Structural and functional advances in the evolutionary studies of cells and viruses

Phylogenomics aims to describe evolutionary relatedness between organisms by analyzing genomic data. The common practice is to produce phylogenomic trees from molecular information in the sequence, order and content of genes in genomes. These phylogenies describe the evolution of life and have become valuable tools for taxonomy. The recent availability of structural and functional data for hundreds of genomes now offer the opportunity to study evolution using more conserved sets of molecular features. Here we report a phylogenomic (i.e. historical) and comparative (ahistorical) analysis that yields novel insights into the origin of cells (Chapters 1-3) and viruses (Chapters 4-6). We utilized conserved protein domain structure information (fold families [FFs] and fold superfamilies [FSFs]) and ontological definitions of gene products (Gene Ontology [GO]) to reconstruct rooted trees of life (ToL), taking advantage of a genomic census of molecular structure and function in the genomes of sampled organisms and viruses. The analysis revealed a global tendency in the proteomic repertories of cellular organisms to increase domain abundance. ToLs built directly from the census of molecular functions confirmed an early origin of Archaea relative to Bacteria and Eukarya, a conclusion further supported by comparative analysis. The analysis further revealed an ancient history of viruses and their evolution by gene loss. Despite the very high levels of variability seen in the replication strategies, morphologies, and host preferences of extant viruses, we recovered a conserved and ancient structural core of protein domains that was shared between cellular organisms and distantly related viruses. This core together with an analysis of the evolution of virion morphotypes strongly suggests an ancient origin for the viral supergroup. Moreover, a large number of viral proteins lacked cellular homologs and strongly negated the idea that viruses merely evolve by acquiring cellular genes. These virus-specific proteins confer pathogenic abilities to viruses and appeared late in evolution suggesting that the shift to parasitic mode of life happened later in viral evolution. The strong evolutionary association between viruses and cells is likely reminiscent of their ancient co-existence inside primordial cells. Moreover, the crucial dependency of viruses to replicate in an intracellular environment creates fertile grounds for genetic innovation. Interestingly, protein domains shared with viruses were widespread in the proteomes of all three cellular superkingdoms suggesting that viruses mediate gene transfer and crucially enhance biodiversity. The phylogenomic trees identify viruses as a ‘fourth supergroup’ along with cellular superkingdoms, Archaea, Bacteria, and Eukarya. The new model for the origin and evolution of viruses and cells is backed by strong molecular data and is compatible with the existing models of viral evolution. Our experiments indicate that structure and functionomic data represent a useful addition to the set of molecular characters used for tree reconstruction and that ToLs carry in deep branches considerable predictive power to explain the evolution of living organisms and viruses

Structure and mechanism of two type III CRISPR defence nucleases activated by cyclic oligoadenylate

Prokaryotes have a wide range of antiviral strategies to defend against invading mobile genetic elements (MGEs). Type III CRISPR-Cas systems typically synthesise cyclic oligoadenylate (cOA) second messengers upon binding to cognate foreign RNA. These second messengers allosterically activate type III CRISPR ancillary proteins, potentiating a powerful immune response. Following the discovery of cOA signalling pathway, several ancillary proteins from Csx1/Csm6 family had been described. They sense cOA molecules with their CARF (CRISPR associated Rossman fold) domains and non-specifically cleave RNA with their effector domains. Here, we describe the structure and mechanism of two novel ancillary proteins Can1 and Can2. Can1 has a unique monomeric architecture that contains two CARF domains, a PD-(D/E)XK nuclease domain and a nuclease-like domain. It favours nicking scDNA in the presence of cyclic tetra-adenylate (cA₄) and metal ions. Can2 forms a canonical homodimer and each monomer contains a CARF domain and a PD-(D/E)XK nuclease domain. It exhibits both DNase and RNase activity in the presence of cA₄ and metal ions. It also provides effective immunity against plasmid and bacteriophage infection in a recombinant type III CRISPR-Cas system."This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BB/S000313/1 to M.F.W., BB/R008035/1 to T.M.G. and BB/T004789/1 to M.F.W. and T.M.G.); grants from Wellcome Trust Institutional Strategic Support Funding (204821/Z/16/Z to M.F.W. and T.M.G.); grants from China Scholarship Council (201703780015 to W.Z.). Funding for open access charge: RCUK block grant." -- Fundin

