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In metazoan, regulatory molecules tightly control gene expression. Among them, microRNAs (miRNAs) are key regulators of several important features, like cell proliferation, differentiation, and homeostasis. During miRNA biogenesis, the canonical strand that loads onto RISC can be switched, in a process called “arm switching.” Due to the miRNA-to-target pairing peculiarities, switching events can lead to changes on the gene-targeted repertoire, promoting the modulation of a distinct set of biological routes. To understand how these events affect cell regulation, we conducted an extensive and detailed in silico analysis of RNA-seq datasets from several tissues and key developmental stages of zebrafish. We identified interesting patterns of miRNA arm switching occurrence, mainly associated with the control of protein coding genes during embryonic development. Additionally, our data show that miRNA isoforms (isomiRs) play an important role in differential arm usage. Our findings provide new insights on how such events emerge and coordinate gene expression regulation, opening perspectives for novel investigations in the area.

MicroRNAs (miRNAs) are small noncoding RNAs that are recognized as posttranscriptional regulators of gene expression. These molecules have been shown to play important roles in several cellular processes. MiRNAs act on their target by guiding the RISC complex and binding to the mRNA molecule. Thus, it is recognized that the function of a miRNA is determined by the function of its target (s). By using high-throughput methodologies, novel miRNAs are being identified, but their functions remain uncharted. Target validation is crucial to properly understand the specific role of a miRNA in a cellular pathway. However, molecular techniques for experimental validation of miRNA-target interaction are expensive, time-consuming, laborious, and can be not accurate in inferring true interactions. Thus, accurate miRNA target predictions are helpful to understand the functions of miRNAs. There are several algorithms proposed for target prediction and databases containing miRNA-target information. However, these available computational tools for prediction still generate a large number of false positives and fail to detect a considerable number of true targets, which indicates the necessity of highly confident approaches to identify bona fide miRNA-target interactions. This chapter focuses on tools and strategies used for miRNA target prediction, by providing practical insights and outlooks.

MicroRNAs (miRNAs) are small noncoding RNAs that are recognized as posttranscriptional regulators of gene expression. These molecules have been shown to play important roles in several cellular processes. MiRNAs act on their target by guiding the RISC complex and binding to the mRNA molecule. Thus, it is recognized that the function of a miRNA is determined by the function of its target (s). By using high-throughput methodologies, novel miRNAs are being identified, but their functions remain uncharted. Target validation is crucial to properly understand the specific role of a miRNA in a cellular pathway. However, molecular techniques for experimental validation of miRNA-target interaction are expensive, time-consuming, laborious, and can be not accurate in inferring true interactions. Thus, accurate miRNA target predictions are helpful to understand the functions of miRNAs. There are several algorithms proposed for target prediction and databases containing miRNA-target information. However, these available computational tools for prediction still generate a large number of false positives and fail to detect a considerable number of true targets, which indicates the necessity of highly confident approaches to identify bona fide miRNA-target interactions. This chapter focuses on tools and strategies used for miRNA target prediction, by providing practical insights and outlooks.

