ACE2 polymorphisms as potential players in COVID-19 outcome.

The clinical condition COVID-19, caused by SARS-CoV-2, was declared a pandemic by the WHO in March 2020. Currently, there are more than 5 million cases worldwide, and the pandemic has increased exponentially in many countries, with different incidences and death rates among regions/ethnicities and, intriguingly, between sexes. In addition to the many factors that can influence these discrepancies, we suggest a biological aspect, the genetic variation at the viral S protein receptor in human cells, ACE2 (angiotensin I-converting enzyme 2), which may contribute to the worse clinical outcome in males and in some regions worldwide. We performed exomics analysis in native and admixed South American populations, and we also conducted in silico genomics databank investigations in populations from other continents. Interestingly, at least ten polymorphisms in coding, noncoding and regulatory sites were found that can shed light on this issue and offer a plausible biological explanation for these epidemiological differences. In conclusion, there are ACE2 polymorphisms that could influence epidemiological discrepancies observed among ancestry and, moreover, between sexes.

Advances in the Bioinformatics Knowledge of mRNA Polyadenylation in Baculovirus Genes.

Baculoviruses are a group of insect viruses with large circular dsDNA genomes exploited in numerous biotechnological applications, such as the biological control of agricultural pests, the expression of recombinant proteins or the gene delivery of therapeutic sequences in mammals, among others. Their genomes encode between 80 and 200 proteins, of which 38 are shared by all reported species. Thanks to multi-omic studies, there is remarkable information about the baculoviral proteome and the temporality in the virus gene expression. This allows some functional elements of the genome to be very well described, such as promoters and open reading frames. However, less information is available about the transcription termination signals and, consequently, there are still imprecisions about what are the limits of the transcriptional units present in the baculovirus genomes and how is the processing of the 3' end of viral mRNA. Regarding to this, in this review we provide an update about the characteristics of DNA signals involved in this process and we contribute to their correct prediction through an exhaustive analysis that involves bibliography information, data mining, RNA structure and a comprehensive study of the core gene 3' ends from 180 baculovirus genomes.

Estudo descritivo molecular de pacientes com RASopatia / Descriptive molecular study of patients with RASopathy

As RASopatias são um grupo de doenças cujos pacientes apresentam alterações constitucionais em genes que participam de uma mesma via de sinalização celular denominada Ras/MAPK, que desempenha um papel importante na proliferação, diferenciação, migração celular e apoptose, além de estar associada a processos carcinogênicos. Apesar dos avanços em métodos diagnósticos, cerca de 20 a 25% dos casos permanecem inconclusivos, o que impulsiona pesquisas que buscam alterações em outros níveis de regulação da via, como RNAs não codificantes e proteínas. O primeiro capítulo deste estudo, avalia um grupo de 6 pacientes diagnosticados clinicamente com RASopatia e com exomas sequenciados. Foram obtidos dados sobre mutações em seus miRNAs. O segundo capítulo relata o caso de um paciente com suspeita clínica de síndrome de Costello, mas sem mutações detectadas. Foram avaliados, in silico, dados sobre miRNAs reguladores do gene HRAS, bem como os diferentes tecidos nos quais o HRAS é expresso. Para o capítulo 3 foi realizado um estudo comparativo entre gêmeas com diagnóstico clínico de síndrome de Noonan, mas com fenótipo discordante. Foi realizado um array por RT-qPCR para diferentes RNAs reguladores e um estudo comparativo de proteoma com análise de vias biológicas, processos biológicos e genes alvo da regulação de fatores de transcrição putativos. No estudo de miRNAs foram encontradas mutações em heterozigose, que são de difícil avaliação em ensaios de expressão. No estudo de caso do paciente S4 (síndrome de Costello), não foram encontradas mutações nos miR-181d-5p, let-7a-5p, miR-143-3p, miR-181a-5p, miR-139-5p, miR-663a e let-7b-5p, descritos como reguladores do HRAS. Também não foram encontrados tecidos viáveis para coleta e análise da expressão do HRAS. Na expressão de RNAs reguladores nas gêmeas (S16 e S17) foram encontrados níveis de expressão aumentados em S17 para Lnc-C21orf33-1, ERBS3/SBNO2e, miR-200be, CTBP1-AS e Lnc_DC. Na análise do proteoma, foram encontradas diferenças de expressão em vias de integrinas, proteoglicanos e trombinas, além de diferenças em processos de transdução de sinal, crescimento e manutenção celular e metabolismo. Os genes com sítio de ligação para os fatores de transcrição como RREB1, ETS1, EGR1 e TBX5 também possuíam expressão diferente entre as gêmeas. Os resultados aqui apresentados apontam novos caminhos para estudos moleculares das RASopatias que possam preencher as lacunas diagnósticas ainda pendentes.

