Extraction of Transcript Diversity from Scientific Literature

Transcript diversity generated by alternative splicing and associated mechanisms contributes heavily to the functional complexity of biological systems. The numerous examples of the mechanisms and functional implications of these events are scattered throughout the scientific literature. Thus, it is crucial to have a tool that can automatically extract the relevant facts and collect them in a knowledge base that can aid the interpretation of data from high-throughput methods. We have developed and applied a composite text-mining method for extracting information on transcript diversity from the entire MEDLINE database in order to create a database of genes with alternative transcripts. It contains information on tissue specificity, number of isoforms, causative mechanisms, functional implications, and experimental methods used for detection. We have mined this resource to identify 959 instances of tissue-specific splicing. Our results in combination with those from EST-based methods suggest that alternative splicing is the preferred mechanism for generating transcript diversity in the nervous system. We provide new annotations for 1,860 genes with the potential for generating transcript diversity. We assign the MeSH term “alternative splicing” to 1,536 additional abstracts in the MEDLINE database and suggest new MeSH terms for other events. We have successfully extracted information about transcript diversity and semiautomatically generated a database, LSAT, that can provide a quantitative understanding of the mechanisms behind tissue-specific gene expression. LSAT (Literature Support for Alternative Transcripts) is publicly available at http://www.bork.embl.de/LSAT/

Cu,Zn superoxide dismutase genes in Tribolium castaneum: evolution, molecular characterisation and gene expression during immune priming.

The production of reactive oxygen species (ROS) is a normal consequence of the aerobic cell metabolism. Despite their high and potentially detrimental reactivity with various biomolecules, the endogenous production of ROS is a vital part of physiological, immunological, and molecular processes that contribute to fitness. The role of ROS in host\u2013parasite interactions is frequently defined by their contribution to innate immunity as effectors, promoting parasite death during infections. In vertebrates, ROS and antioxidant system enzymes, such as superoxide dismutase (SOD) are also involved in acquired immune memory, where they are responsible for T-cell signalling, activation, proliferation, and viability. Based on recent findings, ROS are now also assumed to play a role in immune priming, i.e., a form of memory in invertebrates. In this study, the potential involvement of Cu,Zn SODs in immunity of the red flour beetle Tribolium castaneum is described for the first time, applying an approach that combines an in\ua0silico gene characterisation with an in\ua0vivo immune priming experiment using the Gram-positive entomopathogen Bacillus thuringiensis. We identified an unusually high number of three different transcripts for extracellular SOD and found that priming leads to a fine-tuned modulation of SOD expression, highlighting the potential of physiological co-adaptations for immune phenotypes

Ab Initio Identification of Novel Regulatory Elements in the Genome of Trypanosoma brucei by Bayesian Inference on Sequence Segmentation

Background: The rapid increase in the availability of genome information has created considerable demand for both comparative and ab initio predictive bioinformatic analyses. The biology laid bare in the genomes of many organisms is often novel, presenting new challenges for bioinformatic interrogation. A paradigm for this is the collected genomes of the kinetoplastid parasites, a group which includes Trypanosoma brucei the causative agent of human African trypanosomiasis. These genomes, though outwardly simple in organisation and gene content, have historically challenged many theories for gene expression regulation in eukaryotes. Methodology/Principle Findings: Here we utilise a Bayesian approach to identify local changes in nucleotide composition in the genome of T. brucei. We show that there are several elements which are found at the starts and ends of multicopy gene arrays and that there are compositional elements that are common to all intergenic regions. We also show that there is a composition-inversion element that occurs at the position of the trans-splice site. Conclusions/Significance: The nature of the elements discovered reinforces the hypothesis that context dependant RN

Co-expressed mitochondrial genomes: recently masculinized, recombinant mitochondrial genome is co-expressed with the female – transmitted mtDNA genome in a male Mytilus trossulus mussel from the Baltic Sea