Made available in DSpace on 2021-06-25T10:55:23Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Background: During vertebrate evolution, the heart has undergone remarkable changes that lead to morphophysiological differences in the fully formed heart of these species, such as chamber septation, heart rate frequency, blood pressure, and cardiac output volume. Despite these differences, the heart developmental process is guided by a core gene set conserved across vertebrates. Nonetheless, the regulatory mechanisms controlling the expression of genes involved in heart development and maintenance are largely uncharted. MicroRNAs (miRNAs) have been described as important regulatory elements in several biological processes, including heart biology. These small RNA molecules are broadly conserved in sequence and genomic context in metazoans. Mutations may occur in miRNAs and/or genes that contribute to the establishment of distinct repertoires of miRNA-target interactions, thereby favoring the differential control of gene expression and, consequently, the origin of novel phenotypes. In fact, several studies showed that miRNAs are integrated into genetic regulatory networks (GRNs) governing specific developmental programs and diseases. However, studies integrating miRNAs in vertebrate heart GRNs under an evolutionary perspective are still scarce. Results: We comprehensively examined and compared the heart miRNome of 20 species representatives of the five major vertebrate groups. We found 54 miRNA families with conserved expression and a variable number of miRNA families with group-specific expression in fishes, amphibians, reptiles, birds, and mammals. We also detected that conserved miRNAs present higher expression levels and a higher number of targets, whereas the group-specific miRNAs present lower expression levels and few targets. Conclusions: Both the conserved and group-specific miRNAs can be considered modulators orchestrating the core and peripheral genes of heart GRNs of vertebrates, which can be related to the morphophysiological differences and similarities existing in the heart of distinct vertebrate groups. We propose a hypothesis to explain evolutionary differences in the putative functional roles of miRNAs in the heart GRNs analyzed. Furthermore, we present new insights into the molecular mechanisms that could be helping modulate the diversity of morphophysiology in the heart organ of vertebrate species. Laboratório Especial de Toxinologia Aplicada (LETA) CeTICS Instituto Butantan Department of Chemical and Biological Sciences Institute of Biosciences of Botucatu São Paulo State University (UNESP) Department of Biophysics and Pharmacology Institute of Biosciences of Botucatu São Paulo State University (UNESP) Department of Biological Sciences Vanderbilt University Department of Molecular Biosciences The Wenner-Gren Institute (MBW) Stockholm University Department of Chemical and Biological Sciences Institute of Biosciences of Botucatu São Paulo State University (UNESP) Department of Biophysics and Pharmacology Institute of Biosciences of Botucatu São Paulo State University (UNESP) FAPESP: 2012/15589-7 FAPESP: 2013/06864-7

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Made available in DSpace on 2022-04-29T08:36:39Z (GMT). No. of bitstreams: 0 Previous issue date: 2021-11-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Nile tilapia (Oreochromis niloticus) is a species of worldwide importance for aquaculture. A crossbred lineage was developed through introgressive backcross breeding techniques and combines the high growth performance of the Chitralada (CHIT) lwith attractive reddish color of the Red Stirling (REDS) strains. Since the crossbreed has an unknown genetically improved background, the objective of this work was to characterize expression signatures that portray the advantageous phenotype of the crossbreeds. We characterized the microRNA transcriptome by high throughput sequencing (RNA-seq) and the proteome through mass spectrometry (ESI-Q-TOF-MS) and applied bioinformatics for the comparative analysis of such molecular data on the three strains. Crossbreed expressed a distinct set of miRNAs and proteins compared to the parents. They comprised several microRNAs regulate traits of economic interest. Proteomic profiles revealed differences between parental and crossbreed in expression of proteins associated with glycolisis. Distinctive miRNA and protein signatures contribute to the phenotype of crossbreed. Department of Chemical and Biological Sciences Institute of Biosciences of Botucatu São Paulo State University (UNESP) Department of Biophysics and Pharmacology Institute of Biosciences of Botucatu São Paulo State University (UNESP) Division Molecular Biotechnology and Functional Genomics Technical University of Applied Sciences Wildau Center for the Study of Venoms and Venomous Animals (CEVAP) São Paulo State University (UNESP) Department of Structural and Functional Biology Institute of Biosciences of Botucatu São Paulo State University (UNESP) Unit of Biotechnology University of Mogi das Cruzes Department of Animal Sciences Federal University of Lavras Department of Chemical and Biological Sciences Institute of Biosciences of Botucatu São Paulo State University (UNESP) Department of Biophysics and Pharmacology Institute of Biosciences of Botucatu São Paulo State University (UNESP) Center for the Study of Venoms and Venomous Animals (CEVAP) São Paulo State University (UNESP) Department of Structural and Functional Biology Institute of Biosciences of Botucatu São Paulo State University (UNESP)

Nile tilapia is the third most cultivated fish worldwide and a novel model species for evolutionary studies. Aiming to improve productivity and contribute to the selection of traits of economic impact, biotechnological approaches have been intensively applied to species enhancement. In this sense, recent studies have focused on the multiple roles played by microRNAs (miRNAs) in the post-transcriptional regulation of protein-coding genes involved in the emergence of phenotypes with relevance for aquaculture. However, there is still a growing demand for a reference resource dedicated to integrating Nile Tilapia miRNA information, obtained from both experimental and in silico approaches, and facilitating the analysis and interpretation of RNA sequencing data. Here, we present an open repository dedicated to Nile Tilapia miRNAs: the &ldquo, miRTil database&rdquo, The database stores data on 734 mature miRNAs identified in 11 distinct tissues and five key developmental stages. The database provides detailed information about miRNA structure, genomic context, predicted targets, expression profiles, and relative 5p/3p arm usage. Additionally, miRTil also includes a comprehensive pre-computed miRNA-target interaction network containing 4936 targets and 19,580 interactions.

