Dysregulation of REST-regulated coding and non-coding RNAs in a cellular model of Huntington's disease

Huntingtin (Htt) protein interacts with many transcriptional regulators, with widespread disruption to the transcriptome in Huntington's disease (HD) brought about by altered interactions with the mutant Htt (muHtt) protein. Repressor Element-1 Silencing Transcription Factor (REST) is a repressor whose association with Htt in the cytoplasm is disrupted in HD, leading to increased nuclear REST and concomitant repression of several neuronal-specific genes, including brain-derived neurotrophic factor (Bdnf). Here, we explored a wide set of HD dysregulated genes to identify direct REST targets whose expression is altered in a cellular model of HD but that can be rescued by knock-down of REST activity. We found many direct REST target genes encoding proteins important for nervous system development, including a cohort involved in synaptic transmission, at least two of which can be rescued at the protein level by REST knock-down. We also identified several microRNAs (miRNAs) whose aberrant repression is directly mediated by REST, including miR-137, which has not previously been shown to be a direct REST target in mouse. These data provide evidence of the contribution of inappropriate REST-mediated transcriptional repression to the widespread changes in coding and non-coding gene expression in a cellular model of HD that may affect normal neuronal function and survival.

Extremely high conservation in the untranslated Region as well as the coding region of CNP mRNAs throughout elasmobranch species

C-type natriuretic peptide (CNP) is a crucial osmoregulatory hormone in elasmobranchs, participating in salt secretion and drinking. In contrast to teleosts and tetrapods in which the NP family is composed of a group of structurally related peptides, we have shown that CNP is the sole NP in sharks. In the present study, CNP cDNAs were cloned from four species of batoids, another group of elasmobranchs. The cloned batoid CNP precursors contained a plausible mature peptide of 22 amino acid residues that is identical to most shark CNP-22s, but five successive amino acids were consistently deleted in the prosegment compared with shark precursors, supporting the diphyletic classification of sharks and rays. In addition, molecular phylogenetic trees of CNP precursors were consistent with a diphyletic interpretation. Except for the deletion, the nucleotide and deduced amino acid sequences of the CNP cDNAs are extremely well-conserved among all elasmobranch species, even between sharks and rays. Surprisingly, high conservation is evident not only for the coding region, but also for the untranslated regions. It is most likely that the high conservation is due to the low nucleotide substitution rate in the elasmobranch genome, and high selection pressure. The 3′-untranslated region of the elasmobranch CNP cDNAs contained three to six repeats of the ATTTA motif that is associated with the regulation of mRNA stability and translation efficiency. Alternative polyadenylation sites were also found; the long 3′-untranslated region contains a core of ATTTA motifs while the short form has only one or no ATTTA motif, indicating that the post-transcriptional modification of mRNA is important for regulation of CNP synthesis. These characteristics in the 3′-untranslated region were conserved among all elasmobranch CNP cDNAs. Since CNP has been implicated as a fast-acting hormone to facilitate salt secretion from the rectal gland, the conserved 3′-untranslated region most likely contributes to rapid regulation of CNP synthesis in elasmobranchs in response to acute changes in internal and external environments.

Genetic diversity of the HLA-G coding region in Amerindian populations from the Brazilian Amazon: a possible role of natural selection

HLA-G has an important role in the modulation of the maternal immune system during pregnancy, and evidence that balancing selection acts in the promoter and 3′UTR regions has been previously reported. To determine whether selection acts on the HLA-G coding region in the Amazon Rainforest, exons 2, 3 and 4 were analyzed in a sample of 142 Amerindians from nine villages of five isolated tribes that inhabit the Central Amazon. Six previously described single-nucleotide polymorphisms (SNPs) were identified and the Expectation-Maximization (EM) and PHASE algorithms were used to computationally reconstruct SNP haplotypes (HLA-G alleles). A new HLA-G allele, which originated in Amerindian populations by a crossing-over event between two widespread HLA-G alleles, was identified in 18 individuals. Neutrality tests evidenced that natural selection has a complex part in the HLA-G coding region. Although balancing selection is the type of selection that shapes variability at a local level (Native American populations), we have also shown that purifying selection may occur on a worldwide scale. Moreover, the balancing selection does not seem to act on the coding region as strongly as it acts on the flanking regulatory regions, and such coding signature may actually reflect a hitchhiking effect.Genes and Immunity advance online publication, 3 October 2013; doi:10.1038/gene.2013.47.

