNA levels, and that NO down-regulates sGC activity by decreasing subunit mRNA levels through a cGMP-dependent mechanism, and by reducing β1 subunit stability.

Background: mRNA interactions with each other and other signaling molecules define different biological pathways and functions. Researchers have been investigating various tools to analyze these types of interactions. In particular gene co-expression network methods have proved useful in finding and analyzing these molecular interactions. Many different analytical pipelines to identify these interactions networks have been proposed with the aim of identifying an optimal partition of the network where the individual modules are neither too small to make any general inference or too large to be biologically interpretable. Results: In this study we propose a new pipeline to perform gene co-expression network analysis. The proposed pipeline uses WGCNA a widely used software to perform different aspects of gene co-expression network analysis and modularity maximization algorithm to analyze novel RNA-Seq data to understand the effects of low-dose 56Fe ion irradiation on the formation of hepatocellular carcinoma in mice. The network results along with experimental validation show that using WGCNA combined with Modularity provide a more biologically interpretable network in our dataset. Our pipeline showed better performance than the existing clustering algorithm in WGCNA in finding modules and identified a module with mitochondrial subunits that are supported by mitochondrial complex assay. Conclusions: We present a pipeline that can reduce the problem of parameter selection with the existing algorithm in WGCNA for comparable RNA-Seq datasets which may assist in future research to discover novel mRNA interactions and their downstream molecular effects. C57BL16 males were placed into 2 treatment groups and received the following irradiation treatments at Brookhaven National Laboratories (Long Island NY): 600 MeV/n 56Fe (0.2 Gy) and no irradiation. Left liver lobes were collected at 30 60 120 270 and 360 days post-irradiation flash frozen and stored at -80 xc2 xb0C until they could be processed for RNA-Seq. Livers were sampled by taking two 40-micron thick slices using a cryotome at -20 xc2 xb0C. This allowed multiple sampling of the tissue without the tissue going through multiple freeze/thaw cycles. Total RNA was isolated from the liver slices using RNAqueousTM Total RNA Isolation Kit (ThermoFisher Scientific Waltham MA) and rRNA was removed via Ribo-ZeroTM rRNA Removal Kit (Illumina San Diego CA) prior to library preparation with the Illumina TruSeq RNA Library kit. Samples were sequenced in a paired-end 50 base format on an Illumina HiSeq 1500. Reads were aligned to the mouse GRCm38 reference genome using the STAR alignment program version 2.5.3a with the recommended ENCODE options. The -quantMode GeneCounts option was used to obtain read counts per gene based on the Gencode release M14 annotation file. Total number of reads used in analysis varies between 23-35 millions of reads.

Background Previous work, by us and others, has shown that mammalian galectins-1 have a growth-inhibitory activity for mammalian cells which is apparently independent of their β-galactoside binding site. Results We have made recombinant human galectin-1 as a bacterial fusion protein with an N-terminal hexahistidine tag. This protein displays both haemagglutination and growth-inhibitory activities, even in the presence of the hexahistidine tag. Site-directed mutagenesis of this protein has confirmed the independent nature of the protein sites responsible for the two biological activities. Mutant proteins were created, which displayed each activity in the absence of the other. Conclusions Human galectin-1 possesses a growth-inhibitory site, which is not part of the β-galactoside binding site. A surface loop, comprising amino acid residues 25–30, and joining two internal β-strands, forms part of the growth-inhibitory site. This region is relatively close to the N-terminus of the protein, and N-terminal substitutions or extensions also affect growth-inhibitory activity. Further experiments will be necessary to fully define this site.

Background Nuclear pore complexes (NPCs) are essential for facilitated, directional nuclear transport; however, the mechanism by which ~30 different nucleoporins (nups) are assembled into NPCs is unknown. We combined a genetic strategy in Saccharomyces cerevisiae with Green Fluorescence Protein (GFP) technology to identify mutants in NPC structure, assembly, and localization. To identify such mutants, a bank of temperature sensitive strains was generated and examined by fluorescence microscopy for mislocalization of GFP-tagged nups at the non-permissive temperature. Results A total of 121 mutant strains were isolated, with most showing GFP-Nic96 and Nup170-GFP mislocalized to discrete, cytoplasmic foci. By electron microscopy, several mutants also displayed an expansion of the endoplasmic reticulum (ER). Complementation analysis identified several mutant groups with defects in components required for ER/Golgi trafficking (sec13, sec23, sec27, and bet3). By directed testing, we found that mutant alleles of all COPII components resulted in altered GFP-Nup localization. Finally, at least nine unknown complementation groups were identified that lack secretion defects. Conclusion The isolation of sec mutants in the screen could reflect a direct role for vesicle fusion or the COPII coat during NPC assembly; however, only those sec mutants that altered ER structure affected Nup localization. This suggests that the GFP-Nup mislocalization phenotypes observed in these mutants were the indirect result of overproliferation of the ER and connected outer nuclear envelope. The identification of potentially novel mutants with no secretory defects suggests the distinct GFP-Nup localization defects in other mutants in the collection will provide insights into NPC structure and assembly.