Made available in DSpace on 2020-12-12T02:09:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2019-09-18 MicroRNAs (miRNAs) are non-coding RNAs that regulate a wide range of biological pathways by post-transcriptionally modulating gene expression levels. Given that even a single miRNA may simultaneously control several genes enrolled in multiple biological functions, one would expect that these tiny RNAs have the ability to properly sort among distinctive cellular processes to drive protein production. To test this hypothesis, we scrutinized previously published microarray datasets and clustered protein-coding gene expression profiles according to the intensity of fold-change levels caused by the exogenous transfection of 10 miRNAs (miR-1, miR-7, miR-9, miR-124, miR-128a, miR-132, miR-133a, miR-142, miR-148b, miR-181a) in a human cell line. Through an in silico functional enrichment analysis, we discovered non-randomic regulatory patterns, proper of each cluster identified. We demonstrated that miRNAs are capable of equivalently modulate the expression signatures of target genes in regulatory clusters according to the biological function they are assigned to. Moreover, target prediction analysis applied to ten vertebrate species, suggest that such miRNA regulatory modus operandi is evolutionarily conserved within vertebrates. Overall, we discovered a complex regulatory cluster-module strategy driven by miRNAs, which relies on the controlled intensity of the repression over distinct targets under specific biological contexts. Our discovery helps to clarify the mechanisms underlying the functional activity of miRNAs and makes it easier to take the fastest and most accurate path in the search for the functions of miRNAs in any distinct biological process of interest. Institute of Biosciences of Botucatu Department of Genetics Sao Paulo State University (UNESP) Department of Physics and Biophysics Sao Paulo State University (UNESP) Laboratory of Structural Genomics (GenEstrut) Technology Developmental Center Graduate Program of Biotechnology Federal University of Pelotas Institute of Biosciences of Botucatu Department of Genetics Sao Paulo State University (UNESP) Department of Physics and Biophysics Sao Paulo State University (UNESP)

Germination of the coffee (Coffea arabica L.) seed is the result of events that occur simultaneously in the embryo and endosperm. To understand the molecular mechanisms responsible for these events, we undertook a transcriptome analysis of embryo, micropylar and lateral endosperms from 10-day-imbibed seeds. The sequencing yielded contigs coding for 16,813 proteins. From those, 14,005 (~ 83%) were highly similar to at least one protein sequence in the nr database. 162 genes were significantly expressed in the embryo, 36 in the micropylar endosperm and 72 in the lateral endosperm. The tissue specificity analysis of the significantly expressed genes showed that the embryo had the highest proportion of specific genes (113/162, ~70%), while 11 were expressed in the micropylar and lateral endosperms. In the embryo, genes were mainly associated with abiotic stress, cell growth, and intercellular communication. In the micropylar and lateral endosperms, they were associated with abiotic stress and cell wall degradation. The accuracy of RNA-seq data was confirmed by RT-qPCR. This work adds new information about the molecular mechanism involved in coffee seed germination. Resumo: A germinação da semente do café (Coffea arabica L.) é resultado de eventos que ocorrem simultaneamente no embrião e endosperma. Para entender os mecanismos moleculares responsáveis por estes eventos, nós realizamos uma análise transcricional do embrião e dos endospermas micropilar e lateral de sementes embebidas por 10 dias. O sequenciamento obteve contigs que codificam 16.813 proteínas. Destas, 14.005 (~ 83%) foram altamente similares a pelo menos uma sequência de proteína na base de dados nr. 162 genes foram significativamente expressos em embrião, 36 em endosperma micropilar e 72 em endosperma lateral. A análise de tecido-especificidade dos genes significativamente expressos mostrou que a maior proporção de genes específicos está no embrião (113/162, ~70%), enquanto 11 genes foram expressos nos endospermas lateral e micropilar. No embrião, os genes foram associados principalmente com estresse abiótico, crescimento celular e comunicação intercelular. Nos endospermas micropilar e lateral, eles foram associados com estresse abiótico e degradação da parede celular. A acurácia da análise de RNA-seq foi confirmada por RT-qPCR. Este trabalho acrescenta novas informações sobre os mecanismos moleculares envolvidos na germinação de sementes de café.