Non-Coding RNA Features Critical to the Replication of HIV-1

The HIV-1 genome contains RNA sequences and structures that control many aspects of viral replication including, but not limited to transcription, splicing, nuclear export, translation, packaging and reverse transcription. Despite this extensive existing catalogue of RNA sequences that are critical to its replication, chemical probing and targeting mutagenesis studies suggest that the HIV-1 genome may contain many more RNA elements of unknown important function. To determine whether there are additional, undiscovered cis-acting RNA elements in the HIV-1 genome that are important for viral replication, we conducted a global synonymous mutagenesis experiment. Sixteen mutant proviruses containing clusters of ~50 to ~200 synonymous mutations covering nearly the entire HIV-1 protein coding sequence were designed and synthesized. Analyses of these mutant viruses resulted in their division into three phenotypic groups. Group 1 mutants exhibited near wild-type replication, Group 2 mutants exhibited replication defects accompanied by perturbed RNA splicing, and Group 3 mutants had replication defects in the absence of obvious splicing perturbation. The three phenotypes were caused by mutations that exhibited a clear regional bias in their distribution along the viral genome, and those that caused replication defects all caused reductions in the level of unspliced RNA. We characterized in detail the underlying defects for Group 2 mutants. Second-site revertants that enabled viral replication could be derived for Group 2 mutants, and generally contained point mutations that reduced the utilization of proximal splice sites. Mapping of the changes responsible for splicing perturbations in Group 2 viruses revealed the presence of several RNA sequences that apparently suppressed the use of cryptic or canonical splice sites. Some sequences that affected splicing were diffusely distributed, while others could be mapped to discrete elements, proximal or distal to the affected splice sites. This data from the Group 2 mutants indicates complex negative regulation of HIV-1 splicing by RNA elements in various regions of the HIV-1 genome that enable balanced splicing and viral replication. In silico analysis of the Group 3 mutants revealed that our mutagenesis had significantly increased the frequency of CG dinucleotides in sections of the viral genome to that of random sequence. This is important due to the remarkable CG suppression in both the HIV-1 and human genomes, and we had therefore disrupted the dinucleotide congruence that exists between HIV-1 and the genome of its host. We recoded these mutants to selectively remove either only the CG dinucleotides or only remove the mutations that did not encode a CG dinucleotide. Analysis of these mutants clearly demonstrated that the addition of CG dinucleotides were the causative mutations entirely responsible for the observed replication defects. qPCR analysis and smFISH microscopy revealed that the addition of CG dinucleotides to HIV-1 resulted in a depletion of the cytoplasmic mRNA molecules where the CG-dinucleotides were encoded as exons. A targeted siRNA screen for proteins that destabilize cytoplasmic RNA identified the Zinc-finger Antiviral Protein (ZAP) as responsible for the restriction of the CG-high HIV-1, specifically by targeting CGhigh viral RNA. CLIP-Seq experiments demonstrate that ZAP binds directly to CG dinucleotides in both cellular and viral RNA. Collectively these studies implicate ZAP as a cellular protein that can recognize CG-high viral RNA and is possibly a cellular mechanism for determining self from non-self RNA based on the CG composition. TRIM25 has previously been identified as a cofactor for two cytosolic RNA binding proteins that have antiviral functions, RIG-I where it is an essential cofactor, and ZAP where it functions as an enhancing cofactor. The mechanism by which TRIM25 enhances the antiviral activity of ZAP currently remains unclear. Through CLIP-Seq experiments in cells knocked out for TRIM25, we determined that ZAP does not require TRIM25 to recognize CG-high RNA. Using full length mutants of TRIM25 that are deficient for either RNA binding, E3 ligase activity, or formation of higher order multimers, our data suggest that the key biological activity required for TRIM25 to enhance ZAP is the formation of higher order multimers. Analyzing the replication of CG-high HIV-1 in different cell lines indicates that ZAP is not equally potent across all cell lines. The degree of potency ZAP possess against CG-high HIV-1 does not correlate with TRIM25 expression, suggesting the possibility of an additional ZAP cofactor that is heterogeneously expressed in varying cell lines. siRNA screens have been used in an attempt to identify a yet undiscovered cofactor, but so far these experiments have not yielded any such factor