Alterações epigenéticas do gene RASSF1 e investigação de modulação gene-específica por non-coding RNA em linhagens celulares derivadas de carcinomas mamários

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Expression of Mitochondrial Non-coding RNAs (ncRNAs) Is Modulated by High Risk Human Papillomavirus (HPV) Oncogenes

The study of RNA and DNA oncogenic viruses has proved invaluable in the discovery of key cellular pathways that are rendered dysfunctional during cancer progression. An example is high risk human papillomavirus (HPV), the etiological agent of cervical cancer. The role of HPV oncogenes in cellular immortalization and transformation has been extensively investigated. We reported the differential expression of a family of human mitochondrial non-coding RNAs (ncRNAs) between normal and cancer cells. Normal cells express a sense mitochondrial ncRNA (SncmtRNA) that seems to be required for cell proliferation and two antisense transcripts (ASncmtRNAs). In contrast, the ASncmtRNAs are down-regulated in cancer cells. To shed some light on the mechanisms that trigger down-regulation of the ASncmtRNAs, we studied human keratinocytes (HFK) immortalized with HPV. Here we show that immortalization of HFK with HPV-16 or 18 causes down-regulation of the ASncmtRNAs and induces the expression of a new sense transcript named SncmtRNA-2. Transduction of HFK with both E6 and E7 is sufficient to induce expression of SncmtRNA-2. Moreover, E2 oncogene is involved in down-regulation of the ASncmtRNAs. Knockdown of E2 in immortalized cells reestablishes in a reversible manner the expression of the ASncmtRNAs, suggesting that endogenous cellular factors(s) could play functions analogous to E2 during non-HPV-induced oncogenesis.

Androgen responsive intronic non-coding RNAs

Abstract Background Transcription of large numbers of non-coding RNAs originating from intronic regions of human genes has been recently reported, but mechanisms governing their biosynthesis and biological functions are largely unknown. In this work, we evaluated the existence of a common mechanism of transcription regulation shared by protein-coding mRNAs and intronic RNAs by measuring the effect of androgen on the transcriptional profile of a prostate cancer cell line. Results Using a custom-built cDNA microarray enriched in intronic transcribed sequences, we found 39 intronic non-coding RNAs for which levels were significantly regulated by androgen exposure. Orientation-specific reverse transcription-PCR indicated that 10 of the 13 were transcribed in the antisense direction. These transcripts are long (0.5–5 kb), unspliced and apparently do not code for proteins. Interestingly, we found that the relative levels of androgen-regulated intronic transcripts could be correlated with the levels of the corresponding protein-coding gene (asGAS6 and asDNAJC3) or with the alternative usage of exons (asKDELR2 and asITGA6) in the corresponding protein-coding transcripts. Binding of the androgen receptor to a putative regulatory region upstream from asMYO5A, an androgen-regulated antisense intronic transcript, was confirmed by chromatin immunoprecipitation. Conclusion Altogether, these results indicate that at least a fraction of naturally transcribed intronic non-coding RNAs may be regulated by common physiological signals such as hormones, and further corroborate the notion that the intronic complement of the transcriptome play functional roles in the human gene-expression program.

Identification of protein-coding and non-coding RNA expression profiles in CD34+ and in stromal cells in refractory anemia with ringed sideroblasts

Abstract Background Myelodysplastic syndromes (MDS) are a group of clonal hematological disorders characterized by ineffective hematopoiesis with morphological evidence of marrow cell dysplasia resulting in peripheral blood cytopenia. Microarray technology has permitted a refined high-throughput mapping of the transcriptional activity in the human genome. Non-coding RNAs (ncRNAs) transcribed from intronic regions of genes are involved in a number of processes related to post-transcriptional control of gene expression, and in the regulation of exon-skipping and intron retention. Characterization of ncRNAs in progenitor cells and stromal cells of MDS patients could be strategic for understanding gene expression regulation in this disease. Methods In this study, gene expression profiles of CD34+ cells of 4 patients with MDS of refractory anemia with ringed sideroblasts (RARS) subgroup and stromal cells of 3 patients with MDS-RARS were compared with healthy individuals using 44 k combined intron-exon oligoarrays, which included probes for exons of protein-coding genes, and for non-coding RNAs transcribed from intronic regions in either the sense or antisense strands. Real-time RT-PCR was performed to confirm the expression levels of selected transcripts. Results In CD34+ cells of MDS-RARS patients, 216 genes were significantly differentially expressed (q-value ≤ 0.01) in comparison to healthy individuals, of which 65 (30%) were non-coding transcripts. In stromal cells of MDS-RARS, 12 genes were significantly differentially expressed (q-value ≤ 0.05) in comparison to healthy individuals, of which 3 (25%) were non-coding transcripts. Conclusions These results demonstrated, for the first time, the differential ncRNA expression profile between MDS-RARS and healthy individuals, in CD34+ cells and stromal cells, suggesting that ncRNAs may play an important role during the development of myelodysplastic syndromes.

