Potential global cis and trans regulation of lncRNAs in Saccharomyces cerevisiae subjected to ethanol stress.

Long noncoding RNAs (lncRNAs) are regulatory RNAs. Saccharomyces cerevisiae strains transcribe hundreds of lncRNAs. LncRNAs can regulate the expression of adjacent genes (cis-regulation) or distant genes from lncRNAs (trans-regulation). Here, we analyzed the potential global cis and trans-regulation of lncRNAs of yeast subjected to ethanol stress. For potential cis regulation, for BMA641-A and S288C strains, we observed that most lncRNA-neighbor gene pairs increased the expression at a certain point followed by a decrease, and vice versa. Based on the transcriptome profile and triple helix prediction between lncRNAs and promoters of coding genes, we observed nine different ways of potential trans regulation that work in a strain-specific manner. Our data provide an initial landscape of potential cis and trans regulation in yeast, which seems to be strain-specific.

Integrative Analysis of the Ethanol Tolerance of Saccharomyces cerevisiae.

Ethanol (EtOH) alters many cellular processes in yeast. An integrated view of different EtOH-tolerant phenotypes and their long noncoding RNAs (lncRNAs) is not yet available. Here, large-scale data integration showed the core EtOH-responsive pathways, lncRNAs, and triggers of higher (HT) and lower (LT) EtOH-tolerant phenotypes. LncRNAs act in a strain-specific manner in the EtOH stress response. Network and omics analyses revealed that cells prepare for stress relief by favoring activation of life-essential systems. Therefore, longevity, peroxisomal, energy, lipid, and RNA/protein metabolisms are the core processes that drive EtOH tolerance. By integrating omics, network analysis, and several other experiments, we showed how the HT and LT phenotypes may arise: (1) the divergence occurs after cell signaling reaches the longevity and peroxisomal pathways, with CTA1 and ROS playing key roles; (2) signals reaching essential ribosomal and RNA pathways via SUI2 enhance the divergence; (3) specific lipid metabolism pathways also act on phenotype-specific profiles; (4) HTs take greater advantage of degradation and membraneless structures to cope with EtOH stress; and (5) our EtOH stress-buffering model suggests that diauxic shift drives EtOH buffering through an energy burst, mainly in HTs. Finally, critical genes, pathways, and the first models including lncRNAs to describe nuances of EtOH tolerance are reported here.

First characterization of PIWI-interacting RNA clusters in a cichlid fish with a B chromosome.

BACKGROUND: B chromosomes are extra elements found in several eukaryote species. Usually, they do not express a phenotype in the host. However, advances in bioinformatics over the last decades have allowed us to describe several genes and molecular functions related to B chromosomes. These advances enable investigations of the relationship between the B chromosome and the host to understand how this element has been preserved in genomes. However, considering that transposable elements (TEs) are highly abundant in this supernumerary chromosome, there is a lack of knowledge concerning the dynamics of TE control in B-carrying cells. Thus, the present study characterized PIWI-interacting RNA (piRNA) clusters and pathways responsible for silencing the mobilization of TEs in gonads of the cichlid fish Astatotilapia latifasciata carrying the B chromosome. RESULTS: Through small RNA-seq and genome assembly, we predicted and annotated piRNA clusters in the A. latifasciata genome for the first time. We observed that these clusters had biased expression related to sex and the presence of the B chromosome. Furthermore, three piRNA clusters, named curupira, were identified in the B chromosome. Two of them were expressed exclusively in gonads of samples with the B chromosome. The composition of these curupira sequences was derived from LTR, LINE, and DNA elements, representing old and recent transposition events in the A. latifasciata genome and the B chromosome. The presence of the B chromosome also affected the expression of piRNA pathway genes. The mitochondrial cardiolipin hydrolase-like (pld6) gene is present in the B chromosome, as previously reported, and an increase in its expression was detected in gonads with the B chromosome. CONCLUSIONS: Due to the high abundance of TEs in the B chromosome, it was possible to investigate the origin of piRNA from these jumping genes. We hypothesize that the B chromosome has evolved its own genomic guardians to prevent uncontrolled TE mobilization. Furthermore, we also detected an expression bias in the presence of the B chromosome over A. latifasciata piRNA clusters and pathway genes.