RASopathies are a group of diseases whose patients present constitutional changes in genes that participate in the same cellular signaling pathway called the Ras/MAPK, which plays an important role in proliferation, differentiation, cell migration and apoptosis, in addition to being associated with carcinogenic processes. Despite advances in diagnostic methods, about 20 to 25% of cases remain inconclusive, which drives research that seeks changes in other levels of pathway regulation, such as non-coding RNAs and proteins. The first chapter of this study evaluates a group of 6 patients clinically diagnosed with RASopathy and sequenced exomes. Data were obtained on mutations in their miRNAs. The second chapter reports the case of a patient with clinical suspicion of Costello syndrome, but without mutations detected. Data on the miRNAs regulating the HRAS gene, as well as the different tissues in which HRAS is expressed, were evaluated in silico. For chapter 3 a comparative study was performed between twins with clinical diagnosis of Noonan syndrome, but with a discordant phenotype. An array was performed by RT-qPCR for different regulatory RNAs and a comparative proteome study with analysis of biological pathways, biological processes and genes targeting the regulation of putative transcription factors. In the study of miRNAs, mutations were found in heterozygosis, which are difficult to evaluate in expression assays. In the case study of S4 patient (Costello syndrome), no mutations were found in miR-181d-5p, let-7a-5p, miR-143-3p, miR-181a-5p, miR-139-5p, miR-663a and let-7b -5p, described as HRAS regulators. No available tissues were also found for collection and analysis of HRAS expression. In expression of regulatory RNAs in the S16 and S17 twins, increased levels of S17 expression were found for Lnc-C21orf33-1, ERBS3 / SBNO2e, miR-200b, CTBP1-AS and Lnc_DC. In the proteome analysis, expression differences were found in integrins, proteoglycans and thrombin pathways, as well as differences in signal transduction processes, cell growth and maintenance, and metabolism. Genes with binding site for transcription factors such as RREB1, ETS1, EGR1 and TBX5 also had different expression between the twins. The results presented here point out new ways for molecular studies of RASopathies that may close the remaining diagnostic gaps.

Regulation of Tacaribe Mammarenavirus Translation: Positive 5' and Negative 3' Elements and Role of Key Cellular Factors.

Mammarenaviruses are enveloped viruses with a bisegmented negative-stranded RNA genome that encodes the nucleocapsid protein (NP), the envelope glycoprotein precursor (GPC), the RNA polymerase (L), and a RING matrix protein (Z). Viral proteins are synthesized from subgenomic mRNAs bearing a capped 5' untranslated region (UTR) and lacking 3' poly(A) tail. We analyzed the translation strategy of Tacaribe virus (TCRV), a prototype of the New World mammarenaviruses. A virus-like transcript that carries a reporter gene in place of the NP open reading frame and transcripts bearing modified 5' and/or 3' UTR were evaluated in a cell-based translation assay. We found that the presence of the cap structure at the 5' end dramatically increases translation efficiency and that the viral 5' UTR comprises stimulatory signals while the 3' UTR,specifically the presence of a terminal C+G-rich sequence and/or a stem-loop structure, down-modulates translation. Additionally, translation was profoundly reduced in eukaryotic initiation factor (eIF) 4G-inactivated cells, whereas depletion of intracellular levels of eIF4E had less impact on virus-like mRNA translation than on a cell-like transcript. Translation efficiency was independent of NP expression or TCRV infection. Our results indicate that TCRV mRNAs are translated using a cap-dependent mechanism, whose efficiency relies on the interplay between stimulatory signals in the 5' UTR and a negative modulatory element in the 3' UTR. The low dependence on eIF4E suggests that viral mRNAs may engage yet-unknown noncanonical host factors for a cap-dependent initiation mechanism.IMPORTANCE Several members of the Arenaviridae family cause serious hemorrhagic fevers in humans. In the present report, we describe the mechanism by which Tacaribe virus, a prototypic nonpathogenic New World mammarenavirus, regulates viral mRNA translation. Our results highlight the impact of untranslated sequences and key host translation factors on this process. We propose a model that explains how viral mRNAs outcompete cellular mRNAs for the translation machinery. A better understanding of the mechanism of translation regulation of this virus can provide the bases for the rational design of new antiviral tools directed to pathogenic arenaviruses.

Immunopathogenesis of IBD: current state of the art.

