Ribosomal oxygenases are structurally conserved from prokaryotes to humans

2-Oxoglutarate (2OG)-dependent oxygenases have important roles in the regulation of gene expression via demethylation of N-methylated chromatin components1,2 and in the hydroxylation of transcription factors3 and splicing factor proteins4. Recently, 2OG-dependent oxygenases that catalyse hydroxylation of transfer RNA5,6,7 and ribosomal proteins8 have been shown to be important in translation relating to cellular growth, TH17-cell differentiation and translational accuracy9,10,11,12. The finding that ribosomal oxygenases (ROXs) occur in organisms ranging from prokaryotes to humans8 raises questions as to their structural and evolutionary relationships. In Escherichia coli, YcfD catalyses arginine hydroxylation in the ribosomal protein L16; in humans, MYC-induced nuclear antigen (MINA53; also known as MINA) and nucleolar protein 66 (NO66) catalyse histidine hydroxylation in the ribosomal proteins RPL27A and RPL8, respectively. The functional assignments of ROXs open therapeutic possibilities via either ROX inhibition or targeting of differentially modified ribosomes. Despite differences in the residue and protein selectivities of prokaryotic and eukaryotic ROXs, comparison of the crystal structures of E. coli YcfD and Rhodothermus marinus YcfD with those of human MINA53 and NO66 reveals highly conserved folds and novel dimerization modes defining a new structural subfamily of 2OG-dependent oxygenases. ROX structures with and without their substrates support their functional assignments as hydroxylases but not demethylases, and reveal how the subfamily has evolved to catalyse the hydroxylation of different residue side chains of ribosomal proteins. Comparison of ROX crystal structures with those of other JmjC-domain-containing hydroxylases, including the hypoxia-inducible factor asparaginyl hydroxylase FIH and histone Nε-methyl lysine demethylases, identifies branch points in 2OG-dependent oxygenase evolution and distinguishes between JmjC-containing hydroxylases and demethylases catalysing modifications of translational and transcriptional machinery. The structures reveal that new protein hydroxylation activities can evolve by changing the coordination position from which the iron-bound substrate-oxidizing species reacts. This coordination flexibility has probably contributed to the evolution of the wide range of reactions catalysed by oxygenases

Structural Organization of DNA in Chlorella Viruses

Chlorella viruses have icosahedral capsids with an internal membrane enclosing their large dsDNA genomes and associated proteins. Their genomes are packaged in the particles with a predicted DNA density of ca. 0.2 bp nm−3. Occasionally infection of an algal cell by an individual particle fails and the viral DNA is dynamically ejected from the capsid. This shows that the release of the DNA generates a force, which can aid in the transfer of the genome into the host in a successful infection. Imaging of ejected viral DNA indicates that it is intimately associated with proteins in a periodic fashion. The bulk of the protein particles detected by atomic force microscopy have a size of ∼60 kDa and two proteins (A278L and A282L) of about this size are among 6 basic putative DNA binding proteins found in a proteomic analysis of DNA binding proteins packaged in the virion. A combination of fluorescence images of ejected DNA and a bioinformatics analysis of the DNA reveal periodic patterns in the viral DNA. The periodic distribution of GC rich regions in the genome provides potential binding sites for basic proteins. This DNA/protein aggregation could be responsible for the periodic concentration of fluorescently labeled DNA observed in ejected viral DNA. Collectively the data indicate that the large chlorella viruses have a DNA packaging strategy that differs from bacteriophages; it involves proteins and share similarities to that of chromatin structure in eukaryotes

Molecular basis for DNA repair synthesis on short gaps by mycobacterial Primase-Polymerase C

Cells utilise specialized polymerases from the Primase-Polymerase (Prim-Pol) superfamily to maintain genome stability. Prim-Pol’s function in genome maintenance pathways including replication, repair and damage tolerance. Mycobacteria contain multiple Prim-Pols required for lesion repair, including Prim-PolC that performs short gap repair synthesis during excision repair. To understand the molecular basis of Prim-PolC’s gap recognition and synthesis activities, we elucidated crystal structures of pre- and post-catalytic complexes bound to gapped DNA substrates. These intermediates explain its binding preference for short gaps and reveal a distinctive modus operandi called Synthesis-dependent Template Displacement (STD). This mechanism enables Prim-PolC to couple primer extension with template base dislocation, ensuring that the unpaired templating bases in the gap are ushered into the active site in an ordered manner. Insights provided by these structures establishes the molecular basis of Prim-PolC’s gap recognition and extension activities, while also illuminating the mechanisms of primer extension utilised by closely related Prim-Pols

