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- Quality control of gene expression in the mammalian cell nucleusPublication . Custódio, Noélia; Fonseca, M. Carmo, 1959-Protein-encoding genes are transcribed in the nucleus by RNA polymerase II as precursor RNAs that undergo extensive processing before being translocated to the cytoplasm for translation by the ribosomes. This spatial and temporal separation between RNA and protein synthesis offers an immense opportunity for regulation and quality control.When this study was initiated it was known from biochemical studies that human ß-globin (HBB) transcripts with mutations that impaired splicing or 3' end formation were unable to be exported to the cytoplasm, a phenotype identical to that seen in ß-thalassemia patients harbouring similar mutations (Antoniou et al., 1998). This result was consistent with the retention in the nucleus of incorrectly processed transcripts. Based on this initial observation we hypothesised the existence of a quality control mechanism to retain the incorrectly processed transcripts in the nucleus and proposed to elucidate the mechanism responsible for the observed retention. The first goal of this work was to determine the intranuclear localisation of the retained transcripts. To address this we used as a model system murine erythroleukemia (MEL) cells stably transfected with either wild-type or processing mutant HBB genes. The experimental approach was based on the direct visualisation of both normal and defective HBB transcripts using fluorescence in situ hybridisation and confocal microscopy. Nuclear transcripts of both wild-type and mutant HBB genes were detected only as intranuclear foci co-localising with the template gene locus. To determine the kinetics of transcript release from the site of transcription the cells were treated with the transcriptional inhibitors actinomycin D, α-amanitin and DRB. These drugs induced the rapid disappearance of nuclear foci corresponding to wild-type HBB RNA. In contrast, pre-mRNA mutants defective in either splicing or 3' end formation and which fail to be transported to the cytoplasm were retained at the site of transcription. These results indicated that the quality control mechanism responsible for the nuclear retention of incorrectly processed transcripts operates at the site of transcription and suggest that splicing and 3' end processing are rate limiting for release of mRNA from the transcription site. The next goal of this work was to determine the molecular players involved in the quality control mechanism that operates at the transcription site. Studies over the past years have indicated that there is extensive coupling between transcription, pre-mRNA processing and nuclear export of mRNA (reviewed by Bentley, 2005). One hypothesis to explain the retention of the mutant transcripts could be the absence of recruitment of proteins essential for the release and/or transport of the transcripts from the gene locus. Alternatively, the retention could be mediated by proteins that are bound to the nascent transcripts and become stalled due to incomplete processing To test the first hypothesis we decided to study the recruitment of exon junction complex (EJC) proteins and mRNA export factors to the retention sites in the nucleus. To achieve this we visualised the distribution of EJC proteins and RNA export factors relative to the sites HBB transcription in the nucleus. Using in situ hybridisation and confocal microscopy, we observed accumulation of EJC proteins (REF/Aly, Y14, SRm160, UAP56, RNPS1 and Magoh) and core spliceosome components (U snRNPs) at sites of wild-type HBB transcription. This suggests that EJC proteins bind stably to pre-mRNA co-transcriptionally. No concentration of the export factors NXF1/TAP or p15 was detected on nascent transcripts, arguing that in mammalian cells these proteins bind the mRNA shortly before or after release from the sites of transcription. Contrasting with the results obtained in MEL cells expressing wild-type HBB transcripts, mutant pre-mRNA defective in splicing and 3' end processing do not co-localise with SRm160, REF, UAP56 or Sm proteins. These results suggest that the accumulation of EJC proteins at transcription sites requires efficient processing of the nascent pre-mRNA. Although the results obtained do not discard the hypothesis that efficient recruitment of EJC proteins and/or export factors may contribute to the release of the transcripts from the transcription site, further studies are required to conclusively show or discard the involvement of these proteins in transcription site release. In the second hypothesis the best candidate to mediate the retention is the carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II (RNA Pol II LS). The mammalian CTD comprises 52 heptad repeats followed by a terminal 10 amino acid motif (Corden et al., 1985). Several reports in the last decade showed that both splicing and 3' end processing