Gonzalo Riadi

Histidine Triad-like Motif of the Rotavirus NSP2 Octamer Mediates both RTPase and NTPase Activities

Abstract Rotavirus NSP2 is an abundant non-structural RNA-binding protein essential for forming the viral factories that support replication of the double-stranded RNA genome. NSP2 exists as stable doughnut-shaped octamers within the infected cell, representing the tail-to-tail interaction of two tetramers. Extending diagonally across the surface of each octamer are four highly basic grooves that function as binding sites for single-stranded RNA. Between the N and C-terminal domains of each monomer is a deep electropositive cleft containing a catalytic site that hydrolyzes the γ-β phosphoanhydride bond of any NTP. The catalytic site has similarity to those of the histidine triad (HIT) family of nucleotide-binding proteins. Due to the close proximity of the grooves and clefts, we investigated the possibility that the RNA-binding activity of the groove promoted the insertion of the 5′-triphosphate moiety of the RNA into the cleft, and the subsequent hydrolysis of its γ-β phosphoanhydride bond. Our results show that NSP2 hydrolyzes the γP from RNAs and NTPs through Mg2+-dependent activities that proceed with similar reaction velocities, that require the catalytic His225 residue, and that produce a phosphorylated intermediate. Competition assays indicate that although both substrates enter the active site, RNA is the preferred substrate due to its higher affinity for the octamer. The RNA triphosphatase (RTPase) activity of NSP2 may account for the absence of the 5′-terminal γP on the (−) strands of the double-stranded RNA genome segments. This is the first report of a HIT-like protein with a multifunctional catalytic site, capable of accommodating both NTPs and RNAs during γP hydrolysis.

Architecture and Gene Repertoire of the Flexible Genome of the Extreme Acidophile Acidithiobacillus caldus

Background Acidithiobacillus caldus is a sulfur oxidizing extreme acidophile and the only known mesothermophile within the Acidithiobacillales. As such, it is one of the preferred microbes for mineral bioprocessing at moderately high temperatures. In this study, we explore the genomic diversity of A. caldus strains using a combination of bioinformatic and experimental techniques, thus contributing first insights into the elucidation of the species pangenome. Principal Findings Comparative sequence analysis of A. caldus ATCC 51756 and SM-1 indicate that, despite sharing a conserved and highly syntenic genomic core, both strains have unique gene complements encompassing nearly 20% of their respective genomes. The differential gene complement of each strain is distributed between the chromosomal compartment, one megaplasmid and a variable number of smaller plasmids, and is directly associated to a diverse pool of mobile genetic elements (MGE). These include integrative conjugative and mobilizable elements, genomic islands and insertion sequences. Some of the accessory functions associated to these MGEs have been linked previously to the flexible gene pool in microorganisms inhabiting completely different econiches. Yet, others had not been unambiguously mapped to the flexible gene pool prior to this report and clearly reflect strain-specific adaption to local environmental conditions. Significance For many years, and because of DNA instability at low pH and recurrent failure to genetically transform acidophilic bacteria, gene transfer in acidic environments was considered negligible. Findings presented herein imply that a more or less conserved pool of actively excising MGEs occurs in the A. caldus population and point to a greater frequency of gene exchange in this econiche than previously recognized. Also, the data suggest that these elements endow the species with capacities to withstand the diverse abiotic and biotic stresses of natural environments, in particular those associated with its extreme econiche.

Novel missense mutation of the FAM83H gene causes retention of amelogenin and a mild clinical phenotype of hypocalcified enamel