Reticulate Evolution: Symbiogenesis, Lateral Gene Transfer, Hybridization and Infectious heredity

info:eu-repo/semantics/publishedVersio

RNA, the Epicenter of Genetic Information

The origin story and emergence of molecular biology is muddled. The early triumphs in bacterial genetics and the complexity of animal and plant genomes complicate an intricate history. This book documents the many advances, as well as the prejudices and founder fallacies. It highlights the premature relegation of RNA to simply an intermediate between gene and protein, the underestimation of the amount of information required to program the development of multicellular organisms, and the dawning realization that RNA is the cornerstone of cell biology, development, brain function and probably evolution itself. Key personalities, their hubris as well as prescient predictions are richly illustrated with quotes, archival material, photographs, diagrams and references to bring the people, ideas and discoveries to life, from the conceptual cradles of molecular biology to the current revolution in the understanding of genetic information. Key Features Documents the confused early history of DNA, RNA and proteins - a transformative history of molecular biology like no other. Integrates the influences of biochemistry and genetics on the landscape of molecular biology. Chronicles the important discoveries, preconceptions and misconceptions that retarded or misdirected progress. Highlights major pioneers and contributors to molecular biology, with a focus on RNA and noncoding DNA. Summarizes the mounting evidence for the central roles of non-protein-coding RNA in cell and developmental biology. Provides a thought-provoking retrospective and forward-looking perspective for advanced students and professional researchers

Functional and metagenomic analysis of the human tongue dorsum using phage display

It is well established that mixed microbial communities contain organisms which have not been studied by conventional culture-based methods. In the human oral cavity this number is estimated at around 50%. Commensal bacteria develop and maintain an intimate relationship with human cells without triggering proinflammatory mechanisms and this study aims to explore this by searching for bacterial proteins which facilitate binding to the human tongue dorsum and wider oral cavity. Metagenomic DNA from the human tongue dorsum of 9 volunteers was extracted and a phage display library created, to our knowledge the first to incorporate metagenomic DNA. Phage display is an elegant molecular technique involving fusion of fragmented DNA to a phagemid coat protein, such that inserted DNA is encoded by the phage and displayed on the phage surface. The affinity selection technique panning, then exploited the natural affinity and specificity of the fusion proteins to identify bacterial binding proteins using, in this case, three ligands: IgA, Fibronectin and BSA. IgA is of special interest to this group as it interacts with bacterial proteins and is poised to respond to bacterial numbers in human secretions such as saliva. Proteins from panning were analysed in silico, however, the majority were discarded due to the presence of stop codons in the protein sequences. Remaining phagemid displaying fusion proteins of interest were assessed for function and binding assays carried out to confirm binding specificity. Due to the biased nature of phage display library production, a 16S rRNA gene analysis was also carried out in order to assess metagenomic DNA diversity prior to library construction. Because phage display was used successfully by colleagues with the genomes of single organisms, it was believed that including metagenomic DNA in a phage display library would cast a wide net over the tongue dorsum allowing capture of many more binding proteins occurring in this environment from a wide range of bacteria

Identification and characterisation of rumen bacteria with prominent roles in the ruminal metabolism of forages : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy (Microbiology and Genetics) at Massey University, Palmerston North, New Zealand

Foigures 1.2, 1.4, 1.5 & 1.6 are re-used with permission.This thesis documents the characterisation of two groups of rumen bacteria that are both prominent in forage-fed ruminants, with the aim to better understand their roles in ruminal metabolism. The first group, referred to as the R-7 group, has in recent years been shown to be one of the most abundant rumen bacterial groups, though the few isolated representative strains available were uncharacterised. Two strains of the group included in the Hungate1000 culture collection, R-7 and WTE2008, were selected for characterisation. To facilitate phylogenetic analyses of this group, the complete genomes of an additional three previously isolated R-7 group strains were sequenced. Genomic, phylogenetic and phenotypic characterisation of R-7 and WTE2008 demonstrated that despite their 16S rRNA gene sequences sharing 98.6-99.0% nucleotide identity, their genome-wide average nucleotide identity of 84% assigned them as separate species of a novel genus and family of the proposed order ‘Christensenellales’ using the Genome Taxonomy Database. Phenotypic characterisation showed that the strains were identical in morphology, and both possessed the ability to degrade plant cell wall polysaccharides xylan and pectin, but not cellulose. Acetate, ethanol, hydrogen and lactate were produced by both strains, though R-7 produced greater amounts of hydrogen than WTE2008, which instead produced more lactate. Based on these analyses, it is proposed that R-7 and WTE2008 belong to separate species (Aristaeella gen. nov. hokkaidonensis sp. nov. and Aristaeella lactis sp. nov., respectively) of a newly proposed family (Aristaeellaceae fam. nov.). The second bacterial group of interest, due to their dominant role in ruminal propionate production, was the Prevotella 1 group, following analyses of metatranscriptome datasets of rumen microbial communities of lucerne-fed sheep for dominant community members that express propionate pathway genes from succinate. Screening of 14 strains spanning the diversity of Prevotella 1 found that all except one P. brevis strain produced propionate in a cobalamin (vitamin B12)-dependent manner. To better understand the pathway and regulation of propionate production from succinate, a comparative multi-omics approach was used to test the hypothesis that propionate production is regulated by a cobalamin-binding riboswitch. Scanning of a completed genome assembly of Prevotella ruminicola KHP1 identified four ‘cobalamin’ family riboswitches. However, the riboswitches were not in close proximity to genes putatively involved in converting succinate to propionate, nor were these genes arranged in a single operon. Comparative genomics of the 14 screened strains found that all strains possessed all homologues of candidate propionate pathway genes identified in the KHP1 genome. However, the 13 propionate-producing strains possessed a putative transporter and three subunits encoding a putative methylmalonyl-CoA decarboxylase upstream but antisense to two genes encoding methylmalonyl-CoA mutase subunits, whereas the non-producing strain did not. Comparative transcriptomics and proteomics of KHP1 cultures in the presence and absence of cobalamin demonstrated that some gene candidates were upregulated by cobalamin at the transcriptome level, including co-located genes annotated as phosphate butyryltransferase and butyrate kinase, despite the strain not producing butyrate, suggesting that propionate production may occur via propionyl phosphate. However, only both subunits of methylmalonyl-CoA mutase showed greater transcript and protein abundances in the presence of cobalamin. These results show that while some propionate pathway candidate genes were differentially expressed between cobalamin treatments, they did not appear to be under direct control of a cobalamin-binding riboswitch. This study has contributed to our understanding of the roles of both Aristaeellaceae fam. nov. and Prevotella 1 in ruminal metabolism