IBD is a chronic inflammatory condition of the gastrointestinal tract encompassing two main clinical entities: Crohn's disease and ulcerative colitis. Although Crohn's disease and ulcerative colitis have historically been studied together because they share common features (such as symptoms, structural damage and therapy), it is now clear that they represent two distinct pathophysiological entities. Both Crohn's disease and ulcerative colitis are associated with multiple pathogenic factors including environmental changes, an array of susceptibility gene variants, a qualitatively and quantitatively abnormal gut microbiota and a broadly dysregulated immune response. In spite of this realization and the identification of seemingly pertinent environmental, genetic, microbial and immune factors, a full understanding of IBD pathogenesis is still out of reach and, consequently, treatment is far from optimal. An important reason for this unsatisfactory situation is the currently limited comprehension of what are the truly relevant components of IBD immunopathogenesis. This article will comprehensively review current knowledge of the classic immune components and will expand the concept of IBD immunopathogenesis to include various cells, mediators and pathways that have not been traditionally associated with disease mechanisms, but that profoundly affect the overall intestinal inflammatory process.

Genetic features of a translation initiation system composed of IRES element, nucleotide context surrounding the initiation codon, and translation initiation region of classical swine fever virus RNA.

Nucleotide and codon usage are typically examined to investigate viral evolution. In this study, we analyzed the genetic information of 46 strains of classical swine fever virus (CSFV) RNA, nucleotide usage in the internal ribosome entry site (IRES), the nucleotide context surrounding the initiation codon, and synonymous codon usage in the translation initiation region. Phylogenetic analysis of the IRES element indicated that the genetic diversity of this element is generally similar to the phylogenetic clusters of CSFV genotypes. Nucleotides surrounding the initiation codon of CSFV RNA were generally more stable (ACAUGGCACAUGGAGUUG) compared to the internal AUG in the CSFV coding sequence. The second codon position after the initiation codon was generally selected to be GAG, which has lower tRNA abundance in pigs than its synonymous member (GAA). Regarding the synonymous codon usage bias in the CSFV translation initiation region, some codons showing low tRNA abundance in pigs are more frequently located in the translation initiation region than in the open reading frame of CSFV. Although CSFV, similarly to other RNA viruses, has a high mutation rate in nature, the regulatory features of nucleotide and synonymous codon usage of the IRES element, the nucleotide context surrounding the initiation codon and the translation initiation region in CSFV RNA have been 'branded' in the system of translation initiation to accommodate gene expression mediated by the cap-independent translation mechanism.

A comprehensive survey of non-canonical splice sites in the human transcriptome.

We uncovered the diversity of non-canonical splice sites at the human transcriptome using deep transcriptome profiling. We mapped a total of 3.7 billion human RNA-seq reads and developed a set of stringent filters to avoid false non-canonical splice site detections. We identified 184 splice sites with non-canonical dinucleotides and U2/U12-like consensus sequences. We selected 10 of the herein identified U2/U12-like non-canonical splice site events and successfully validated 9 of them via reverse transcriptase-polymerase chain reaction and Sanger sequencing. Analyses of the 184 U2/U12-like non-canonical splice sites indicate that 51% of them are not annotated in GENCODE. In addition, 28% of them are conserved in mouse and 76% are involved in alternative splicing events, some of them with tissue-specific alternative splicing patterns. Interestingly, our analysis identified some U2/U12-like non-canonical splice sites that are converted into canonical splice sites by RNA A-to-I editing. Moreover, the U2/U12-like non-canonical splice sites have a differential distribution of splicing regulatory sequences, which may contribute to their recognition and regulation. Our analysis provides a high-confidence group of U2/U12-like non-canonical splice sites, which exhibit distinctive features among the total human splice sites.

Activity of the human immunodeficiency virus type 1 cell cycle-dependent internal ribosomal entry site is modulated by IRES trans-acting factors.

The 5' leader of the human immunodeficiency virus type 1 (HIV-1) genomic RNA harbors an internal ribosome entry site (IRES) that is functional during the G2/M phase of the cell cycle. Here we show that translation initiation mediated by the HIV-1 IRES requires the participation of trans-acting cellular factors other than the canonical translational machinery. We used 'standard' chemical and enzymatic probes and an 'RNA SHAPE' analysis to model the structure of the HIV-1 5' leader and we show, by means of a footprinting assay, that G2/M extracts provide protections to regions previously identified as crucial for HIV-1 IRES activity. We also assessed the impact of mutations on IRES function. Strikingly, mutations did not significantly affect IRES activity suggesting that the requirement for pre-formed stable secondary or tertiary structure within the HIV-1 IRES may not be as strict as has been described for other viral IRESes. Finally, we used a proteomic approach to identify cellular proteins within the G2/M extracts that interact with the HIV-1 5' leader. Together, data show that HIV-1 IRES-mediated translation initiation is modulated by cellular proteins.

Alternative splicing and genetic diversity: silencers are more frequently modified by SNVs associated with alternative exon/intron borders.