BACKGROUND: Few exceptions have been described from strict maternal inheritance of mitochondrial DNA in animals, including sea mussels (Mytilidae), clams (Donacidae, Veneridae and Solenidae) and freshwater mussels (Unionoidae) order. In these bivalves mitochondria and their DNA are transferred through two separate routes. The females inherit only the maternal mitochondrial DNA whereas the males inherit maternal as well as paternal mitochondrial DNA, which is usually present only in gonads and sperm. The mechanism controlling this phenomenon is unclear but leads to the existence of two separate mitochondrial DNA lineages in a single species. The lineages are usually well differentiated: up to 20-50% divergence in nucleotide sequence. Occasionally, a maternal mitochondrial DNA can invade the paternal transmission route, eventually replacing the diverged M-type and lowering the divergence. Such role reversal (masculinization) event has happened recently in the Mytilus population of the Baltic Sea which consists of M. edulis × M. trossulus hybrids, but the functional status of the resulting mitochondrial genome was unknown. RESULTS: In this paper we sequenced transcripts from one specimen that was identified as male carrying both the female mitochondrial genome and a recently masculinized mitochondrial genome. Additionally, the analysis of the control region has showed that the recently masculinized, recombinant genome, not only has an M-type control region and all coding regions derived from the F-type, but also is transcriptionally active along side the maternally inherited F-type genome. In the comparative analysis, the two genomes exhibit different substitution patterns, typical for the M vs. F genome comparisons. The genetic distances and ratios of non-synonymous substitutions also suggest that one of the genomes is transitioning from the maternal to the paternal inheritance mode, consistent with its recent masculinization. CONCLUSION: We have shown, for the first time, that the recently masculinized mitochondrial genome is active and that it accumulates excess of non-synonymous substitutions across its coding sequence. This suggests, that, under certain cytonuclear incompatibility conditions, masculinization may serve to restore the endangered functionality of the paternally inherited genome. This is also another example of a mitochondrial genome in which the recombination in the control region predated its transition from paternal to maternal transmission route

Cu,Zn Superoxide Dismutases from Tetrahymena thermophila: Molecular Evolution and Gene Expression of the First Line of Antioxidant Defenses

In the present study, we describe the molecular and functional characterization of two Cu,Zn superoxide dismutase (SOD) genes, named tt-sod1a and tt-sod1b from Tetrahymena thermophila, a free-living ciliated protozoan widely used as model organism in biological research. The cDNAs and the putative amino acid sequences were compared with Cu,Zn SODs from other Alveolata. The primary sequences of T. thermophila Cu,Zn SODs are unusually long if compared to orthologous proteins, but the catalytically important residues are almost fully conserved. Both phylogenetic and preliminary homology modeling analyses provide some indications about the evolutionary relationships between the Cu,Zn SODs of Tetrahymena and the Alveolata orthologous enzymes. Copper-dependent regulation of Cu,Zn SODs expression was investigated by measuring mRNA accumulation and enzyme activity in response to chronic exposure to non-toxic doses of the metal. Our in silico analyses of the tt-sod1a and tt-sod1b promoter regions revealed putative consensus sequences similar to half Antioxidant Responsive Elements (hARE), suggesting that the transcription of these genes directly depends on ROS formation. These data emphasize the importance of complex metal regulation of tt-sod1a and tt-sod1b activation, as components of an efficient detoxification pathway allowing the survival of T. thermophila in continued, elevated presence of metals in the environment

Characterization of 5´ and 3´ UTRs from Bone morphogenetic protein receptor type 1A (Bmpr1A) transcripts under the context of bone metabolism