Rethinking Proteasome Evolution: Two Novel Bacterial Proteasomes

The proteasome is a multisubunit structure that degrades proteins. Protein degradation is an essential component of regulation because proteins can become misfolded, damaged, or unnecessary. Proteasomes and their homologues vary greatly in complexity: from HslV (heat shock locus v), which is encoded by 1 gene in bacteria, to the eukaryotic 20S proteasome, which is encoded by more than 14 genes. Despite this variation in complexity, all the proteasomes are composed of homologous subunits. We searched 238 complete bacterial genomes for structures related to the proteasome and found evidence of two novel groups of bacterial proteasomes. The first, which we name Anbu, is sparsely distributed among cyanobacteria and proteobacteria. We hypothesize that Anbu must be very ancient because of its distribution within the cyanobacteria, and that it has been lost in many more recent species. We also present evidence for a fourth type of bacterial proteasome found in a few β-proteobacteria, which we call β-proteobacteria proteasome homologue (BPH). Sequence and structural analyses show that Anbu and BPH are both distinct from known bacterial proteasomes but have homologous structures. Anbu is encoded by one gene, so we postulate a duplication of Anbu created the 20S proteasome. Anbu’s function appears to be related to transglutaminase activity, not the general stress response associated with HslV. We have found different combinations of Anbu, BPH, and HslV within these bacterial genomes, which raises questions about specialized protein degradation systems

Identification of a Novel Marine Fish Virus, Singapore Grouper Iridovirus-Encoded MicroRNAs Expressed in Grouper Cells by Solexa Sequencing

BACKGROUND: MicroRNAs (miRNAs) are ubiquitous non-coding RNAs that regulate gene expression at the post-transcriptional level. An increasing number of studies has revealed that viruses can also encode miRNAs, which are proposed to be involved in viral replication and persistence, cell-mediated antiviral immune response, angiogenesis, and cell cycle regulation. Singapore grouper iridovirus (SGIV) is a pathogenic iridovirus that has severely affected grouper aquaculture in China and Southeast Asia. Comprehensive knowledge about the related miRNAs during SGIV infection is helpful for understanding the infection and the pathogenic mechanisms. METHODOLOGY/PRINCIPAL FINDINGS: To determine whether SGIV encoded miRNAs during infection, a small RNA library derived from SGIV-infected grouper (GP) cells was constructed and sequenced by Illumina/Solexa deep-sequencing technology. We recovered 6,802,977 usable reads, of which 34,400 represented small RNA sequences encoded by SGIV. Sixteen novel SGIV-encoded miRNAs were identified by a computational pipeline, including a miRNA that shared a similar sequence to herpesvirus miRNA HSV2-miR-H4-5p, which suggests miRNAs are conserved in far related viruses. Generally, these 16 miRNAs are dispersed throughout the SGIV genome, whereas three are located within the ORF057L region. Some SGIV-encoded miRNAs showed marked sequence and length heterogeneity at their 3' and/or 5' end that could modulate their functions. Expression levels and potential biological activities of these viral miRNAs were examined by stem-loop quantitative RT-PCR and luciferase reporter assay, respectively, and 11 of these viral miRNAs were present and functional in SGIV-infected GP cells. CONCLUSIONS: Our study provided a genome-wide view of miRNA production for iridoviruses and identified 16 novel viral miRNAs. To the best of our knowledge, this is the first experimental demonstration of miRNAs encoded by aquatic animal viruses. The results provide a useful resource for further in-depth studies on SGIV infection and iridovirus pathogenesis

Reduced stability of mRNA secondary structure near the translation-initiation site in dsDNA viruses

<p>Abstract</p> <p>Background</p> <p>Recent studies have demonstrated a selection pressure for reduced mRNA secondary-structure stability near the start codon of coding sequences. This selection pressure can be observed in bacteria, archaea, and eukaryotes, and is likely caused by the requirement of efficient translation initiation in cellular organism.</p> <p>Results</p> <p>Here, we surveyed the complete genomes of 650 dsDNA virus strains for signals of reduced stability of mRNA secondary structure near the start codon. Our analysis included viruses infecting eukaryotic, prokaryotic, and archaeic hosts. We found that many viruses showed evidence for reduced mRNA secondary-structure stability near the start codon. The effect was most pronounced in viruses infecting prokaryotes, but was also observed in viruses infecting eukaryotes and archaea. The reduction in stability generally increased with increasing genomic GC content. For bacteriophage, the reduction was correlated with a corresponding reduction of stability in the phage hosts.</p> <p>Conclusions</p> <p>We conclude that reduced stability of the mRNA secondary structure near the start codon is a common feature for dsDNA viruses, likely driven by the same selective pressures that cause it in cellular organisms.</p

Domain-Domain Interactions Underlying Herpesvirus-Human Protein-Protein Interaction Networks

