Transcriptional dynamics of the human DMD locus

The human DMD locus encodes dystrophin protein. Absence or reduced levels of dystrophin (DMD or BMD phenotype, respectively) lead to progressive muscle wasting. Little is known about the complex coordination of dystrophin expression and its transcriptional regulation is a field of intense interest. In this work we found that DMD locus harbours multiple long non coding RNAs which orchestrate and control transcription of muscle dystrophin mRNA isoforms. These lncRNAs are tissue-specific and highly expressed during myogenesis, suggesting a possible role in tissue-specific expression of DMD gene isoforms. Their forced ectopic expression in human muscle and neuronal cells leads to a specific and negative regulation of endogenous dystrophin full lenght isoforms. An intriguing aspect regarding the transcription of the DMD locus is the gene size (2.4Mb). The mechanism that ensures the complete synthesis of the primary transcript and the coordinated splicing of 79 exons is still completely unknown. By ChIP-on-chip analyses, we discovered novel regions never been involved before in the transcription regulation of the DMD locus. Specifically, we observed enrichments for Pol II, P-Ser2, P-Ser5, Ac-H3 and 2Me-H3K4 in an intronic region of 3Kb (approximately 21Kb) downstream of the end of DMD exon 52 and in a region of 4Kb spanning the DMD exon 62. Interestingly, this latter region and the TSS of Dp71 are strongly marked by 3Me-H3K36, an histone modification associated with the regulation of splicing process. Furthermore, we also observed strong presence of open chromatin marks (Ac-H3 and 2Me-H3K4) around intron 34 and the exon 45 without presence of RNA pol II. We speculate that these two regions may exert an enhancer-like function on Dp427m promoter, although further investigations are necessary. Finally, we investigated the nuclear-cytoplasmic compartmentalization of the muscular dystrophin mRNA and, specifically, we verified whether the exon skipping therapy could influence its cellular distribution.

Expression of the vault RNA protects cells from undergoing apoptosis

Non-protein-coding RNAs are a functionally versatile class of transcripts exerting their biological roles on the RNA level. Recently, we demonstrated that the vault complex-associated RNAs (vtRNAs) are significantly upregulated in Epstein-Barr virus (EBV)-infected human B cells. Very little is known about the function(s) of the vtRNAs or the vault complex. Here, we individually express latent EBV-encoded proteins in B cells and identify the latent membrane protein 1 (LMP1) as trigger for vtRNA upregulation. Ectopic expression of vtRNA1-1, but not of the other vtRNA paralogues, results in an improved viral establishment and reduced apoptosis, a function located in the central domain of vtRNA1-1. Knockdown of the major vault protein has no effect on these phenotypes revealing that vtRNA1-1 and not the vault complex contributes to general cell death resistance. This study describes a NF-κB-mediated role of the non-coding vtRNA1-1 in inhibiting both the extrinsic and intrinsic apoptotic pathways.

RNA regulation: a new genetics?

Do non-coding RNAs that are derived from the introns and exons of protein-coding and non-protein-coding genes represent a fundamental advance in the genetic operating system of higher organisms? Recent evidence from comparative genomics and molecular genetics indicates that this might be the case. If so, there will be profound consequences for our understanding of the genetics of these organisms, and in particular how the trajectories of differentiation and development and the differences among individuals and species are genomically programmed. But how might this hypothesis be tested?

Molecular simulations and modeling of pharmaceutically relevant RNA-processing enzymes

The two-metal-ion architecture is a structural feature found in a variety of RNA processing metalloenzymes or ribozymes (RNA-based enzymes), which control the biogenesis and the metabolism of vital RNAs, including non-coding RNAs (ncRNAs). Notably, such ncRNAs are emerging as key players for the regulation of cellular homeostasis, and their altered expression has been often linked to the development of severe human pathologies, from cancer to mental disorders. Accordingly, understanding the biological processing of ncRNAs is foundational for the development of novel therapeutic strategies and tools. Here, we use state-of the-art molecular simulations, complemented with X-ray crystallography and biochemical experiments, to characterize the RNA processing cycle as catalyzed by two two-metal-ion enzymes: the group II intron ribozymes and the RNase H1. We show that multiple and diverse cations are strategically recruited at and timely released from the enzymes’ active site during catalysis. Such a controlled cations’ trafficking leads to the recursive formation and disruption of an extended two-metal ion architecture that is functional for RNA-hydrolysis – from substrate recruitment to product release. Importantly, we found that these cations’ binding sites are conserved among other RNA-processing machineries, including the human spliceosome and CRISPR-Cas systems, suggesting that an evolutionarily-converged catalytic strategy is adopted by these enzymes to process RNA molecules. Thus, our findings corroborate and sensibly extend the current knowledge of two-metal-ion enzymes, and support the design of novel drugs targeting RNA-processing metalloenzymes or ribozymes as well as the rational engineering of novel programmable gene-therapy tools.