LncRNAs of Saccharomyces cerevisiae bypass the cell cycle arrest imposed by ethanol stress.

Ethanol alters many subsystems of Saccharomyces cerevisiae, including the cell cycle. Two ethanol-responsive lncRNAs in yeast interact with cell cycle proteins, and here, we investigated the role of these RNAs in cell cycle. Our network dynamic modeling showed that higher and lower ethanol-tolerant strains undergo cell cycle arrest in mitosis and G1 phases, respectively, during ethanol stress. The higher population rebound of the lower ethanol-tolerant phenotype after stress relief responds to the late phase arrest. We found that the lncRNA lnc9136 of SEY6210 (a lower ethanol-tolerant strain) induces cells to skip mitosis arrest. Simulating an overexpression of lnc9136 and analyzing CRISPR-Cas9 mutants lacking this lncRNA suggest that lnc9136 induces a regular cell cycle even under ethanol stress, indirectly regulating Swe1p and Clb1/2 by binding to Gin4p and Hsl1p. Notably, lnc10883 of BY4742 (a higher ethanol-tolerant strain) does not prevent G1 arrest in this strain under ethanol stress. However, lnc19883 circumvents DNA and spindle damage checkpoints, maintaining a functional cell cycle by interacting with Mec1p or Bub1p even in the presence of DNA/spindle damage. Overall, we present the first evidence of direct roles for lncRNAs in regulating yeast cell cycle proteins, the dynamics of this system in different ethanol-tolerant phenotypes, and a new yeast cell cycle model.

Three topological features of regulatory networks control life-essential and specialized subsystems.

Gene regulatory networks (GRNs) play key roles in development, phenotype plasticity, and evolution. Although graph theory has been used to explore GRNs, associations amongst topological features, transcription factors (TFs), and systems essentiality are poorly understood. Here we sought the relationship amongst the main GRN topological features that influence the control of essential and specific subsystems. We found that the Knn, page rank, and degree are the most relevant GRN features: the ones are conserved along the evolution and are also relevant in pluripotent cells. Interestingly, life-essential subsystems are governed mainly by TFs with intermediary Knn and high page rank or degree, whereas specialized subsystems are mainly regulated by TFs with low Knn. Hence, we suggest that the high probability of TFs be toured by a random signal, and the high probability of the signal propagation to target genes ensures the life-essential subsystems' robustness. Gene/genome duplication is the main evolutionary process to rise Knn as the most relevant feature. Herein, we shed light on unexplored topological GRN features to assess how they are related to subsystems and how the duplications shaped the regulatory systems along the evolution. The classification model generated can be found here: https://github.com/ivanrwolf/NoC/ .

Differential expression of miRNAs in the presence of B chromosome in the cichlid fish Astatotilapia latifasciata.

BACKGROUND: B chromosomes (Bs) are extra elements observed in diverse eukaryotes, including animals, plants and fungi. Although Bs were first identified a century ago and have been studied in hundreds of species, their biology is still enigmatic. Recent advances in omics and big data technologies are revolutionizing the B biology field. These advances allow analyses of DNA, RNA, proteins and the construction of interactive networks for understanding the B composition and behavior in the cell. Several genes have been detected on the B chromosomes, although the interaction of B sequences and the normal genome remains poorly understood. RESULTS: We identified 727 miRNA precursors in the A. latifasciata genome, 66% which were novel predicted sequences that had not been identified before. We were able to report the A. latifasciata-specific miRNAs and common miRNAs identified in other fish species. For the samples carrying the B chromosome (B+), we identified 104 differentially expressed (DE) miRNAs that are down or upregulated compared to samples without B chromosome (B-) (p < 0.05). These miRNAs share common targets in the brain, muscle and gonads. These targets were used to construct a protein-protein-miRNA network showing the high interaction between the targets of differentially expressed miRNAs in the B+ chromosome samples. Among the DE-miRNA targets there are protein-coding genes reported for the B chromosome that are present in the protein-protein-miRNA network. Additionally, Gene Ontology (GO) terms related to nuclear matrix organization and response to stimulus are exclusive to DE miRNA targets of B+ samples. CONCLUSIONS: This study is the first to report the connection of B chromosomes and miRNAs in a vertebrate species. We observed that the B chromosome impacts the miRNAs expression in several tissues and these miRNAs target several mRNAs involved with important biological processes.