Protein-domains play an important role in mediating protein-protein interactions. Furthermore, the same domain-pairs mediate different interactions in different contexts and in various organisms, and therefore domain-pairs are considered as the building blocks of interactome networks. Here we extend these principles to the host-virus interface and find the domain-pairs that potentially mediate human-herpesvirus interactions. Notably, we find that the same domain-pairs used by other organisms for mediating their interactions underlie statistically significant fractions of human-virus protein inter-interaction networks. Our analysis shows that viral domains tend to interact with human domains that are hubs in the human domain-domain interaction network. This may enable the virus to easily interfere with a variety of mechanisms and processes involving various and different human proteins carrying the relevant hub domain. Comparative genomics analysis provides hints at a molecular mechanism by which the virus acquired some of its interacting domains from its human host

A Compact Multiphoton 3D Imaging System for Recording Fast Neuronal Activity

We constructed a simple and compact imaging system designed specifically for the recording of fast neuronal activity in a 3D volume. The system uses an Yb:KYW femtosecond laser we designed for use with acousto-optic deflection. An integrated two-axis acousto-optic deflector, driven by digitally synthesized signals, can target locations in three dimensions. Data acquisition and the control of scanning are performed by a LeCroy digital oscilloscope. The total cost of construction was one order of magnitude lower than that of a typical Ti:sapphire system. The entire imaging apparatus, including the laser, fits comfortably onto a small rig for electrophysiology. Despite the low cost and simplicity, the convergence of several new technologies allowed us to achieve the following capabilities: i) full-frame acquisition at video rates suitable for patch clamping; ii) random access in under ten microseconds with dwelling ability in the nominal focal plane; iii) three-dimensional random access with the ability to perform fast volume sweeps at kilohertz rates; and iv) fluorescence lifetime imaging. We demonstrate the ability to record action potentials with high temporal resolution using intracellularly loaded potentiometric dye di-2-ANEPEQ. Our design proffers easy integration with electrophysiology and promises a more widespread adoption of functional two-photon imaging as a tool for the study of neuronal activity. The software and firmware we developed is available for download at http://neurospy.org/ under an open source license

Ultrastructural Characterization of the Giant Volcano-like Virus Factory of Acanthamoeba polyphaga Mimivirus

Acanthamoeba polyphaga Mimivirus is a giant double-stranded DNA virus defining a new genus, the Mimiviridae, among the Nucleo-Cytoplasmic Large DNA Viruses (NCLDV). We used utrastructural studies to shed light on the different steps of the Mimivirus replication cycle: entry via phagocytosis, release of viral DNA into the cell cytoplasm through fusion of viral and vacuolar membranes, and finally viral morphogenesis in an extraordinary giant cytoplasmic virus factory (VF). Fluorescent staining of the AT-rich Mimivirus DNA showed that it enters the host nucleus prior to the generation of a cytoplasmic independent replication centre that forms the core of the VF. Assembly and filling of viral capsids were observed within the replication centre, before release into the cell cytoplasm where progeny virions accumulated. 3D reconstruction from fluorescent and differential contrast interference images revealed the VF emerging from the cell surface as a volcano-like structure. Its size dramatically grew during the 24 h infectious lytic cycle. Our results showed that Mimivirus replication is an extremely efficient process that results from a rapid takeover of cellular machinery, and takes place in a unique and autonomous giant assembly centre, leading to the release of a large number of complex virions through amoebal lysis

Progressive GAA·TTC Repeat Expansion in Human Cell Lines

Trinucleotide repeat expansion is the genetic basis for a sizeable group of inherited neurological and neuromuscular disorders. Friedreich ataxia (FRDA) is a relentlessly progressive neurodegenerative disorder caused by GAA·TTC repeat expansion in the first intron of the FXN gene. The expanded repeat reduces FXN mRNA expression and the length of the repeat tract is proportional to disease severity. Somatic expansion of the GAA·TTC repeat sequence in disease-relevant tissues is thought to contribute to the progression of disease severity during patient aging. Previous models of GAA·TTC instability have not been able to produce substantial levels of expansion within an experimentally useful time frame, which has limited our understanding of the molecular basis for this expansion. Here, we present a novel model for studying GAA·TTC expansion in human cells. In our model system, uninterrupted GAA·TTC repeat sequences display high levels of genomic instability, with an overall tendency towards progressive expansion. Using this model, we characterize the relationship between repeat length and expansion. We identify the interval between 88 and 176 repeats as being an important length threshold where expansion rates dramatically increase. We show that expansion levels are affected by both the purity and orientation of the repeat tract within the genomic context. We further demonstrate that GAA·TTC expansion in our model is independent of cell division. Using unique reporter constructs, we identify transcription through the repeat tract as a major contributor to GAA·TTC expansion. Our findings provide novel insight into the mechanisms responsible for GAA·TTC expansion in human cells