factors can associate with the CTD of RNA Pol II (reviewed by Bentley, 2005). Since the processing mutants analysed are able to assemble some processing machinery, the release of the transcripts could be blocked by the stalled or abnormal processing machinery associated with the CTD. In order to test this hypothesis we generated cell lines that express either the wild-type HBB gene or a mutant version defective in splicing and an α-amanitin resistant form of RNA Pol II LS with either 52 (wt), 31 (Δ31) or 5 (Δ5) repeats of the CTD. When these cells were treated with actinomycin D to stop transcription the results showed that a large proportion of the wild-type HBB transcripts made by RNA Pol II with only 31 CTD repearts were retained at the site of transcription. Despite this result we observed recruitment of several EJC proteins, includind Aly/REF, Y14 and SRm160 to the transcription sites and RNase protection assays showed that the HBB transcripts produced by RNA Pol II LS Δ31 are correctly spliced and 3' end cleaved. These results show that the form of RNA Pol II LS with only 31 repeats of the CTD is competent in transcription and processing but fails to allow the efficient release of the transcripts from the transcription site. These results provide evidence that mRNA release from the transcription site requires the heptad repeat structure of the CTD and we propose that the missing heptads in the truncated CTD mutant are required for binding of proteins implicated in a final co-transcriptional maturation of spliced and 3' end cleaved mRNAs into export-competent ribonucleoprotein particles. Eukaryotic RNA polymerase II is a complex enzyme composed of 12 distinct subunits that is present in cells in low abundance (reviewed by Shilatifard et al., 2003). Transcription of mRNA by RNA polymerase II involves a phosphorylation/ dephosphorylation cycle of the CTD of the enzyme's largest subunit. The hypophosphorylated form assembles into pre-initiation complexes and the CTD becomes first phosphorylated on Serine at position 5 of the heptad repeat and during elongation on Serine at position 2 (Komarnitsky et al., 2000; Lu et al., 1991). As mentioned before we have generated stable murine cell lines expressing an α-amanitin resistant form of RNA Pol II LS. The cell lines generated were screened for survival and transcription in the presence of α-amanitin ensuring the functionality of the exogenous subunit. To characterise these cell lines we studied the localisation of the exogenous subunit and observed that over-expressed RNA Pol II LS was predominantly hypophosphorylated, soluble and accumulated in the cytoplasm in a CRM1-dependent manner. Our results further showed that the transcriptionally active form of RNA Pol II LS containing phosphoserine in position 2 of the CTD repeats was restricted to the nucleus and its levels remained remarkably constant. These results suggest that the nuclear-cytoplasmic distribution of RNA Pol II LS may be regulated by shuttling and we propose that this may provide a mechanism to control the pool of RNA polymerase subunits that is accessible for assembly of a functional enzyme in the nucleus.
- In vivo recruitment of exon junction complex proteins to transcription sites in mammalian cell nucleiPublication . Custódio, Noélia; Carvalho, Célia; Condado, Inês; Antoniou, Michael; Blencowe, Benjamin J.; Carmo-Fonseca, MariaStudies over the past years indicate that there is extensive coupling between nuclear export of mRNA and pre-mRNA processing. Here, we visualized the distribution of exon junction complex (EJC) proteins and RNA export factors relative to sites of abundant pre-mRNA synthesis in the nucleus. We analyzed both HeLa cells infected with adenovirus and murine erythroleukemia (MEL) cells stably transfected with the human beta-globin gene. Using in situ hybridization and confocal microscopy, we observe accumulation of EJC proteins (REF/Aly, Y14, SRm160, UAP56, RNPS1, and Magoh) and core spliceosome components (U snRNPs) at sites of transcription. This suggests that EJC proteins bind stably to pre-mRNA cotranscriptionally. No concentration of the export factors NXF1/TAP, p15, and Dbp5 was detected on nascent transcripts, arguing that in mammalian cells these proteins bind the mRNA shortly before or after release from the sites of transcription. These results also suggest that binding of EJC proteins to the mRNA is not sufficient to recruit TAP-p15, consistent with recent findings showing that the EJC does not play a crucial role in mRNA export. Contrasting to the results obtained in MEL cells expressing normal human beta-globin transcripts, mutant pre-mRNAs defective in splicing and 3'end processing do not colocalize with SRm160, REF, UAP56, or Sm proteins. This shows that the accumulation of EJC proteins at transcription sites requires efficient processing of the nascent pre-mRNAs, arguing that transcription per se is not sufficient for the stable assembly of the EJC.