During our life our bones are in a constant balance between bone formation by osteoblasts and bone resorption by osteoclasts. Bone morphogenetic proteins are a group of cytokines from the transforming growth factor-β family involved in several processes. Their signal is transduced through two types of serine/threonine kinase receptors, BMPRI and BMPRII. BMPRIA is a type one receptor involved in osteoblast/osteoclast communication and therefore affecting bone metabolism. Upon binding of different ligands, type II phosphorylates the type I and activates one of the signal pathways. Although their function is known, the mechanisms of regulation of expression are still unclear. The objective of this work was to characterize some of the Mus musculus Bmpr1a molecular regulatory mechanisms including upstream open reading frames (uORFs), polyadenylation sites, microRNA binding sites and constitutive decay elements (CDE). Bioinformatically, we were able to identify 11 Mus musculus Bmpr1a transcripts on available databases, 11 polyadenylations and a constitutive decay element on the 3’UTR, while on 5’UTR we were able to identify 3 uORFs. Experimentally, we isolated a new and shorter Bmpr1a 3’UTR with an alternative polyadenylation site from MC3T3-E1 but without the CDE We tried to isolate different fragments from different tissues, but we were capable of isolate a longer transcript only from mouse liver that contains the CDE loop. Within the 5’UTRs we isolated 4 different fragments containing 2 or 3 uORFs and insert them on reporter plasmids for functional analysis. The last step was mutating all the AUG from uORFs and validating their effect on regulation of luciferase expression. We measured a decrease in the expression of luciferase on fragments containing no mutations and an increase in the expression of fragments with mutations, except in one case where the mutation was inserted 7bp after the start of the fragment. Results indicate that there were no different transcripts from proliferating and differentiated cells, and in the 5’ UTR , the uORFs could possibly contribute to regulate the expression of Bmpr1a. However further studies are needed to confirm and expand our data.As Bone morphogenetic proteins (BMP) são um grupo de citocinas pertencentes à família de proteínas do Transforming growth factor β e estão envolvidas em vários processos celulares importantes como o regulamento da divisão celular e de diferenciação de células. Estas proteínas transmitem o sinal para dentro das células através de dois tipos de recetores de serina/treonina diferentes, os Bone morphogenetic proteins receptors (BMPR) tipo I e tipo II. Estes recetores formam homo e hétero dímeros entre si para formar um recetor totalmente funcional; quando isso acontece e após a ligação de uma molécula aos receptores, o tipo II fosforila o tipo I levando a uma cascata de fosforilação no interior da célula podendo ativar diferentes vias se sinalização dependendo da molécula que se liga ao recetor e qual os dímeros de recetores que se formaram. Alguns dos exemplos de vias de sinalização que se ativam aquando da ligação de moléculas a estes recetores são as vias Smad, MAPK, PI3K/Akt, Wnt ou ERK1/2 que são importantes reguladores da expressão de genes e que atuam em processos celulares importantes como diferenciação celular e formação de tecidos. As BMPRs estão envolvidas também nos processos de metabolismo do osso associados à deposição de hidroxiapatite pelos osteoblastos e reabsorção óssea pelos osteoclastos que degradam a matriz extracelular. Apesar de se conhecer a função da BMPR1A, nem todos os mecanismos de regulação pós-transcrição da sua expressão estão estudados. O objetivo deste trabalho é contribuir para a caracterização dos mecanismos de regulação dos diferentes transcritos de Bmpr1a, e determinar quais os elementos regulatórios pós-transcrição presentes nas sequências identificadas, tanto na região 5’ untraslated region (UTR), como na região 3’UTR. Começou-se por identificar os diferentes transcritos de bmpr1a sendo possível identificar 11 diferentes transcritos de Bmpr1a de mus musculus (ratinho) e 5 diferentes transcritos de BMPR1A de homo sapiens (humano) em diferentes bases de dados. Procurou-se também identificar sequencias associadas a locais de poliadenilação sendo que no ratinho foi possível encontrar 11 diferentes locais de poliadenilação, dos quais 6 contêm a sequencia canónica de poliadenilação, enquanto os outros 5 foram obtidos através do uso de ferramentas bioinformáticas como o RegRNA2.0. Nos transcritos de homo sapiens identificou se 8 locais de poliadenilação. De seguida identificou se a presença de um loop de degradação constitutivo (Constitutive Decay Element - CDE). Este loop está presente em 8 das 11 sequencias de ratinho identificadas enquanto no humano está presente em todos os transcritos. Verificou-se igualmente a conservação deste loop em 24 espécies diferentes e foi possível verificar que se encontra totalmente conservado em 18 dessas 24 espécies e em 22 delas contem a sequencia necessária para que seja funcional. Outro dos elementos regulatórios que se identificou foram locais ricos em Adeninas/Uracilos que são associados a um aumento de afinidade de ligação de proteínas reguladoras. Foi possível identificar a sequencia mínima para a ligação dessas proteínas, mas não foi possível encontrar um dos 5 diferentes conjuntos de sequências que estão descritas na literatura. Por último construiu-se uma árvore filogenética da região 3’UTR dos transcritos de Bmpr1a para perceber as relações filogenéticas desta região não codificante entre diferentes espécies de vertebrados. Na região 5’ identificou-se a presença de upstream Open Reading frames (uORFs) tendo sido verificada a presença de 3 uORFs conservadas na maior parte dos transcritos analisados e conservados em várias espécies de mamíferos exceto nos marsupiais. Experimentalmente, foi possível isolar um transcrito da região 3’UTR de Bmpr1a de ratinho com 250 pares de bases tanto em células em proliferação como em células diferenciadas em osteoblastos. Este transcrito contém um local de poliadenilação alternativo antes da cauda de poliadeninas e não contém o loop de degradação constitutivo. Foi-se então tentar isolar transcritos de outros tecidos de ratinho. Começou se por tentar isolar transcritos de Bmpr1a em tecidos de joelho e de articulações não sendo possível isolar transcritos de outros tamanhos além do transcrito de 250 pares de bases acima indicado. No entanto foi possível identificar a partir de fígado um transcrito com 1500 pares de base o qual já contem o loop de degradação constitutivo. Na região 5’ isolamos 4 fragmentos diferentes contendo 2 ou 3 das uORFs e com diferentes distâncias entre estas e o início dos fragmentos. Esses 4 fragmentos foram de seguida inseridos com a orientação correta num vetor de expressão para a luciferase e transfectados em células Hek293 (do inglês Human Embrionic Kidney) e os valores de luminescência registados. Observou-se nos resultados preliminares uma diminuição da expressão da luciferase associada à presença das uORFs dos transcritos sem mutações. Apos a mutação dos AUGs para AAG por SDM verificou-se que no fragmento mais longo (fragmento A) parece haver uma tendência para o aumento da luciferase ao longo das mutações sequenciais dos AUG, confirmado pelas mutações individuais. No segundo fragmento (fragmento B) analisado ocorre um aumento da expressão da luciferase quando se mutou o AUG da uORF1 em comparação com o controlo, contudo na mutação da uORF2 ocorre uma diminuição dramática da expressão da luciferase que é confirmada pela mutação isolada da uORF. Uma das possíveis explicações para esta diminuição da expressão da luciferase é a localização desta uORF no fragmento, pois esta uORF está localizada no início do fragmento, a 7 pares de base do início da mesma, podendo levar a uma destabilização deste fragmento e consequentemente levando ao resultado observado, ou seja, à diminuição da expressão da luciferase. No último fragmento (fragmento D) que não contem a uORF2 é possível observar uma diminuição da expressão da luciferase no fragmento sem nenhuma mutação dos AUG, enquanto um aumento da expressão da luciferase nos fragmentos que contem as mutações sequenciais. Sendo que apenas duas experiências foram efetuadas com este fragmento, mais estudos são necessários para determinar se a ausência da uORF2 tem algum efeito na regulação da expressão deste fragmento Podemos então com este trabalho concluir que na região 3’UTR se encontra um loop de degradação constitutivo que está bastante conservado, podendo assim significar que aquela região pode ser importante para a regulação da expressão do gene. Contudo experimentalmente não foi possível isolar das células MC3T3-E1 um fragmento dessa região, podendo ser devido à necessidade destas células de ratinho expressarem esta proteína que está envolvida na comunicação entre osteoblastos e osteoclastos. Experimentalmente podemos também verificar que existem na região 5’UTR do Bmpr1a 3 uORFs que podem, portanto, vir a ter efeito na regulação da expressão do gene, mas mais estudos precisam de ser feitos para confirmar ou não esta atividade