Background DNA methyltransferases (MTases), unlike MTases acting on other substrates, exhibit sequence permutation. Based on the sequential order of the cofactor-binding subdomain, the catalytic subdomain, and the target recognition domain (TRD), several classes of permutants have been proposed. The majority of known DNA MTases fall into the α, β, and γ classes. There is only one member of the ζ class known and no members of the δ and ε classes have been identified to date. Two mechanisms of permutation have been proposed: one involving gene duplication and in-frame fusion, and the other involving inter- and intragenic shuffling of gene segments. Results Two novel cases of sequence permutation in DNA MTases implicated in restriction-modification systems have been identified, which suggest that members of the δ and ζ classes (M.MwoI and M.TvoORF1413P, respectively) evolved from β-class MTases. This is the first identification of the δ-class MTase and the second known ζ-class MTase (the first ζ-class member among DNA:m4C and m6A-MTases). Conclusions Fragmentation of a DNA MTase gene may result from attack of nucleases, for instance when the RM system invades a new cell. Its reassembly into a functional form, the order of motifs notwithstanding, may be strongly selected for, if the cognate ENase gene remains active and poses a threat to the host's chromosome. The "cut-and-paste" mechanism is proposed for β-δ permutation, which is non-circular and involves relocation of one segment of a gene. The circular β-ζ permutation may be explained both by gene duplication or shuffling of gene fragments. These two mechanisms are not mutually exclusive and probably both played a role in the evolution of permuted DNA MTases.

Nidovirus subgenomic mRNAs contain a leader sequence derived from the 5′ end of the genome fused to different sequences (‘bodies’) derived from the 3′ end. Their generation involves a unique mechanism of discontinuous subgenomic RNA synthesis that resembles copy-choice RNA recombination. During this process, the nascent RNA strand is transferred from one site in the template to another, during either plus or minus strand synthesis, to yield subgenomic RNA molecules. Central to this process are transcription-regulating sequences (TRSs), which are present at both template sites and ensure the fidelity of strand transfer. Here we present results of a comprehensive co-variation mutagenesis study of equine arteritis virus TRSs, demonstrating that discontinuous RNA synthesis depends not only on base pairing between sense leader TRS and antisense body TRS, but also on the primary sequence of the body TRS. While the leader TRS merely plays a targeting role for strand transfer, the body TRS fulfils multiple functions. The sequences of mRNA leader–body junctions of TRS mutants strongly suggested that the discontinuous step occurs during minus strand synthesis.

Background Post-transcriptional gene silencing (PTGS) by short interfering RNA has opened up new directions in the phenotypic mutation of cellular genes. However, its efficacy on non-nuclear genes and its effect on the interferon pathway remain unexplored. Since directed mutation of RNA genomes is not possible through conventional mutagenesis, we have tested sequence-specific 21-nucleotide long double-stranded RNAs (dsRNAs) for their ability to silence cytoplasmic RNA genomes. Results Short dsRNAs were generated against specific mRNAs of respiratory syncytial virus, a nonsegmented negative-stranded RNA virus with a cytoplasmic life cycle. At nanomolar concentrations, the dsRNAs specifically abrogated expression of the corresponding viral proteins, and produced the expected mutant phenotype ex vivo. The dsRNAs did not induce an interferon response, and did not inhibit cellular gene expression. The ablation of the viral proteins correlated with the loss of the specific mRNAs. In contrast, viral genomic and antigenomic RNA, which are encapsidated, were not directly affected. Conclusions Synthetic inhibitory dsRNAs are effective in specific silencing of RNA genomes that are exclusively cytoplasmic and transcribed by RNA-dependent RNA polymerases. RNA-directed RNA gene silencing does not require cloning, expression, and mutagenesis of viral cDNA, and thus, will allow the generation of phenotypic null mutants of specific RNA viral genes under normal infection conditions and at any point in the infection cycle. This will, for the first time, permit functional genomic studies, attenuated infections, reverse genetic analysis, and studies of host-virus signaling pathways using a wild type RNA virus, unencumbered by any superinfecting virus.

Background Nitrogen fixation gene expression in Sinorhizobium meliloti, the alfalfa symbiont, depends on a cascade of regulation that involves both positive and negative control. On top of the cascade, the two-component regulatory system FixLJ is activated under the microoxic conditions of the nodule. In addition, activity of the FixLJ system is inhibited by a specific anti-kinase protein, FixT. The physiological significance of this negative regulation by FixT was so far unknown. Results We have isolated by random Tn5 mutagenesis a S. meliloti mutant strain that escapes repression by FixT. Complementation test and DNA analysis revealed that inactivation of an asparagine synthetase-like gene was responsible for the phenotype of the mutant. This gene, that was named asnO, encodes a protein homologous to glutamine-dependent asparagine synthetases. The asnO gene did not appear to affect asparagine biosynthesis and may instead serve a regulatory function in S. meliloti. We provide evidence that asnO is active during symbiosis . Conclusions Isolation of the asnO mutant argues for the existence of a physiological regulation associated with fixT and makes it unlikely that fixT serves a mere homeostatic function in S. meliloti. Our data suggest that asnO might control activity of the FixT protein, in a way that remains to be elucidated. A proposed role for asnO might be to couple nitrogen fixation gene expression in S. meliloti to the nitrogen needs of the cells.