With the availability of a large amount of genomic data it is expected that the influence of single nucleotide variations (SNVs) in many biological phenomena will be elucidated. Here, we approached the problem of how SNVs affect alternative splicing. First, we observed that SNVs and exonic splicing regulators (ESRs) independently show a biased distribution in alternative exons. More importantly, SNVs map more frequently in ESRs located in alternative exons than in ESRs located in constitutive exons. By looking at SNVs associated with alternative exon/intron borders (by their common presence in the same cDNA molecule), we observed that a specific type of ESR, the exonic splicing silencers (ESSs), are more frequently modified by SNVs. Our results establish a clear association between genetic diversity and alternative splicing involving ESSs.

Analysis of natural variants of the hepatitis C virus internal ribosome entry site reveals that primary sequence plays a key role in cap-independent translation.

The HCV internal ribosome entry site (IRES) spans a region of approximately 340 nt that encompasses most of the 5' untranslated region (5'UTR) of the viral mRNA and the first 24-40 nt of the core-coding region. To investigate the implication of altering the primary sequence of the 5'UTR on IRES activity, naturally occurring variants of the 5'UTR were isolated from clinical samples and analyzed. The impact of the identified mutations on translation was evaluated in the context of RLuc/FLuc bicistronic RNAs. Results show that depending on their location within the RNA structure, these naturally occurring mutations cause a range of effects on IRES activity. However, mutations within subdomain IIId hinder HCV IRES-mediated translation. In an attempt to explain these data, the dynamic behavior of the subdomain IIId was analyzed by means of molecular dynamics (MD) simulations. Despite the loss of function, MD simulations predicted that mutant G266A/G268U possesses a structure similar to the wt-RNA. This prediction was validated by analyzing the secondary structure of the isolated IIId RNAs by circular dichroism spectroscopy in the presence or absence of Mg(2+) ions. These data strongly suggest that the primary sequence of subdomain IIId plays a key role in HCV IRES-mediated translation.

RegulonDB (version 6.0): gene regulation model of Escherichia coli K-12 beyond transcription, active (experimental) annotated promoters and Textpresso navigation.

RegulonDB (http://regulondb.ccg.unam.mx/) is the primary reference database offering curated knowledge of the transcriptional regulatory network of Escherichia coli K12, currently the best-known electronically encoded database of the genetic regulatory network of any free-living organism. This paper summarizes the improvements, new biology and new features available in version 6.0. Curation of original literature is, from now on, up to date for every new release. All the objects are supported by their corresponding evidences, now classified as strong or weak. Transcription factors are classified by origin of their effectors and by gene ontology class. We have now computational predictions for sigma(54) and five different promoter types of the sigma(70) family, as well as their corresponding -10 and -35 boxes. In addition to those curated from the literature, we added about 300 experimentally mapped promoters coming from our own high-throughput mapping efforts. RegulonDB v.6.0 now expands beyond transcription initiation, including RNA regulatory elements, specifically riboswitches, attenuators and small RNAs, with their known associated targets. The data can be accessed through overviews of correlations about gene regulation. RegulonDB associated original literature, together with more than 4000 curation notes, can now be searched with the Textpresso text mining engine.

A 5' RNA element promotes dengue virus RNA synthesis on a circular genome.

The mechanisms of RNA replication of plus-strand RNA viruses are still unclear. Here, we identified the first promoter element for RNA synthesis described in a flavivirus. Using dengue virus as a model, we found that the viral RdRp discriminates the viral RNA by specific recognition of a 5' element named SLA. We demonstrated that RNA-RNA interactions between 5' and 3' end sequences of the viral genome enhance dengue virus RNA synthesis only in the presence of an intact SLA. We propose a novel mechanism for minus-strand RNA synthesis in which the viral polymerase binds SLA at the 5' end of the genome and reaches the site of initiation at the 3' end via long-range RNA-RNA interactions. These findings provide an explanation for the strict requirement of dengue virus genome cyclization during viral replication.

Dynamic behaviour of the B12 riboswitch.

Riboswitches are RNA segments that serve as ligand-responsive genetic control elements. They modulate the expression of certain genes in response to changing concentrations of metabolites. In this paper, we study the dynamic behaviour of the B12 riboswitch in E. coli--perhaps the most widely studied and best known of all riboswitches--through a mathematical model of its regulatory pathway. To carry this out, we simulate dynamic experiments in which the bacterial B12 uptake capacity is measured after being depleted of this vitamin for a long time. The results of these simulations compare favourably with reported experimental data. The model also predicts that an overshoot of intracellular B12 should be observed if the replenishment experiments were to be carried out for longer times. This behaviour is discussed in terms of a possible evolutionary advantage for E. coli, together with the fact that regulation at the transcriptional and translational levels is almost equivalent dynamically.