OBJECTIVE Amelogenesis imperfecta (AI) is a group of clinically and genetically heterogeneous inherited conditions, causing alterations in the structure of enamel and chemical composition of enamel matrix during development. The objective of this study was to compare the clinical, radiographic, histological and immunohistochemical phenotypes of subjects affected with hypocalcified AI from three Chilean families and identify causal mutations in the FAM83H gene. DESIGN The diagnosis was made using clinical, radiographic, histological and genealogical data from the patients, who were evaluated according to the classification criteria by Witkop. PCR and Sanger sequencing of the complete coding sequence and surrounding intron regions of the FAM83H gene were conducted. The structural study of the affected teeth was performed with light microscopy, scanning electron microscopy and immunohistochemistry. RESULTS The probands of the three families were diagnosed with hypocalcified AI, but in only one of them the missense variant p.Gly557Cys was identified. This variant was not present in the SNP database or in 100 healthy controls and segregated with the disease in the affected family. Using light microscopy, a normal prismatic structure was observed in all three cases. However, the ultrastructure was found to be affected in two of the cases, showing persistence of organic matter including amelogenins. CONCLUSIONS These results suggest that FAM83H missense mutation reported in one of the families analyzed in this study might cause a phenotype of hypocalcified enamel more attenuated with retention of amelogenin.

K2P channels in plants and animals

Two-pore domain potassium (K2P) channels are membrane proteins widely identified in mammals, plants, and other organisms. A functional channel is a dimer with each subunit comprising two pore-forming loops and four transmembrane domains. The genome of the model plant Arabidopsis thaliana harbors five genes coding for K2P channels. Homologs of Arabidopsis K2P channels have been found in all higher plants sequenced so far. As with the K2P channels in mammals, plant K2P channels are targets of external and internal stimuli, which fine-tune the electrical properties of the membrane for specialized transport and/or signaling tasks. Plant K2P channels are modulated by signaling molecules such as intracellular H+ and calcium and physical factors like temperature and pressure. In this review, we ask the following: What are the similarities and differences between K2P channels in plants and animals in terms of their physiology? What is the nature of the last common ancestor (LCA) of these two groups of proteins? To answer these questions, we present physiological, structural, and phylogenetic evidence that discards the hypothesis proposing that the duplication and fusion that gave rise to the K2P channels occurred in a prokaryote LCA. Conversely, we argue that the K2P LCA was most likely a eukaryote organism. Consideration of plant and animal K2P channels in the same study is novel and likely to stimulate further exchange of ideas between students of these fields.

The receptor-like pseudokinase MRH1 interacts with the voltage-gated potassium channel AKT2

The potassium channel AKT2 plays important roles in phloem loading and unloading. It can operate as inward-rectifying channel that allows H+-ATPase-energized K+ uptake. Moreover, through reversible post-translational modifications it can also function as an open, K+-selective channel, which taps a ‘potassium battery’, providing additional energy for transmembrane transport processes. Knowledge about proteins involved in the regulation of the operational mode of AKT2 is very limited. Here, we employed a large-scale yeast two-hybrid screen in combination with fluorescence tagging and null-allele mutant phenotype analysis and identified the plasma membrane localized receptor-like kinase MRH1/MDIS2 (AT4G18640) as interaction partner of AKT2. The phenotype of the mrh1-1 knockout plant mirrors that of akt2 knockout plants in energy limiting conditions. Electrophysiological analyses showed that MRH1/MDIS2 failed to exert any functional regulation on AKT2. Using structural protein modeling approaches, we instead gathered evidence that the putative kinase domain of MRH1/MDIS2 lacks essential sites that are indispensable for a functional kinase suggesting that MRH1/MDIS2 is a pseudokinase. We propose that MRH1/MDIS2 and AKT2 are likely parts of a bigger protein complex. MRH1 might help to recruit other, so far unknown partners, which post-translationally regulate AKT2. Additionally, MRH1 might be involved in the recognition of chemical signals.

A Novel Analytical Strategy to Identify Fusion Transcripts between Repetitive Elements and Protein Coding-Exons Using RNA-Seq.

Repetitive elements (REs) comprise 40–60% of the mammalian genome and have been shown to epigenetically influence the expression of genes through the formation of fusion transcript (FTs). We previously showed that an intracisternal A particle forms an FT with the agouti gene in mice, causing obesity/type 2 diabetes. To determine the frequency of FTs genome-wide, we developed a TopHat-Fusion-based analytical pipeline to identify FTs with high specificity. We applied it to an RNA-seq dataset from the nucleus accumbens (NAc) of mice repeatedly exposed to cocaine. Cocaine was previously shown to increase the expression of certain REs in this brain region. Using this pipeline that can be applied to single- or paired-end reads, we identified 438 genes expressing 813 different FTs in the NAc. Although all types of studied repeats were present in FTs, simple sequence repeats were underrepresented. Most importantly, reverse-transcription and quantitative PCR validated the expression of selected FTs in an independent cohort of animals, which also revealed that some FTs are the prominent isoforms expressed in the NAc by some genes. In other RNA-seq datasets, developmental expression as well as tissue specificity of some FTs differed from their corresponding non-fusion counterparts. Finally, in silico analysis predicted changes in the structure of proteins encoded by some FTs, potentially resulting in gain or loss of function. Collectively, these results indicate the robustness of our pipeline in detecting these new isoforms of genes, which we believe provides a valuable tool to aid in better understanding the broad role of REs in mammalian cellular biology.