- Expression profiling in ovarian cancer reveals coordinated regulation of BRCA1/2 and homologous recombination genesPublication . Custódio, Noélia; Savisaar, Rosina; Carvalho, Célia; Bak-Gordon, Pedro; Ribeiro, Maria I.; Almeida-Tavares, Joana; Nunes, Paula B.; Peixoto, Carolina; Pinto, Carla; Escudeiro, Carla; Teixeira, Manuel R.; Carmo-Fonseca, MariaPredictive biomarkers are crucial in clarifying the best strategy to use poly(ADP-ribose) polymerase inhibitors (PARPi) for the greatest benefit to ovarian cancer patients. PARPi are specifically lethal to cancer cells that cannot repair DNA damage by homologous recombination (HR), and HR deficiency is frequently associated with BRCA1/2 mutations. Genetic tests for BRCA1/2 mutations are currently used in the clinic, but results can be inconclusive due to the high prevalence of rare DNA sequence variants of unknown significance. Most tests also fail to detect epigenetic modifications and mutations located deep within introns that may alter the mRNA. The aim of this study was to investigate whether quantitation of BRCA1/2 mRNAs in ovarian cancer can provide information beyond the DNA tests. Using the nCounter assay from NanoString Technologies, we analyzed RNA isolated from 38 ovarian cancer specimens and 11 normal fallopian tube samples. We found that BRCA1/2 expression was highly variable among tumors. We further observed that tumors with lower levels of BRCA1/2 mRNA showed downregulated expression of 12 additional HR genes. Analysis of 299 ovarian cancer samples from The Cancer Genome Atlas (TCGA) confirmed the coordinated expression of BRCA1/2 and HR genes. To facilitate the routine analysis of BRCA1/2 mRNA in the clinical setting, we developed a targeted droplet digital PCR approach that can be used with FFPE samples. In conclusion, this study underscores the potential clinical benefit of measuring mRNA levels in tumors when BRCA1/2 DNA tests are negative or inconclusive.
- Deep intronic mutations and human diseasePublication . Vaz-Drago, Rita; Custódio, Noélia; Carmo-Fonseca, MariaNext-generation sequencing has revolutionized clinical diagnostic testing. Yet, for a substantial proportion of patients, sequence information restricted to exons and exon-intron boundaries fails to identify the genetic cause of the disease. Here we review evidence from mRNA analysis and entire genomic sequencing indicating that pathogenic mutations can occur deep within the introns of over 75 disease-associated genes. Deleterious DNA variants located more than 100 base pairs away from exon-intron junctions most commonly lead to pseudo-exon inclusion due to activation of non-canonical splice sites or changes in splicing regulatory elements. Additionally, deep intronic mutations can disrupt transcription regulatory motifs and non-coding RNA genes. This review aims to highlight the importance of studying variation in deep intronic sequence as a cause of monogenic disorders as well as hereditary cancer syndromes.
- Abundance of the largest subunit of RNA polymerase II in the nucleus is regulated by nucleo-cytoplasmic shuttlingPublication . Custódio, Noélia; Antoniou, Michael; Carmo-Fonseca, MariaEukaryotic RNA polymerase II is a complex enzyme composed of 12 distinct subunits that is present in cells in low abundance. Transcription of mRNA by RNA polymerase II involves a phosphorylation/dephosphorylation cycle of the carboxyl-terminal domain (CTD) of the enzyme's largest subunit. We have generated stable murine cell lines expressing an alpha-amanitin-resistant form of the largest subunit of RNA polymerase II (RNA Pol II LS). These cells maintained transcriptional activity in the presence of alpha-amanitin, indicating that the exogenous protein was functional. We observed that over-expressed RNA Pol II LS was predominantly hypophosphorylated, soluble and accumulated in the cytoplasm in a CRM1-dependent manner. Our results further showed that the transcriptionally active form of RNA Pol II LS containing phosphoserine in position 2 of the CTD repeats was restricted to the nucleus and its levels remained remarkably constant. We propose that nucleo-cytoplasmic shuttling of RNA Pol II LS may provide a mechanism to control the pool of RNA polymerase subunits that is accessible for assembly of a functional enzyme in the nucleus.