Uncovering patterns of the evolution of genomic sequence entropy and complexity.

The lack of consensus concerning the biological meaning of entropy and complexity of genomes and the different ways to assess these data hamper conclusions concerning what are the causes of genomic entropy variation among species. This study aims to evaluate the entropy and complexity of genomic sequences of several species without using homologies to assess relationships among these variables and non-molecular data (e.g., the number of individuals) to seek a trigger of interspecific genomic entropy variation. The results indicate a relationship among genomic entropy, genome size, genomic complexity, and the number of individuals: species with a small number of individuals harbors large genome, and hence, low entropy but a higher complexity. We defined that the complexity of a genome relies on the entropy of each DNA segment within genome. Then, the entropy and complexity of a genome reflects its organization solely. Exons of vertebrates harbor smaller entropies than non-exon regions (likely by the repeats that accumulated from duplications), whereas other taxonomic groups do not present this pattern. Our findings suggest that small initial population might have defined current genomic entropy and complexity: actual genomes are less complex than ancestral ones. Besides, our data disagree with the relationship between phenotype and genomic entropies previously established. Finally, by establishing the relationship between genomic entropy/complexity with the number of individuals and genome size, under an evolutive perspective, ideas concerning the genomic variability may emerge.

A New Method for Next-Generation Sequencing of the Full Hepatitis B Virus Genome from A Clinical Specimen: Impact for Virus Genotyping.

Hepatitis B virus (HBV) is an enveloped virus that induces chronic liver disease. HBV has been classified into eight genotypes (A-H) according to its genome sequence by using Sanger sequencing or reverse hybridization. Sanger sequencing is often restricted to analyzing the S gene and is inaccurate for detecting minority genetic variants, whereas reverse hybridization detects only known mutations. Next-generation sequencing (NGS) is a robust tool for clinical virology with different protocols available. The objective of this study was to develop a new method for the study of viral genetic polymorphisms or more accurate genotyping using genome amplification followed by NGS. Plasma obtained from five chronically infected HBV individuals was used for viral DNA isolation. HBV full-genome PCR amplification was the enrichment method for NGS. Primers were used to amplify all HBV genotypes in three overlapping amplicons, following a tagmentation step and Illumina NGS. For phylogenetic analysis, sequences were extracted from the HBVdb database. We were able to amplify a full HBV genome; further, NGS was shown to be a robust method and allowed better genotyping, mainly in patients carrying mixed genotypes, classified according to other techniques. This new method may be significant for whole genome analyses, including other viruses.

Transcriptional analysis of THP-1 cells infected with Leishmania infantum indicates no activation of the inflammasome platform.

Leishmaniasis is caused by intracellular parasites transmitted to vertebrates by sandfly bites. Clinical manifestations include cutaneous, mucosal or visceral involvement depending upon the host immune response and the parasite species. To assure their survival inside macrophages, these parasites developed a plethora of highly successful strategies to manipulate various immune system pathways. Considering that inflammasome activation is critical for the establishment of a protective immune response in many parasite infections, in this study we determined the transcriptome of THP-1 cells after infection with L. infantum, with a particular focus on the inflammasome components. To this end, the human cell line THP-1, previously differentiated into macrophages by PMA treatment, was infected with L. infantum promastigotes. Differentiated THP-1 cells were also stimulated with LPS to be used as a comparative parameter. The gene expression signature was determined 8 hours after by RNA-seq technique. Infected or uninfected THP-1 cells were stimulated with nigericin (NIG) to measure active caspase-1 and TNF-α, IL-6 and IL-1ß levels in culture supernatants after 8, 24 and 48 hours. L. infantum triggered a gene expression pattern more similar to non-infected THP-1 cells and very distinct from LPS-stimulated cells. Some of the most up-regulated genes in L. infantum-infected cells were CDC20, CSF1, RPS6KA1, CD36, DUSP2, DUSP5, DUSP7 and TNFAIP3. Some up-regulated GO terms in infected cells included cell coagulation, regulation of MAPK cascade, response to peptide hormone stimulus, negative regulation of transcription from RNA polymerase II promoter and nerve growth factor receptor signaling pathway. Infection was not able to induce the expression of genes associated with the inflammasome signaling pathway. This finding was confirmed by the absence of caspase-1 activation and IL-1ß production after 8, 24 and 48 hours of infection. Our results indicate that L. infantum was unable to activate the inflammasomes during the initial interaction with THP-1 cells.