Made available in DSpace on 2018-12-11T17:20:34Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-12-01 MicroRNAs (miRNAs) are key regulators of gene expression in multicellular organisms. The elucidation of miRNA function and evolution depends on the identification and characterization of miRNA repertoire of strategic organisms, as the fast-evolving cichlid fishes. Using RNA-seq and comparative genomics we carried out an in-depth report of miRNAs in Nile tilapia (Oreochromis niloticus), an emergent model organism to investigate evo-devo mechanisms. Five hundred known miRNAs and almost one hundred putative novel vertebrate miRNAs have been identified, many of which seem to be teleost-specific, cichlid-specific or tilapia-specific. Abundant miRNA isoforms (isomiRs) were identified with modifications in both 5p and 3p miRNA transcripts. Changes in arm usage (arm switching) of nine miRNAs were detected in early development, adult stage and even between male and female samples. We found an increasing complexity of miRNA expression during ontogenetic development, revealing a remarkable synchronism between the rate of new miRNAs recruitment and morphological changes. Overall, our results enlarge vertebrate miRNA collection and reveal a notable differential ratio of miRNA arms and isoforms influenced by sex and developmental life stage, providing a better picture of the evolutionary and spatiotemporal dynamics of miRNAs. Department of Genetics Institute of Biosciences of Botucatu Sao Paulo State University (UNESP) Department of Physics and Biophysics Institute of Biosciences of Botucatu Sao Paulo State University (UNESP) School of Biological Sciences University of East Anglia (UEA), Norwich Research Park Department of Clinical and Molecular Medicine Norwegian University of Science and Technology (NTNU) Stevenson Center Vanderbilt University Unit of Biotechnology University of Mogi das Cruzes Department of Morphology Institute of Biosciences of Botucatu Sao Paulo State University (UNESP) Department of Genetics Institute of Biosciences of Botucatu Sao Paulo State University (UNESP) Department of Physics and Biophysics Institute of Biosciences of Botucatu Sao Paulo State University (UNESP) Department of Morphology Institute of Biosciences of Botucatu Sao Paulo State University (UNESP)

Made available in DSpace on 2018-12-11T17:12:20Z (GMT). No. of bitstreams: 0 Previous issue date: 2017-05-09 Target prediction is generally the first step toward recognition of bona fide microRNA (miRNA)-target interactions in living cells. Several target prediction tools are now available, which use distinct criteria and stringency to provide the best set of candidate targets for a single miRNA or a subset of miRNAs. However, there are many false-negative predictions, and consensus about the optimum strategy to select and use the output information provided by the target prediction tools is lacking. We compared the performance of four tools cited in literature-TargetScan (TS), miRanda-mirSVR (MR), Pita, and RNA22 (R22), and we determined the most effective approach for analyzing target prediction data (individual, union, or intersection). For this purpose, we calculated the sensitivity, specificity, precision, and correlation of these approaches using 10 miRNAs (miR-1-3p, miR-17-5p, miR-21-5p, miR-24-3p, miR-29a-3p, miR-34a-5p, miR-124-3p, miR-125b-5p, miR-145-5p, and miR-155-5p) and 1,400 genes (700 validated and 700 non-validated) as targets of these miRNAs. The four tools provided a subset of high-quality predictions and returned few false-positive predictions; however, they could not identify several known true targets. We demonstrate that union of TS/MR and TS/MR/R22 enhanced the quality of in silico prediction analysis of miRNA targets. We conclude that the union rather than the intersection of the aforementioned tools is the best strategy for maximizing performance while minimizing the loss of time and resources in subsequent in vivo and in vitro experiments for functional validation of miRNA-target interactions. Laboratory of Genomics and Molecular Evolution Department of Genetics Institute of Biosciences of Botucatu São Paulo State Univesity (UNESP) Laboratory of Bioinformatics and Computational Biophysics Department of Physics and Biophysics Institute of Biosciences of Botucatu São Paulo State Univesity (UNESP) Laboratory of Genomics and Molecular Evolution Department of Genetics Institute of Biosciences of Botucatu São Paulo State Univesity (UNESP) Laboratory of Bioinformatics and Computational Biophysics Department of Physics and Biophysics Institute of Biosciences of Botucatu São Paulo State Univesity (UNESP)