Mapping new non-genetic dependencies in malignant pleural mesothelioma

Malignant Pleural Mesothelioma (MPM) is a very aggressive cancer whose incidence is growing worldwide. MPM escapes the classical models of carcinogenesis and lacks a distinctive genetic fingerprint, keeping obscure the molecular events that lead to tumorigenesis. This severely impacts on the limited therapeutic options and on the lack of specific biomarkers, concurring to make MPM one of the deadliest cancers. Here we combined a functional genome-wide loss of function CRISPR/Cas9 screening with patients’ transcriptomic and clinical data, to identify genes essential for MPM progression. Besides, we explored the role of non-coding RNAs to MPM progression by analysing gene expression profiles and clinical data from the MESO-TCGA dataset. We identified TRIM28 and the lncRNA LINC00941 as new vulnerabilities of MPM, associated with disease aggressiveness and bad outcome of patients. TRIM28 is a multi-domain protein involved in many processes, including transcription regulation. We showed that TRIM28 silencing impairs MPM cells’ growth and clonogenicity by blocking cells in mitosis. RNA-seq profiling showed that TRIM28 loss abolished the expression of major mitotic players. Our data suggest that TRIM28 is part of the B-MYB/FOXM1-MuvB complex that specifically drives the activation of mitotic genes, keeping the time of mitosis. In parallel, we found LINC00941 as strongly associated with reduced survival probability in MPM patients. LINC00941 KD profoundly reduced MPM cells’ growth, migration and invasion. This is accompanied by changes in morphology, cytoskeleton organization and cell-cell adhesion properties. RNA-seq profiling showed that LINC00941 KD impacts crucial functions of MPM, including HIF1α signalling. Collectively these data provided new insights into MPM biology and demonstrated that the integration of functional screening with patients’ clinical data is a powerful tool to highlight new non-genetic cancer dependencies that associate to a bad outcome in vivo, paving the way to new MPM-oriented targeted strategies and prognostic tools to improve patients risk-based stratification.

Crosstalk Between Kinases, Phosphatases And Mirnas In Cancer.

Reversible phosphorylation of proteins, performed by kinases and phosphatases, is the major post translational protein modification in eukaryotic cells. This intracellular event represents a critical regulatory mechanism of several signaling pathways and can be related to a vast array of diseases, including cancer. Cancer research has produced increasing evidence that kinase and phosphatase activity can be compromised by mutations and also by miRNA silencing, performed by small non-coding and endogenously produced RNA molecules that lead to translational repression. miRNAs are believed to target about one-third of human mRNAs while a single miRNA may target about 200 transcripts simultaneously. Regulation of the phosphorylation balance by miRNAs has been a topic of intense research over the last years, spanning topics going as far as cancer aggressiveness and chemotherapy resistance. By addressing recent studies that have shown miRNA expression patterns as phenotypic signatures of cancers and how miRNA influence cellular processes such as apoptosis, cell cycle control, angiogenesis, inflammation and DNA repair, we discuss how kinases, phosphatases and miRNAs cooperatively act in cancer biology.

Phylogenetic relationships in Solanaceae and related species based on cpDNA sequence from plastid trnE-trnT region

Intergenic spacers of chloroplast DNA (cpDNA) are very useful in phylogenetic and population genetic studies of plant species, to study their potential integration in phylogenetic analysis. The non-coding trnE-trnT intergenic spacer of cpDNA was analyzed to assess the nucleotide sequence polymorphism of 16 Solanaceae species and to estimate its ability to contribute to the resolution of phylogenetic studies of this group. Multiple alignments of DNA sequences of trnE-trnT intergenic spacer made the identification of nucleotide variability in this region possible and the phylogeny was estimated by maximum parsimony and rooted with Convolvulaceae Ipomoea batalas, the most closely related family. Besides, this intergenic spacer was tested for the phylogenetic ability to differentiate taxonomic levels. For this purpose, species from four other families were analyzed and compared with Solanaceae species. Results confirmed polymorphism in the trnE-trnT region at different taxonomic levels.