A novel molecular mechanism to explain mutations of the HCV protease associated with resistance against covalently bound inhibitors.

NS3 is an important therapeutic target for direct-acting antiviral (DAA) drugs. However, many patients treated with DAAs have unsustained virologic response (UVR) due to the high mutation rate of HCV. The aim of this work was to shed some light on the puzzling molecular mechanisms of the virus's of patients who showed high viral loads even under treatment with DAA. Bioinformatics tools, molecular modelling analyses were employed to identify mutations associated with HCV resistance to boceprevir and possible structural features related to this phenomenon. We identified two mutations of NS3 that may be associated with HCV resistance: D168N and L153I. The substitution D168N was previously reported in the literature as related with drug failure. Additionally, we identified that its molecular resistance mechanism can be explained by the destabilization of receptor-ligand hydrogen bonds. For the L153I mutation, the resistance mechanism is different from previous models reported in the literature. The L153I substitution decreases the S139 deprotonation susceptibility, and consequently, this mutation impairs the covalent binding between the residue S139 from NS3 and the electrophilic trap on boceprevir, which can induce drug failure. These results were supported by the time course analysis of the mutations of the NS3 protease, which showed that boceprevir was designed for enzymes with an L residue at position 153; however, the sequences with I153 are predominant nowadays. The results presented here could be used to infer about resistance in others DAA, mainly protease inhibitors.

Transcriptional profile of a bioethanol production contaminant Candida tropicalis.

The fermentation process is widely used in the industry for bioethanol production. Even though it is widely used, microbial contamination is unpredictable and difficult to control. The problem of reduced productivity is directly linked to competition for nutrients during contamination. Yeasts representing the Candida species are frequently isolated contaminants. Elucidating the behavior of a contaminant during the fermentation cycle is essential for combatting the contamination. Consequently, the aim of the current study was to better understand the functional and transcriptional behavior of a contaminating yeast Candida tropicalis. We used a global RNA sequencing approach (RNA-seq/MiSeq) to analyze gene expression. Genes with significantly repressed or induced expression, and related to the fermentations process, such as sugar transport, pyruvate decarboxylase, amino acid metabolism, membrane, tolerance to high concentrations of ethanol and temperatures, nutrient suppression), and transcription-linked processes, were identified. The expression pattern suggested that the functional and transcriptional behavior of the contaminating yeast during fermentation for bioethanol production is similar to that of the standard yeast Saccharomyces cerevisiae. In addition, the analysis confirmed that C. tropicalis is an important contaminant of the alcoholic fermentation process, generating bioethanol and viability through its tolerance to all the adversities of a fermentation process essential for the production of bioethanol. According on the gene expression profile, many of these mechanisms are similar to those of S. cerevisiae strains currently used for bioethanol production. These mechanisms can inform studies on antimicrobials, to combat yeast contamination during industrial bioethanol production.

Functional annotation and distribution overview of RNA families in 27 Streptococcus agalactiae genomes.

