Profiling RNA editing in human tissues: towards the inosinome Atlas

Adenine to Inosine RNA editing is a widespread co- and post-transcriptional mechanism mediated by ADAR enzymes acting on double stranded RNA. It has a plethora of biological effects, appears to be particularly pervasive in humans with respect to other mammals, and is implicated in a number of diverse human pathologies. Here we present the first human inosinome atlas comprising 3,041,422 A-to-I events identified in six tissues from three healthy individuals. Matched directional total-RNA-Seq and whole genome sequence datasets were generated and analysed within a dedicated computational framework, also capable of detecting hyper-edited reads. Inosinome profiles are tissue specific and edited gene sets consistently show enrichment of genes involved in neurological disorders and cancer. Overall frequency of editing also varies, but is strongly correlated with ADAR expression levels. The inosinome database is available at: http://srv00.ibbe.cnr.it/editing/

Management at the service of research: ReOmicS, a quality management system for omics sciences

AbstractManagement and research represent a binomial almost unknown, whose potentialities and requirements have not yet been fully exploited even if, recently, the scientific and social communities have felt the burden of producing results and data requiring at the same time reproducibility, reliability, safety and efficacy of the discoveries, as well as a profitable use of resources. A Quality Management System (QMS) could represent a valid tool for these purposes, improving the quality of the research. The research community could ask whether and how it is possible to apply this approach in a research laboratory without hindering their creativity, and what the possible benefits might be. On the other hand, an international standard for a quality management system appropriate for a research laboratory is yet to come. The choice, the design and the application of a QMS, inspired by the Good Laboratory Practices, in a research laboratory specialized on "omics" sciences, is fully described in this paper. Its application has already shown good outcomes as testified by specific metric of efficiency and effectiveness. The approach is innovative as there is no obvious requirement for research laboratories to develop and define quality objectives. The paper highlights how the QMS approach enhances the relationship with public and private sectors by increasing customer confidence and loyalty, as well as improving the overall performance of the laboratory in terms of throughput and value of research. These results encourage proposing it as a QMS model providing a new and scalable operational strategy to be applied in a research environment with the same target and even in a generic research laboratory

MTERF3, the most conserved member of the mTERF-family, is a modular factor involved in mitochondrial protein synthesis

AbstractThe MTERF-family is a wide family of proteins identified in Metazoa and plants which includes the known mitochondrial transcription termination factors. With the aim to shed light on the function of MTERF-family members in Drosophila, we performed the cloning and characterization of D-MTERF3, a component of the most conserved group of this family. D-MTERF3 is a mitochondrial protein of 323 amino acids. Sequence analysis in seven different organisms showed that the protein contains five conserved “mTERF-motifs”, three of which include a leucine zipper-like domain. D-MTERF3 knock-down, obtained by RNAi in D.Mel-2 cells, did not affect mitochondrial replication and transcription. On the contrary, it decreased to a variable extent the rate of labelling of about half of the mitochondrial polypeptides, with ND1 being the most affected by D-MTERF3 depletion. These results indicate that D-MTERF3 is involved in mitochondrial translation. This role, likely based on protein–protein interactions, may be exerted either through a direct interaction with the translation machinery or by bridging the mitochondrial transcription and translation apparatus

Identification of an Amylomaltase from the Halophilic Archaeon Haloquadratum walsbyi by Functional Metagenomics: Structural and Functional Insights

Amylomaltases are prokaryotic 4-α-glucanotransferases of the GH77 family. Thanks to the ability to modify starch, they constitute a group of enzymes of great interest for biotechnological applications. In this work we report the identification, by means of a functional metagenomics screening of the crystallization waters of the saltern of Margherita di Savoia (Italy), of an amylomaltase gene from the halophilic archaeon Haloquadratum walsbyi, and its expression in Escherichia coli cells. Sequence analysis indicated that the gene has specific insertions yet unknown in homologous genes in prokaryotes, and present only in amylomaltase genes identified in the genomes of other H. walsbyi strains. The gene is not part of any operon involved in the metabolism of maltooligosaccharides or glycogen, as it has been found in bacteria, making it impossible currently to assign a precise role to the encoded enzyme. Sequence analysis of the H. walsbyi amylomaltase and 3D modelling showed a common structure with homologous enzymes characterized in mesophilic and thermophilic bacteria. The recombinant H. walsbyi enzyme showed starch transglycosylation activity over a wide range of NaCl concentrations, with maltotriose as the best acceptor substrate compared to other maltooligosaccharides. This is the first study of an amylomaltase from a halophilic microorganism