Mitochondrial gene content, arrangement and composition compared in African and Asian schistosomes

Complete sequences were obtained for the coding portions of the mitochondrial (mt) genomes of Schistosoma mansoni (NMRI strain, Puerto Rico; 14415 bp), S. japonicum (Anhui strain, China; 14085 bp) and S. mekongi (Khong Island, Laos; 14072 bp). Each comprises 36 genes: 12 protein-encoding genes (cox1-3, nad1-6, nad4L, atp6 and cob); two ribosomal RNAs, rrnL (large subunit rRNA or 16S) and rrnS (small subunit rRNA or 12S); as well as 22 transfer RNA (tRNA) genes. The atp8 gene is absent. A large segment (9.6 kb) of the coding region (comprising 14 tRNAs, eight complete and two incomplete protein-encoding genes) for S. malayensis (Baling, Malaysian Peninsula) was also obtained. Each genome also possesses a long non-coding region that is divided into two parts (a small and a large non-coding region, the latter not fully sequenced in any species) by one or more tRNAs. The protein-encoding genes are similar in size, composition and codon usage in all species except for cox1 in S. mansoni (609 aa) and cox2 in S. mekongi (219 an), both of which are longer than homologues in other species. An unexpected finding in all the Schistosoma species was the presence of a leucine zipper motif in the nad4L gene. The gene order in S. mansoni is strikingly different from that seen in the S. japonicum group and other flatworms. There is a high level of identity (87-94% at both the nucleotide and amino acid levels) for all protein-encoding genes of S. mekongi and S. malayensis. The identity between genes of these two species and those of S. japonicum is less (56-83% for amino acids and 73-79 for nucleotides). The identity between the genes of S. mansoni and the Asian schistosomes is far less (33-66% for amino acids and 54-68% for nucleotides), an observation consistent with the known phylogenetic distance between S. mansoni and the other species. (C) 2001 Elsevier Science B.V. All rights reserved.

Complete DNA sequence and gene organization of the mitochondrial genome of the liverfluke, Fasciola hepatica L. (Platyhelminthes; Trematoda)

The complete nucleotide sequence of the mitochondrial (mt) DNA molecule of the liverfluke, Fasciola hepatica (phylum Platyhelminthes, class Trematoda, family Fasciolidae), was determined, It comprises 14462 bp, contains 12 protein-encoding, 2 ribosomal and 22 transfer RNA genes, and is the second complete flatworm (and the first trematode) mitochondrial sequence to be described in detail. All of the genes are transcribed from the same strand. Of the genes typically found in mitochondrial genomes of eumetazoans, only atp8 is absent. The nad4L and nad4 genes overlap by 40 nt. Most intergenic sequences are very short. Two larger non-coding regions are present. The longer one (817 nt) is located between trnG and cox3 and consists of 8 identical tandem repeats of 85 nt, rich in G and C, followed by 1 imperfect repeat. The shorter non-coding region (187 nt) exhibits no special features and is separated from the longer region by trnG. The gene arrangement resembles that of some other trematodes including the eastern Asian Schistosoma species (and cyclophyllidean cestode species) but it is strikingly different from that of the African schistosomes, represented by Schistosoma mansoni. The genetic code is as inferred previously for flatworms. Transfer RNA genes range in length from 58 to 70 nt, their products producing characteristic 'clover leaf' structures, except for tRNA(S-VON) and tRNA(S-AGN) lacking the DHU arm.

Brazil and the nuclear issues in the years of the Luiz Inácio Lula da Silva government (2003-2010)

The present article is an analysis of Brazilian nuclear diplomacy and of the international relevance of the national nuclear program adopted during the two Presidential terms of Luiz Inácio Lula da Silva. Particular attention will be given to the Brazilian position in the agreement on nuclear non-proliferation, to the role of mediation between Iran and the nuclear powers, and finally to the national ambitions concerning the industrial and military uses of the nuclear technologies.