Sequencing and Analysis of Full-Length cDNAs, 5′-ESTs and 3′-ESTs from a Cartilaginous Fish, the Elephant Shark (Callorhinchus milii)

10.1371/journal.pone.0047174PLoS ONE710

A comprehensive analysis of 3' end sequencing data sets reveals novel polyadenylation signals and the repressive role of heterogeneous ribonucleoprotein C on cleavage and polyadenylation

Alternative polyadenylation (APA) is a general mechanism of transcript diversification in mammals, which has been recently linked to proliferative states and cancer. Different 3' untranslated region (3' UTR) isoforms interact with different RNA-binding proteins (RBPs), which modify the stability, translation, and subcellular localization of the corresponding transcripts. Although the heterogeneity of pre-mRNA 3' end processing has been established with high-throughput approaches, the mechanisms that underlie systematic changes in 3' UTR lengths remain to be characterized. Through a uniform analysis of a large number of 3' end sequencing data sets, we have uncovered 18 signals, six of which are novel, whose positioning with respect to pre-mRNA cleavage sites indicates a role in pre-mRNA 3' end processing in both mouse and human. With 3' end sequencing we have demonstrated that the heterogeneous ribonucleoprotein C (HNRNPC), which binds the poly(U) motif whose frequency also peaks in the vicinity of polyadenylation (poly(A)) sites, has a genome-wide effect on poly(A) site usage. HNRNPC-regulated 3' UTRs are enriched in ELAV-like RBP 1 (ELAVL1) binding sites and include those of the CD47 gene, which participate in the recently discovered mechanism of 3' UTR-dependent protein localization (UDPL). Our study thus establishes an up-to-date, high-confidence catalog of 3' end processing sites and poly(A) signals, and it uncovers an important role of HNRNPC in regulating 3' end processing. It further suggests that U-rich elements mediate interactions with multiple RBPs that regulate different stages in a transcript's life cycle