Transcritos quiméricos em bovinos.

Trans-splicing é um fenômeno no qual exons dos transcritos primários de genes distintos se unem para formar um novo e único transcrito quimérico, sendo comum em nematóides e kinetoplastideos, mas raro em organismos superiores. Embora não seja tão bem compreendido quanto o cis-splicing, o trans-splicing tem despertado interesse devido às suas possíveis aplicações biotecnológicas, principalmente aquelas envolvendo regulação da expressão gênica ou formação de novas proteínas. Recentemente, desenvolvemos uma metodologia de Bioinformática para a detecção de transcritos quiméricos em bases de FlcDNA (Herai & Yamagishi, 2010a), e estendemos essa metodologia para organismos cujo genoma não tenha uma alta qualidade de montagem (Herai & Yamagishi, 2010b). Como prova de conceito, aplicamos essa metodologia ao genoma do Bos taurus UMD 3.0 e identificamos 13 transcritos quiméricos candidatos a trans-splicing. O foco da pesquisa é a confirmação biológica do trans-splicing em bovinos, particularmente, nas fases embrionária e fetal onde, a formação de transcritos quiméricos pode ter função biológica importante. Se confirmados, esses seriam os primeiros casos biologicamente comprovados de trans-splicing em bovinos, pois o único caso até agora reportado (Roux et al., 2006) envolve dois genes muito próximos, o que pode ser interpretado como um caso de splicing alternativo, ao invés de trans-splicing.Pôster 77

Apparent non-canonical trans-splicing is generated by reverse transcriptase in vitro

Trans-splicing, the in vivo joining of two RNA molecules, is well characterized in several groups of simple organisms but was long thought absent from fungi, plants and mammals. However, recent bioinformatic analyses of expressed sequence tag (EST) databases suggested widespread trans-splicing in mammals^1-2^. Splicing, including the characterised trans-splicing systems, involves conserved sequences at the splice junctions. Our analysis of a yeast non-coding RNA revealed that around 30% of the products of reverse transcription lacked an internal region of 117 nt, suggesting that the RNA was spliced. The junction sequences lacked canonical splice-sites but were flanked by direct repeats, and further analyses indicated that the apparent splicing actually arose because reverse transcriptase can switch templates during transcription^3^. Many newly identified, apparently trans-spliced, RNAs lacked canonical splice sites but were flanked by short regions of homology, leading us to question their authenticity. Here we report that all reported categories of non-canonical splicing could be replicated using an in vitro reverse transcription system with highly purified RNA substrates. We observed the reproducible occurrence of ostensible trans-splicing, exon shuffling and sense-antisense fusions. The latter generate apparent antisense non-coding RNAs, which are also reported to be abundant in humans^4^. Different reverse transcriptases can generate different products of template switching, providing a simple diagnostic. Many reported examples of splicing in the absence of canonical splicing signals may be artefacts of cDNA preparation

Synthetic RNA Silencing of Actinorhodin Biosynthesis in Streptomyces coelicolor A3(2)

We demonstrate the first application of synthetic RNA gene silencers in Streptomyces coelicolor A3(2). Peptide nucleic acid and expressed antisense RNA silencers successfully inhibited actinorhodin production. Synthetic RNA silencing was target-specific and is a new tool for gene regulation and metabolic engineering studies in Streptomyces.Peer reviewe

Is a Genome a Codeword of an Error-Correcting Code?

Since a genome is a discrete sequence, the elements of which belong to a set of four letters, the question as to whether or not there is an error-correcting code underlying DNA sequences is unavoidable. The most common approach to answering this question is to propose a methodology to verify the existence of such a code. However, none of the methodologies proposed so far, although quite clever, has achieved that goal. In a recent work, we showed that DNA sequences can be identified as codewords in a class of cyclic error-correcting codes known as Hamming codes. In this paper, we show that a complete intron-exon gene, and even a plasmid genome, can be identified as a Hamming code codeword as well. Although this does not constitute a definitive proof that there is an error-correcting code underlying DNA sequences, it is the first evidence in this direction

The Genome Sequence of Leishmania (Leishmania) amazonensis: Functional Annotation and Extended Analysis of Gene Models