Non-coding keratin variants associate with liver fibrosis progression in patients with hemochromatosis

Background Keratins 8 and 18 (K8/K18) are intermediate filament proteins that protect the liver from various forms of injury. Exonic K8/K18 variants associate with adverse outcome in acute liver failure and with liver fibrosis progression in patients with chronic hepatitis C infection or primary biliary cirrhosis. Given the association of K8/K18 variants with end-stage liver disease and progression in several chronic liver disorders, we studied the importance of keratin variants in patients with hemochromatosis. Methods The entire K8/K18 exonic regions were analyzed in 162 hemochromatosis patients carrying homozygous C282Y HFE (hemochromatosis gene) mutations. 234 liver-healthy subjects were used as controls. Exonic regions were PCR-amplified and analyzed using denaturing high-performance liquid chromatography and DNA sequencing. Previously-generated transgenic mice overexpressing K8 G62C were studied for their susceptibility to iron overload. Susceptibility to iron toxicity of primary hepatocytes that express K8 wild-type and G62C was also assessed. Results We identified amino-acid-altering keratin heterozygous variants in 10 of 162 hemochromatosis patients (6.2%) and non-coding heterozygous variants in 6 additional patients (3.7%). Two novel K8 variants (Q169E/R275W) were found. K8 R341H was the most common amino-acid altering variant (4 patients), and exclusively associated with an intronic KRT8 IVS7+10delC deletion. Intronic, but not amino-acid-altering variants associated with the development of liver fibrosis. In mice, or ex vivo, the K8 G62C variant did not affect iron-accumulation in response to iron-rich diet or the extent of iron-induced hepatocellular injury. Conclusion In patients with hemochromatosis, intronic but not exonic K8/K18 variants associate with liver fibrosis development.

Endothelium-specific replacement of the connexin43 coding region by a lacZ reporter gene

The murine gap junction protein connexin43 (Cx43) is expressed in blood vessels, with vastly different contribution by endothelial and smooth muscle cells. We have used the Cre recombinase under control of TIE2 transcriptional elements to inactivate a floxed Cx43 gene specifically in endothelial cells. Cre-mediated deletion led to replacement of the Cx43 coding region by a lacZ reporter gene. This allowed us to monitor the extent of deletion and to visualize the endothelial expression pattern of Cx43. We found widespread endothelial expression of the Cx43 gene during embryonic development, which became restricted largely to capillaries and small vessels in all adult organs examined. Mice lacking Cx43 in endothelium did not exhibit altered blood pressure, in contrast to mice deficient in Cx40. Our results show that lacZ activation after deletion of the target gene allows us to determine the extent of cell type-specific deletion after phenotypical investigation of the same animal.

A non-coding genomic duplication at the HMX1 locus is associated with crop ears in highland cattle.

Highland cattle with congenital crop ears have notches of variable size on the tips of both ears. In some cases, cartilage deformation can be seen and occasionally the external ears are shortened. We collected 40 cases and 80 controls across Switzerland. Pedigree data analysis confirmed a monogenic autosomal dominant mode of inheritance with variable expressivity. All affected animals could be traced back to a single common ancestor. A genome-wide association study was performed and the causative mutation was mapped to a 4 Mb interval on bovine chromosome 6. The H6 family homeobox 1 (HMX1) gene was selected as a positional and functional candidate gene. By whole genome re-sequencing of an affected Highland cattle, we detected 6 non-synonymous coding sequence variants and two variants in an ultra-conserved element at the HMX1 locus with respect to the reference genome. Of these 8 variants, only a non-coding 76 bp genomic duplication (g.106720058_106720133dup) located in the conserved region was perfectly associated with crop ears. The identified copy number variation probably results in HMX1 misregulation and possible gain-of-function. Our findings confirm the role of HMX1 during the development of the external ear. As it is sometimes difficult to phenotypically diagnose Highland cattle with slight ear notches, genetic testing can now be used to improve selection against this undesired trait.