Plant Health and Rhizosphere Microbiome: Effects of the Bionematicide Aphanocladium album in Tomato Plants Infested by Meloidogyne javanica

The artificial introduction in the soil of antagonistic microorganisms can be a successful strategy, alternative to agrochemicals, for the control of the root-knot nematodes (Meloidogyne spp.) and for preserving plant health. On the other hand, plant roots and the associated rhizosphere constitute a complex system in which the contribution of microbial community is fundamental to plant health and development, since microbes may convert organic and inorganic substances into available plant nutrients. In the present study, the potential nematicidal activity of the biopesticide Aphanocladium album (A. album strain MX-95) against the root-knot nematode Meloidogyne javanica in infected tomato plants was investigated. Specifically, the effect of the A. album treatment on plant fitness was evaluated observing the plant morphological traits and also considering the nematode propagation parameters, the A. album MX-95 vitality and population density. In addition, the treatment effects on the rhizosphere microbiome were analysed by a metabarcoding procedure. Treatments with A. album isolate MX-95 significantly decreased root gall severity index and soil nematode population. The treatment also resulted in increased rhizosphere microbial populations. A. album MX-95 can be favourably considered as a new bionematicide to control M. javanica infestation

Whole transcriptome profiling of Late-Onset Alzheimer's Disease patients provides insights into the molecular changes involved in the disease

Alzheimer's Disease (AD) is the most common cause of dementia affecting the elderly population worldwide. We have performed a comprehensive transcriptome profiling of Late-Onset AD (LOAD) patients using second generation sequencing technologies, identifying 2,064 genes, 47 lncRNAs and 4 miRNAs whose expression is specifically deregulated in the hippocampal region of LOAD patients. Moreover, analyzing the hippocampal, temporal and frontal regions from the same LOAD patients, we identify specific sets of deregulated miRNAs for each region, and we confirm that the miR-132/212 cluster is deregulated in each of these regions in LOAD patients, consistent with these miRNAs playing a role in AD pathogenesis. Notably, a luciferase assay indicates that miR-184 is able to target the 3'UTR NR4A2 - which is known to be involved in cognitive functions and long-term memory and whose expression levels are inversely correlated with those of miR-184 in the hippocampus. Finally, RNA editing analysis  reveals a general RNA editing decrease in LOAD hippocampus, with 14 recoding sites significantly and differentially edited in 11 genes. Our data underline specific transcriptional changes in LOAD brain and provide an important source of information for understanding the molecular changes characterizing LOAD progression

YAP contributes to DNA methylation remodeling upon mouse embryonic stem cell differentiation

The Yes-associated protein YAP, one of the major effectors of the Hippo pathway together with its related protein TAZ, mediates a range of cellular processes from proliferation and death to morphogenesis. YAP and TAZ regulate a large number of target genes, acting as co-activators of DNA-binding transcription factors or as negative regulators of transcription by interacting with the nucleosome remodeling and histone deacetylase complexes. YAP is expressed in self-renewing embryonic stem cells (ESCs), although it is still debated whether it plays any crucial roles in the control of either stemness or differentiation. Here we show that the transient downregulation of YAP in mouse ESCs perturbs cellular homeostasis, leading to the inability to differentiate properly. Bisulfite genomic sequencing revealed that this transient knockdown caused a genome-wide alteration of the DNA methylation remodeling that takes place during the early steps of differentiation, suggesting that the phenotype we observed might be due to the dysregulation of some of the mechanisms involved in regulation of ESC exit from pluripotency. By gene expression analysis we identified two molecules which could have a role in the altered genome-wide methylation profile: the long non-coding RNA Ephemeron, whose rapid upregulation is crucial for ESCs transition into epiblast, and the methyltransferase-like protein Dnmt3l, which, during the embryo development, cooperates with Dnmt3a and Dnmt3b to contribute to the de novo DNA methylation that governs early steps of ESC differentiation. These data suggest a new role for YAP in the governance of the epigenetic dynamics of exit from pluripotency

The influence of invasive jellyfish blooms on the aquatic microbiome in a coastal lagoon (Varano, SE Italy) detected by an Illumina-based deep sequencing strategy