We present the sequencing and annotation of the Leishmania (Leishmania) amazonensis genome, an etiological agent of human cutaneous leishmaniasis in the Amazon region of Brazil. L. (L.) amazonensis shares features with Leishmania (L.) mexicana but also exhibits unique characteristics regarding geographical distribution and clinical manifestations of cutaneous lesions (e.g. borderline disseminated cutaneous leishmaniasis). Predicted genes were scored for orthologous gene families and conserved domains in comparison with other human pathogenic Leishmania spp. Carboxypeptidase, aminotransferase, and 3'-nucleotidase genes and ATPase, thioredoxin, and chaperone-related domains were represented more abundantly in L. (L.) amazonensis and L. (L.) mexicana species. Phylogenetic analysis revealed that these two species share groups of amastin surface proteins unique to the genus that could be related to specific features of disease outcomes and host cell interactions. Additionally, we describe a hypothetical hybrid interactome of potentially secreted L. (L.) amazonensis proteins and host proteins under the assumption that parasite factors mimic their mammalian counterparts. the model predicts an interaction between an L. (L.) amazonensis heat-shock protein and mammalian Toll-like receptor 9, which is implicated in important immune responses such as cytokine and nitric oxide production. the analysis presented here represents valuable information for future studies of leishmaniasis pathogenicity and treatment.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, EPM UNIFESP, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilLNBio CNPEM, Lab Nacl Biociencias, Campinas, SP, BrazilLGE UNICAMP, Lab Genom & Expressao, Campinas, SP, BrazilInst Agron Campinas, Ctr Pesquisa & Desenvolvimento Recursos Geneti Ve, Campinas, SP, BrazilUniv Calif San Diego, Sch Med, Dept Pediat, San Diego, CA 92103 USAUniversidade Federal de São Paulo, UNIFESP, Dept Ciencia & Tecnol, Sao Jose Dos Campos, BrazilUniv N Carolina, Sch Med, Dept Genet, Chapel Hill, NC USAUniv Fed Minas Gerais, ICB UFMG, Inst Ciencias Biol, Dept Biol Geral, Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, EPM UNIFESP, Escola Paulista Med, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, BrazilUniversidade Federal de São Paulo, UNIFESP, Dept Ciencia & Tecnol, Sao Jose Dos Campos, BrazilFAPESP: 07/50551-2FAPESP: 10/19335-4Web of Scienc

Loss of GTF2I promotes neuronal apoptosis and synaptic reduction in human cellular models of neurodevelopment

Individuals with Williams syndrome (WS), a neurodevelopmental disorder caused by hemizygous loss of 26-28 genes at 7q11.23, characteristically portray a hypersocial phenotype. Copy-number variations and mutations in one of these genes, GTF2I, are associated with altered sociality and are proposed to underlie hypersociality in WS. However, the contribution of GTF2I to human neurodevelopment remains poorly understood. Here, human cellular models of neurodevelopment, including neural progenitors, neurons, and three-dimensional cortical organoids, are differentiated from CRISPR-Cas9-edited GTF2I-knockout (GTF2I-KO) pluripotent stem cells to investigate the role of GTF2I in human neurodevelopment. GTF2I-KO progenitors exhibit increased proliferation and cell-cycle alterations. Cortical organoids and neurons demonstrate increased cell death and synaptic dysregulation, including synaptic structural dysfunction and decreased electrophysiological activity on a multielectrode array. Our findings suggest that changes in synaptic circuit integrity may be a prominent mediator of the link between alterations in GTF2I and variation in the phenotypic expression of human sociality

Loss of GTF2I promotes neuronal apoptosis and synaptic reduction in human cellular models of neurodevelopment

Summary: Individuals with Williams syndrome (WS), a neurodevelopmental disorder caused by hemizygous loss of 26–28 genes at 7q11.23, characteristically portray a hypersocial phenotype. Copy-number variations and mutations in one of these genes, GTF2I, are associated with altered sociality and are proposed to underlie hypersociality in WS. However, the contribution of GTF2I to human neurodevelopment remains poorly understood. Here, human cellular models of neurodevelopment, including neural progenitors, neurons, and three-dimensional cortical organoids, are differentiated from CRISPR-Cas9-edited GTF2I-knockout (GTF2I-KO) pluripotent stem cells to investigate the role of GTF2I in human neurodevelopment. GTF2I-KO progenitors exhibit increased proliferation and cell-cycle alterations. Cortical organoids and neurons demonstrate increased cell death and synaptic dysregulation, including synaptic structural dysfunction and decreased electrophysiological activity on a multielectrode array. Our findings suggest that changes in synaptic circuit integrity may be a prominent mediator of the link between alterations in GTF2I and variation in the phenotypic expression of human sociality

Modeling anorexia nervosa: transcriptional insights from human iPSC-derived neurons

Anorexia nervosa (AN) is a complex and multifactorial disorder occurring predominantly in women. Despite having the highest mortality among psychiatric conditions, it still lacks robust and effective treatment. Disorders such as AN are most likely syndromes with multiple genetic contributions, however, genome-wide studies have been underpowered to reveal associations with this uncommon illness. Here, we generated induced pluripotent stem cells (iPSCs) from adolescent females with AN and unaffected controls. These iPSCs were differentiated into neural cultures and subjected to extensive transcriptome analysis. Within a small cohort of patients who presented for treatment, we identified a novel gene that appears to contribute to AN pathophysiology, TACR1 (tachykinin 1 receptor). The participation of tachykinins in a variety of biological processes and their interactions with other neurotransmitters suggest novel mechanisms for how a disrupted tachykinin system might contribute to AN symptoms. Although TACR1 has been associated with psychiatric conditions, especially anxiety disorders, we believe this report is its first association with AN. Moreover, our human iPSC approach is a proof-of-concept that AN can be modeled in vitro with a full human genetic complement, and represents a new tool for understanding the elusive molecular and cellular mechanisms underlying the disease