BACKGROUND: Streptococcus agalactiae, also known as Group B Streptococcus (GBS), is a Gram-positive bacterium that colonizes the gastrointestinal and genitourinary tract of humans. This bacterium has also been isolated from various animals, such as fish and cattle. Non-coding RNAs (ncRNAs) can act as regulators of gene expression in bacteria, such as Streptococcus pneumoniae and Streptococcus pyogenes. However, little is known about the genomic distribution of ncRNAs and RNA families in S. agalactiae. RESULTS: Comparative genome analysis of 27 S. agalactiae strains showed more than 5 thousand genomic regions identified and classified as Core, Exclusive, and Shared genome sequences. We identified 27 to 89 RNA families per genome distributed over these regions, from these, 25 were in Core regions while Shared and Exclusive regions showed variations amongst strains. We propose that the amount and type of ncRNA present in each genome can provide a pattern to contribute in the identification of the clonal types. CONCLUSIONS: The identification of RNA families provides an insight over ncRNAs, sRNAs and ribozymes function, that can be further explored as targets for antibiotic development or studied in gene regulation of cellular processes. RNA families could be considered as markers to determine infection capabilities of different strains. Lastly, pan-genome analysis of GBS including the full range of functional transcripts provides a broader approach in the understanding of this pathogen.

Draft Genome Sequence of Burkholderia gladioli Coa14, a Bacterium with Petroleum Bioremediation Potential Isolated from Coari Lake, Amazonas, Brazil.

Burkholderia gladioli Coa14 is a bacterium isolated from water collected from Coari Lake (Amazonas, Brazil) that shows a capacity for survival in a medium containing only oil as a carbon source. Here, we report its draft genome sequence, highlighting some genes involved with petroleum derivative degradation.

Repetitive DNA in the Catfish Genome: rDNA, Microsatellites, and Tc1-Mariner Transposon Sequences in Imparfinis Species (Siluriformes, Heptapteridae).

Physical mapping of repetitive DNA families in the karyotypes of fish is important to understand the organization and evolution of different orders, families, genera, or species. Fish in the genus Imparfinis show diverse karyotypes with various diploid numbers and ribosomal DNA (rDNA) locations. Here we isolated and characterized Tc1-mariner nucleotide sequences from Imparfinis schubarti, and mapped their locations together with 18S rDNA, 5S rDNA, and microsatellite probes in Imparfinis borodini and I. schubarti chromosomes. The physical mapping of Tc1/Mariner on chromosomes revealed dispersed signals in heterochromatin blocks with small accumulations in the terminal and interstitial regions of I. borodini and I. schubarti. Tc1/Mariner was coincident with rDNA chromosomes sites in both species, suggesting that this transposable element may have participated in the dispersion and evolution of these sequences in the fish genome. Our analysis suggests that different transposons and microsatellites have accumulated in the I. borodini and I. schubarti genomes and that the distribution patterns of these elements may be related to karyotype evolution within Imparfinis.

Isolation and characterization of 5S rDNA sequences in catfishes genome (Heptapteridae and Pseudopimelodidae): perspectives for rDNA studies in fish by C0t method.

Sequences of 5S ribosomal RNA (rRNA) are extensively used in fish cytogenomic studies, once they have a flexible organization at the chromosomal level, showing inter- and intra-specific variation in number and position in karyotypes. Sequences from the genome of Imparfinis schubarti (Heptapteridae) were isolated, aiming to understand the organization of 5S rDNA families in the fish genome. The isolation of 5S rDNA from the genome of I. schubarti was carried out by reassociation kinetics (C0t) and PCR amplification. The obtained sequences were cloned for the construction of a micro-library. The obtained clones were sequenced and hybridized in I. schubarti and Microglanis cottoides (Pseudopimelodidae) for chromosome mapping. An analysis of the sequence alignments with other fish groups was accomplished. Both methods were effective when using 5S rDNA for hybridization in I. schubarti genome. However, the C0t method enabled the use of a complete 5S rRNA gene, which was also successful in the hybridization of M. cottoides. Nevertheless, this gene was obtained only partially by PCR. The hybridization results and sequence analyses showed that intact 5S regions are more appropriate for the probe operation, due to conserved structure and motifs. This study contributes to a better understanding of the organization of multigene families in catfish's genomes.