Characterization of a polyubiquitin gene in T. thermophila and of ubiquitin gene expression during sexual reproduction and under stress conditions

A 5-unit polyubiquitin gene, TTU3, was isolated from a T. thermophila genomic library and sequenced. This gene presents an extra triplet coding for Phe, a AGAGA motif and a putative HSE element in its 5'-non-coding region. The ubiquitin gene expression in this ciliate was investigated by Northern blot hybridization in conjugating cells or cells under stress conditions. Exponentially growing cells express two ubiquitin mRNAs of 0.75 and 1.8 kb and a new species of 1.4 kb is induced under hyperthermic stress. During sexual reproduction of the cells (conjugation) the 1.8-kb mRNA is still transcribed whereas the steady-state population of the 0.75 mRNA transcripts is strongly diminished. Southern blot analysis suggests that ubiquitin in T. thermophila constitutes a large family of about ten members.

The Role of Small RNAs and Ribonucleases in the Control of Gene Expression in Salmonella Typhimurium

Dissertation presented to obtain the Ph.D degree in Biology

Interplay of Exoribonucleases, Hfq and Small RNAs Structural Determinants in the Control of Gene Expression

Dissertation presented to obtain the Ph.D degree in Biology

Functional genomic analysis of heat stress in Vitis vinifera

Grapevine (Vitis vinifera) is one of most agro-economically important fruit crops worldwide, with a special relevance in Portugal where over 300 varieties are used for wine production. Due to global warming, temperature stress is currently a serious issue affecting crop production especially in temperate climates. Mobile genetic elements such as retrotransposons have been shown to be involved in environmental stress induced genetic and epigenetic modifications. In this study, sequences related to Grapevine Retrotransposon 1 (Gret1) were utilized to determine heat induced genomic and transcriptomic modifications in Touriga Nacional, a traditional Portuguese grapevine variety. For this purpose, growing canes were treated to 42 oC for four hours and leaf genomic DNA and RNA was utilized for various techniques to observe possible genomic alterations and variation in transcription levels of coding and non-coding sequences between non-treated plants and treated plants immediately after heat stress (HS-0 h) or after a 24 hour recovery period (HS-24 h). Heat stress was found to induce a significant decrease in Gret1 related sequences in HS-24 h leaves, indicating an effect of heat stress on genomic structure. In order to identify putative heat induced DNA modifications, genome wide approaches such as Amplified Fragment Length Polymorphism were utilized. This resulted in the identification of a polymorphic DNA fragment in HS-0 h and HS-24 h leaves whose sequence mapped to a genomic region flanking a house keeping gene (NADH) that is represented in multiple copies in the Vitis vinifera genome. Heat stress was also found to affect the transcript levels of various non-coding and gene coding sequences. Accordingly, quantitative real time PCR results established that Gret1 related sequences are up regulated immediately after heat stress whereas the level of transcript of genes involved in identification and repair of double strand breaks are significantly down regulated in HS-0 h plants. Taken together, the results of this work demonstrated heat stress affects both genomic integrity and transcription levels.

Revisiting the specificity of the MHC class II transactivator CIITA in vivo.

CIITA is a master regulatory factor for the expression of MHC class II (MHC-II) and accessory genes involved in Ag presentation. It has recently been suggested that CIITA also regulates numerous other genes having diverse functions within and outside the immune system. To determine whether these genes are indeed relevant targets of CIITA in vivo, we studied their expression in CIITA-transgenic and CIITA-deficient mice. In contrast to the decisive control of MHC-II and related genes by CIITA, nine putative non-MHC target genes (Eif3s2, Kpna6, Tap1, Yars, Col1a2, Ctse, Ptprr, Tnfsf6 and Plxna1) were found to be CIITA independent in all cell types examined. Two other target genes, encoding IL-4 and IFN-gamma, were indeed found to be up- and down-regulated, respectively, in CIITA-transgenic CD4(+) T cells. However, there was no correlation between MHC-II expression and this Th2 bias at the level of individual transgenic T cells, indicating an indirect control by CIITA. These results show that MHC-II-restricted Ag presentation, and its indirect influences on T cells, remains the only pathway under direct control by CIITA in vivo. They also imply that precisely regulated MHC-II expression is essential for maintaining a proper Th1-Th2 balance.