Easy Identification of Residues Involved on Structural Differences Between Nonphosphorylated and Phosphorylated CDK2Cyclin A Complexes Using Two-Dimensional Networks

The structures of proteins in Protein Data Bank (PDB) contain a lot of information that can be revealed through the use of tools to facilitate their organization and analysis. The increase in available structural data of nonphosphorylated and phosphorylated CDK2cyclin A (npCDK2cycA and pCDK2cycA) complexes has enabled a more realistic description of the fine structural details of the interface residues of these proteins. This work reports the application of two‐dimensional network representations (TDNRs) to the structures deposited in PDB to distinguish the differences in the surface between both complexes due to phosphorylation. As a result, a detailed map of the hydrogen bonds (HBs) and hydrophobic interactions between the T‐loop residues of CDK2 and the residues of cycA that are different among nonphosphorylated and phosphorylated complexes were described. In addition, we found some interesting subtle differences in the CDK2cycA interface between nonphosphorylated and phosphorylated complexes due to residues that are not located at the T‐loop of CDK2. We noted that some HB interactions in CDK2cycA complex are reinforced when the CDK2 is phosphorylated.

Mitochondrial acetyl-CoA reversibly regulates locus-specific histone acetylation and gene expression

The impact of mitochondria in epigenetics is emerging but our understanding of this relationship and its impact on gene expression remain incomplete. We previously showed that acute mitochondrial DNA (mtDNA) loss leads to histone hypoacetylation. It remains to be defined if these changes are maintained when mitochondrial dysfunction is chronic and, importantly, if they are sufficient to alter gene expression. To fill these gaps, we here studied both a progressive and a chronic model of mtDNA depletion using biochemical, pharmacological, genomics and genetic assays. We show that histones are hypoacetylated in both models. We link these effects to decreased histone acetyltransferase (HAT) activity independent of changes in ATP citrate lyase function, which can be reversibly modulated by altering specifically the mitochondrial pool of acetyl-CoA. Also, we determined that these changes regulate locus-specific gene expression and physiological outcomes, including the production of prostaglandins. These results may be relevant to the pathophysiology of mtDNA depletion syndromes and to understanding the effects of environmental agents, such as AZT or antibiotics, that lead to physical or functional mtDNA loss.

TEcandidates: prediction of genomic origin of expressed transposable elements using RNA-seq data

Motivation In recent years, Transposable Elements (TEs) have been related to gene regulation. However, estimating the origin of expression of TEs through RNA‐seq is complicated by multi‐mapping reads coming from their repetitive sequences. Current approaches that address multi‐mapping reads are focused in expression quantification and not in finding the origin of expression. Addressing the genomic origin of expressed TEs could further aid in understanding the role that TEs might have in the cell. Results We have developed a new pipeline called TEcandidates, based on de novo transcriptome assembly to assess the instances of TEs being expressed, along with their location, to include in downstream DE analysis. TEcandidates takes as input the RNA‐seq data, the genome sequence and the TE annotation file and returns a list of coordinates of candidate TEs being expressed, the TEs that have been removed and the genome sequence with removed TEs as masked. This masked genome is suited to include TEs in downstream expression analysis, as the ambiguity of reads coming from TEs is significantly reduced in the mapping step of the analysis. Availability and implementation The script which runs the pipeline can be downloaded at http://www.mobilomics.org/tecandidates/downloads or http://github.com/TEcandidates/TEcandidates. Supplementary information Supplementary data are available at Bioinformatics online.