White Paper 2: Origins, (Co)Evolution, Diversity & Synthesis Of Life

Publicado en Madrid, 185 p. ; 17 cm.How life appeared on Earth and how then it diversified into the different and currently existing forms of life are the unanswered questions that will be discussed this volume. These questions delve into the deep past of our planet, where biology intermingles with geology and chemistry, to explore the origin of life and understand its evolution, since “nothing makes sense in biology except in the light of evolution” (Dobzhansky, 1964). The eight challenges that compose this volume summarize our current knowledge and future research directions touching different aspects of the study of evolution, which can be considered a fundamental discipline of Life Science. The volume discusses recent theories on how the first molecules arouse, became organized and acquired their structure, enabling the first forms of life. It also attempts to explain how this life has changed over time, giving rise, from very similar molecular bases, to an immense biological diversity, and to understand what is the hylogenetic relationship among all the different life forms. The volume further analyzes human evolution, its relationship with the environment and its implications on human health and society. Closing the circle, the volume discusses the possibility of designing new biological machines, thus creating a cell prototype from its components and whether this knowledge can be applied to improve our ecosystem. With an effective coordination among its three main areas of knowledge, the CSIC can become an international benchmark for research in this field

Training Manual ICAR Short course on Application of advanced molecular methods in marine fishery resource management, conservation and sustainable mariculture

Molecular Biology and Biotechnology has undergone incredible progress in this decade mainly due to the rapid advancements in DNA sequencing technologies. Marine biology and fishery science also reaped the fruits of these modern inventions improving our understanding regarding complex adaptations in aquatic organisms. Fish Genetics have evolved into genomics incorporating knowledge about neutral and non-neutral markers. A project called Genome 10k was started by the international community of scientists for sequencing the genome of 10000 vertebrates. Whole genomes of many marine organisms are now available which provided insights into the evolution of many important traits. Transcriptome sequencing provides insights into expressed genes and metagenome sequencing provides information regarding the microbes present in environment. All these technologies are rapid and cost effective. Over years, these technologies provided exciting opportunities for understanding ecology and evolution. Genomic information can also be sustainably utilized to enhance productivity of mariculture activities by selective breeding, genetic improvement and manipulation of economically important traits. ICAR-Central Marine Fisheries Research Institute has contributed significantly to marine biotechnology research in the country and played a pivotal role in development of marine fisheries sector. The short course on “Application of advanced molecular methods in marine fisheries resource management, conservation and sustainable mariculture” conducted in ICAR-CMFRI from 24th October, 2018 to 2nd November, 2018 is specially designed to provide exposure to various applications of molecular tools in fisheries resource management, conservation of biodiversity and mariculture. I hope this compendium of lectures and protocols will be extremely useful for the participants to effectively utilize the knowledge in their own area of research. Simultaneously, on behalf of ICAR-CMFRI, I warmly welcome all the participants from various institutions and wish them all success in their future endeavors. I am sure that this training will result in new knowledge, collaborations and friendships