Mechanism of translationally coupled mRNA turnover mediated by c-fos protein-coding-region determinant

Proto-oncogene c-fos is a member of the class of early-response genes whose transient expression plays a crucial role in cell proliferation, differentiation, and apoptosis. Degradation of c- fos mRNA is an important mechanism for controlling c-fos expression. Rapid mRNA turnover mediated by the protein-coding-region determinant (mCRD) of the c-fos transcript illustrates a functional interplay between mRNA turnover and translation that coordinately influences the fate of cytoplasmic mRNA. It is suggested that mCRD communicates with the 3′ poly(A) tail via an mRNP complex comprising mCRD-associated proteins, which prevents deadenylation in the absence of translation. Ribosome transit as a result of translation is required to alter the conformation of the mRNP complex, thereby eliciting accelerated deadenylation and mRNA decay. To gain further insight into the mechanism of mCRD-mediated mRNA turnover, Unr was identified as an mCRD-binding protein, and its binding site within mCRD was characterized. Moreover, the functional role for Unr in mRNA decay was demonstrated. The result showed that elevation of Unr protein level in the cytoplasm led to inhibition of mRNA destabilization by mCRD. In addition, GST pull-down assay and immuno-precipitation analysis revealed that Unr interacted with PABP in an RNA-independent manner, which identified Unr as a novel PABP-interacting protein. Furthermore, the Unr interacting domain in PABP was characterized. In vivo mRNA decay experiments demonstrated a role for Unr-PABP interaction in mCRD-mediated mRNA decay. In conclusion, the findings of this study provide the first evidence that Unr plays a key role in mCRD-mediated mRNA decay. It is proposed that Unr is recruited by mCRD to initiate the formation of a dynamic mRNP complex for communicating with poly(A) tail through PABP. This unique mRNP complex may couple translation to mRNA decay, and perhaps to recruit the responsible nuclease for deadenylation. ^