The rapid expansion of multicellular native and alien species outbreaks in aquatic and terrestrial ecosystems (bioinvasions) may produce significant impacts on bacterial community dynamics and nutrient pathways with major ecological implications. In aquatic ecosystems, bioinvasions may cause adverse effects on the water quality resulting from changes in biological, chemical and physical properties linked to significant transformations of the microbial taxonomic and functional diversity. Here we used an effective and highly sensitive experimental strategy, bypassing the efficiency bottleneck of the traditional bacterial isolation and culturing method, to identify changes of the planktonic microbial community inhabiting a marine coastal lagoon (Varano, Adriatic Sea) under the influence of an outbreak-forming alien jellyfish species. Water samples were collected from two areas that differed in their level of confinement inside in the lagoon and jellyfish densities (W, up to 12.4 medusae m−3; E, up to 0.03 medusae m−3) to conduct a snapshot microbiome analysis by a metagenomic approach. After extraction of the genetic material in the environmental water samples, we deep-sequenced metagenomic amplicons of the V5–V6 region of the 16S rRNA bacterial gene by an Illumina MiSeq platform. Experiments were carried out in triplicates, so six libraries of dual indexed amplicons of 420 bp were successfully sequenced on the MiSeq platform using a 2 × 250 bp paired-end sequencing strategy. Approximately 7.5 million paired-end reads (i.e. 15 million total reads) were generated, with an average of 2.5 million reads (1.25 M pairs) per sample replicate. The sequence data, analyzed through a novel bioinformatics pipeline (BioMaS), showed that the structure of the resident bacterial community was significantly affected by the occurrence of jellyfish outbreaks. Clear qualitative and quantitative differences were found between the western and eastern areas (characterized by many or few jellyfish), with 84 families, 153 genera and 324 species in the W samples, and 104 families, 199 genera and 331 species in the E samples. Significant differences between the two sampling areas were particularly detected in the occurrence of 16 families, 22 genera and 61 species of microbial taxa. This is the first time that a NGS platform has been used to screen the impact of jellyfish bioinvasions on the aquatic microbiome, providing a preliminary assessment of jellyfish-driven changes of the functional and structural microbial biodiversity

A platform independent RNA-Seq protocol for the detection of transcriptome complexity

Background: Recent studies have demonstrated an unexpected complexity of transcription in eukaryotes. The majority of the genome is transcribed and only a little fraction of these transcripts is annotated as protein coding genes and their splice variants. Indeed, most transcripts are the result of antisense, overlapping and non-coding RNA expression. In this frame, one of the key aims of high throughput transcriptome sequencing is the detection of all RNA species present in the cell and the first crucial step for RNA-seq users is represented by the choice of the strategy for cDNA library construction. The protocols developed so far provide the utilization of the entire library for a single sequencing run with a specific platform. Results: We set up a unique protocol to generate and amplify a strand-specific cDNA library representative of all RNA species that may be implemented with all major platforms currently available on the market (Roche 454, Illumina, ABI/SOLiD). Our method is reproducible, fast, easy-to-perform and even allows to start from low input total RNA. Furthermore, we provide a suitable bioinformatics tool for the analysis of the sequences produced following this protocol. Conclusion: We tested the efficiency of our strategy, showing that our method is platform-independent, thus allowing the simultaneous analysis of the same sample with different NGS technologies, and providing an accurate quantitative and qualitative portrait of complex whole transcriptomes

Tissue-specific mtDNA abundance from exome data and its correlation with mitochondrial transcription, mass and respiratory activity.

Eukaryotic cells contain a population of mitochondria, variable in number and shape, which in turn contain multiple copies of a tiny compact genome (mtDNA) whose expression and function is strictly coordinated with the nuclear one. mtDNA copy number varies between different cell or tissues types, both in response to overall metabolic and bioenergetics demands and as a consequence or cause of specific pathological conditions. Here we present a novel and reliable methodology to assess the effective mtDNA copy number per diploid genome by investigating off-target reads obtained by whole-exome sequencing (WES) experiments. We also investigate whether and how mtDNA copy number correlates with mitochondrial mass, respiratory activity and expression levels. Analyzing six different tissues from three age- and sex-matched human individuals, we found a highly significant linear correlation between mtDNA copy number estimated by qPCR and the frequency of mtDNA off target WES reads. Furthermore, mtDNA copy number showed highly significant correlation with mitochondrial gene expression levels as measured by RNA-Seq as well as with mitochondrial mass and respiratory activity. Our methodology makes thus feasible, at a large scale, the investigation of mtDNA copy number in diverse cell-types, tissues and pathological conditions or in response to specific treatments.This work was supported by Ministero dell'Istruzione, Università e Ricerca (projects PRIN-2009, Micromap [PON01_02589], Virtualab [PON01_01297]) and by Consiglio Nazionale delle Ricerche (progetto strategico “Medicina personalizzata”, progetto strategico “Invecchiamento”, progetto bandiera “Epigen”)