- CD155/PVR determines acute myeloid leukemia targeting by Delta One T cellsPublication . Mensurado, Sofia; Condeço, Carolina; Sánchez-Martínez, Diego; Shirley, Sara; Coelho, Rui; Tirado, Néstor; Vinyoles, Meritxell; Blanco Dominguez, Rafael; Barros, Leandro; Galvão, Beatriz; Custódio, Noélia; Gomes da Silva, Maria; Menéndez, Pablo; Silva-Santos, BrunoRelapsed or refractory acute myeloid leukemia (AML) remains a major therapeutic challenge. We have recently developed a Vδ1+ γδ T cell-based product for adoptive immunotherapy, named Delta One T (DOT) cells, and demonstrated their cytolytic capacity to eliminate AML cell lines and primary blasts in vitro and in vivo. However, the molecular mechanisms responsible for the broad DOT-cell recognition of AML cells remain poorly understood. Here, we dissected the role of natural killer (NK) cell receptor ligands in AML cell recognition by DOT cells. Screening of multiple AML cell lines highlighted a strong upregulation of the DNAM-1 ligands, CD155/pulmonary vascular resistance (PVR), CD112/nectin-2, as well as the NKp30 ligand, B7-H6, in contrast with NKG2D ligands. CRISPR-mediated ablation revealed key nonredundant and synergistic contributions of PVR and B7-H6 but not nectin-2 to DOT-cell targeting of AML cells. We further demonstrate that PVR and B7-H6 are critical for the formation of robust immunological synapses between AML and DOT cells. Importantly, PVR but not B7-H6 expression in primary AML samples predicted their elimination by DOT cells. These data provide new mechanistic insight into tumor targeting by DOT cells and suggest that assessing PVR expression levels may be highly relevant to DOT cell-based clinical trials.
- Genetic modulation of RNA splicing rescues BRCA2 function in mutant cellsPublication . Lima, Beatriz Anjo; Pais, Ana Carolina; Dupont, Juliette; Dias, Patrícia; Custódio, Noélia; Sousa, Ana Berta; Carmo-Fonseca, Maria; Carvalho, CéliaVariants in the hereditary cancer-associated BRCA1 and BRCA2 genes can alter RNA splicing, producing transcripts that encode internally truncated yet potentially functional proteins. However, few studies have quantitatively analyzed variant-specific splicing isoforms. Here, we investigated cells heterozygous and homozygous for the BRCA2:c.681+5G>C variant. Using droplet digital RT-PCR, we identified two variant-specific mRNA isoforms. The predominant transcript is out-of-frame, contains a premature termination codon, and is degraded via the nonsense-mediated mRNA decay pathway. In addition, we detected a novel minor isoform encoding an internally truncated protein lacking non-essential domains. Homozygous mutant cells expressed low levels of BRCA2 protein and were defective in DNA repair. Using CRISPR-Cas9 gene editing, we induced the production of in-frame transcripts in mutant cells, which resulted in increased protein expression, enhanced RAD51 focus formation, and reduced chromosomal breaks after exposure to genotoxic agents. Our findings highlight the therapeutic potential of splicing modulation to restore BRCA2 function in mutant cells, offering a promising strategy to prevent cancer development.
- Co-transcriptional splicing and the CTD codePublication . Custódio, Noélia; Carmo-Fonseca, MariaTranscription and splicing are fundamental steps in gene expression. These processes have been studied intensively over the past four decades, and very recent findings are challenging some of the formerly established ideas. In particular, splicing was shown to occur much faster than previously thought, with the first spliced products observed as soon as splice junctions emerge from RNA polymerase II (Pol II). Splicing was also found coupled to a specific phosphorylation pattern of Pol II carboxyl-terminal domain (CTD), suggesting a new layer of complexity in the CTD code. Moreover, phosphorylation of the CTD may be scarcer than expected, and other post-translational modifications of the CTD are emerging with unanticipated roles in gene expression regulation.