The role of non-coding RNA in the development of pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a progressive disease of the small pulmonary arteries, characterised by pulmonary vascular remodelling due to excessive proliferation and resistance to apoptosis of pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth muscle cells (PASMCs). The increased pulmonary vascular resistance and elevated pulmonary artery pressures result in right heart failure and premature death. Germline mutations of the bone morphogenetic protein receptor-2 (bmpr2) gene, a receptor of the transforming growth factor beta (TGF-β) superfamily, account for approximately 75%-80% of the cases of heritable form of PAH (HPAH) and 20% of sporadic cases or idiopathic PAH (IPAH). IPAH patients without known bmpr2 mutations show reduced expression of BMPR2. However only ~ 20% of bmpr2-mutation carriers will develop the disease, due to an incomplete penetrance, thus the need for a ‘second hit’ including other genetic and/or environmental factors is accepted. Diagnosis of PAH occurs most frequently when patients have reached an advanced stage of disease. Although modern PAH therapies can markedly improve a patient’s symptoms and slow the rate of clinical deterioration, the mortality rate from PAH remains unacceptably high. Therefore, the development of novel therapeutic approaches is required for the treatment of this multifaceted disease. Noncoding RNAs (ncRNAs) include microRNAs (miRNAs) and long noncoding RNAs (lncRNAs). MiRNAs are ~ 22 nucleotide long and act as negative regulators of gene ex-pression via degradation or translational inhibition of their target mRNAs. Previous studies showed extensive evidence for the role of miRNAs in the development of PAH. LncRNAs are transcribed RNA molecules greater than 200 nucleotides in length. Similar to classical mRNA, lncRNAs are translated by RNA polymerase II and are generally alternatively spliced and polyadenylated. LncRNAs are highly versatile and function to regulate gene expression by diverse mechanisms. Unlike miRNAs, which exhibit well-defined actions in negatively regulating gene expression via the 3’-UTR of mRNAs, lncRNAs play more diverse and unpredictable regulatory roles. Although a number of lncRNAs have been intensively investigated in the cancer field, studies of the role of lncRNAs in vascular diseases such as PAH are still at a very early stage. The aim of this study was to investigate the involvement of specific ncRNAs in the development of PAH using experimental animal models and cell culture. The first ncRNA we focused on was miR-143, which is up-regulated in the lung and right ventricle tissues of various animal models of PH, as well as in the lungs and PASMCs of PAH patients. We show that genetic ablation of miR-143 is protective against the development of chronic hypoxia induced PH in mice, assessed via measurement of right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH) and pulmonary vascular remodelling. We further report that knockdown of miR-143-3p in WT mice via anti-miR-143-3p administration prior to exposure of mice to chronic hypoxia significantly decreases certain indices of PH (RVSP) although no significant changes in RVH and pulmo-nary vascular remodelling were observed. However, a reversal study using antimiR-143-3p treatment to modulate miR-143-3p demonstrated a protective effect on RVSP, RVH, and muscularisation of pulmonary arteries in the mouse chronic hypoxia induced PH model. In vitro experiments showed that miR-143-3p overexpression promotes PASMC migration and inhibits PASMC apoptosis, while knockdown miR-143-3p elicits the opposite effect, with no effects observed on cellular proliferation. Interestingly, miR-143-3p-enriched exosomes derived from PASMCs mediated cell-to-cell communication between PASMCs and PAECs, contributing to the pro-migratory and pro-angiogenic phenotype of PAECs that underlies the pathogenesis of PAH. Previous work has shown that miR-145-5p expression is upregulated in the chronic hypoxia induced mouse model of PH, as well as in PAH patients. Genetic ablation and pharmacological inhibition (subcutaneous injection) of miR-145-5p exert a protective against the de-velopment of PAH. In order to explore the potential for alternative, more lung targeted delivery strategies, miR-145-5p expression was inhibited in WT mice using intranasal-delivered antimiR-145-5p both prior to and post exposure to chronic hypoxia. The decreased expression of miR-145-5p in lung showed no beneficial effect on the development of PH compared with control antimiRNA treated mice exposed to chronic hypoxia. Thus, miR-143-3p modulated both cellular and exosome-mediated responses in pulmonary vascular cells, while the inhibition of miR-143-3p prevented the development of experimental pulmonary hypertension. We focused on two lncRNAs in this project: Myocardin-induced Smooth Muscle Long noncoding RNA, Inducer of Differentiation (MYOSLID) and non-annotated Myolnc16, which were identified from RNA sequencing studies in human coronary artery smooth muscle cells (HCASMCs) that overexpress myocardin. MYOSLID was significantly in-creased in PASMCs from patients with IPAH compared to healthy controls and increased in circulating endothelial progenitor cells (EPCs) from bmpr2 mutant PAH patients. Exposure of PASMCs to hypoxia in vitro led to a significant upregulation in MYOSLID expres-sion. MYOSLID expression was also induced by treatment of PASMC with BMP4, TGF-β and PDGF, which are known to be triggers of PAH in vitro. Small interfering RNA (siR-NA)-mediated knockdown MYOSLID inhibited migration and induced cell apoptosis without affecting cell proliferation and upregulated several genes in the BMP pathway in-cluding bmpr1α, bmpr2, id1, and id3. Modulation of MYOSLID also affected expression of BMPR2 at the protein level. In addition, MYOSLID knockdown affected the BMP-Smad and BMP-non-Smad signalling pathways in PASMCs assessed by phosphorylation of Smad1/5/9 and ERK1/2, respectively. In PAECs, MYOSLID expression was also induced by hypoxia exposure, VEGF and FGF2 treatment. In addition, MYOSLID knockdown sig-nificantly decreased the proliferation of PAECs. Thus, MYOSLID may be a novel modulator in pulmonary vascular cell functions, likely through the BMP-Smad and –non-Smad pathways. Treatment of PASMCs with inflammatory cytokines (IL-1 and TNF-α) significantly in-duced the expression of Myolnc16 at a very early time point. Knockdown of Myolnc16 in vitro decreased the expression of il-6, and upregulated the expression of il-1 and il-8 in PASMCs. Moreover, the expression levels of chemokines (cxcl1, cxcl6 and cxcl8) were sig-nificantly decreased with Myolnc16 knockdown. In addition, Myolnc16 knockdown decreased the MAP kinase signalling pathway assessed by phosphorylation of ERK1/2 and p38 MAPK and inhibited cell migration and proliferation in PASMCs. Thus, Myolnc16 may a novel modulator of PASMCs functions through anti-inflammatory signalling pathways. In summary, in this thesis we have demonstrated how miR-143-3p plays a protective role in the development of PH both in vivo animal models and patients, as well as in vitro cell cul-ture. Moreover, we have showed the role of two novel lncRNAs in pulmonary vascular cells. These ncRNAs represent potential novel therapeutic targets for the treatment of PAH with further work addressing to investigate the target genes, and the pathways modulated by these ncRNAs during the development of PAH.