Calreticulin (CALR) is a multifaceted protein primarily involved in intracellular protein control... more Calreticulin (CALR) is a multifaceted protein primarily involved in intracellular protein control processes. The identification of CALR mutations in essential thrombocythemia (ET) and primary myelofibrosis that are mutually exclusive with the JAK2 V617F mutation has stirred an intensive research interest about the molecular functions of CALR and its mutants in myeloproliferative neoplasms (MPNs) and its diagnostic/prognostic value. The recently characterized protein–protein interaction of CALR mutants and MPL receptor has advanced our knowledge on the functional role of CALR mutants in thrombocythemia but it has also uncovered limitations of the current established research models. Human cell lines and mouse models provide useful information but they lack the advantages provided by ex vivo primary cultures of physiologically relevant to the disease cell types [i.e., megakaryocytes (MKs), platelets]. The results from gene expression and chromatin occupancy analysis have focused on the JAK-STAT pathway activated in both JAK2 V617F-and CALR-mutated MPN patient groups, although a more complete analysis is needed to be performed in MKs. Stress related processes seem to be affected in CALR mutant ET-MKs, but the precise mechanism is not known yet. Herein, we describe a culture method for human MKs from peripheral blood progenitors, which could help further toward an unbiased characterization of the role of CALR in ET and MK differentiation.
Gene expression activity is heterogeneous in a population of isogenic cells. Identifying the mole... more Gene expression activity is heterogeneous in a population of isogenic cells. Identifying the molecular basis of this variability will improve our understanding of phenomena like tumor resistance to drugs, virus infection, or cell fate choice. The complexity of the molecular steps and machines involved in transcription and translation could introduce sources of randomness at many levels, but a common constraint to most of these processes is its energy dependence. In eukaryotic cells, most of this energy is provided by mitochondria. A clonal population of cells may show a large variability in the number and functionality of mitochondria. Here, we discuss how differences in the mitochondrial content of each cell contribute to heterogeneity in gene products. Changes in the amount of mitochondria can also entail drastic alterations of a cell's gene expression program, which ultimately leads to phenotypic diversity. Also watch the Video Abstract.
Noise in gene expression is a main determinant of phenotypic variability. Increasing experimental... more Noise in gene expression is a main determinant of phenotypic variability. Increasing experimental evidence suggests that genome-wide cellular constraints largely contribute to the heterogeneity observed in gene products. It is still unclear, however, which global factors mostly affect gene expression noise, and to which extent. Since eukaryotic gene expression is an energy demanding process, differences in the energy budget of each cell could determine gene expression differences. Here, we quantify the contribution of mitochondrial variability (a natural source of ATP variation) to global variability in gene expression. We find that changes in mitochondrial content can account for ~50% of the variability observed in protein levels. This is the combined result of the effect of mitochondria dosage on transcription and translation apparatus content and activities. Moreover, we find that mitochondrial levels have a large impact on alternative splicing, thus modulating both the abundance...
RESUMEN Objetivos Describimos un nuevo método para visualizar y cuantificar la actividad transcri... more RESUMEN Objetivos Describimos un nuevo método para visualizar y cuantificar la actividad transcripcional en la retina, como alternativa a los métodos autorradiográficos hasta ahora existentes y que nos ha permitido detectar" in vivo" la síntesis y transporte del ácido ribonucleico (ARN) con una alta resolución (Iborra y cols. J. Cell Science'98).
Este trabajo ha sido subvencionado por un proyecto de investigación FIS-FEDER (P1020191) del Inst... more Este trabajo ha sido subvencionado por un proyecto de investigación FIS-FEDER (P1020191) del Instituto de Salud Carlos III (Investigador Principal Dra. Pinazo-Durán) y beca de formación en investigación oftalmológica asociada a este proyecto otorgada a Sheila Pons Vázquez, y por un proyecto de investigación Fundaçao para a ciencia e tecnologia, Portugal (praxis XXI/BD/3395, praxis P/SAU/12287/1998) (Investigador Principal Prof.
La vitreorretinopatía proliferante (VRP) es la causa más frecuente de fracaso en la cirugía del d... more La vitreorretinopatía proliferante (VRP) es la causa más frecuente de fracaso en la cirugía del desprendimiento de retina (1-3). La VRP está causada por una proliferación fibrocelular que constituye membranas con capacidad contráctil, en la superficie y debajo de la retina (4).
Genes on different chromosomes can be spatially associated in the nucleus in several transcriptio... more Genes on different chromosomes can be spatially associated in the nucleus in several transcriptional and regulatory situations; however, the functional significance of such associations remains unclear. Using human erythropoiesis as a model, we show that five cotranscribed genes, which are found on four different chromosomes, associate with each other at significant but variable frequencies. Those genes most frequently in association lie in decondensed stretches of chromatin.
Summary Transcription factories are nuclear domains where gene transcription takes place although... more Summary Transcription factories are nuclear domains where gene transcription takes place although the molecular basis for their formation and maintenance are unknown. In this study, we explored how the properties of chromatin as a polymer may contribute to the structure of transcription factories. We found that transcriptional active chromatin contains modifications like histone H4 acetylated at Lysine 16 (H4K16ac). Single fibre analysis showed that this modification spans the entire body of the gene.
Summary The route taken by transcripts from synthetic sites in the nucleus to the cytoplasm has b... more Summary The route taken by transcripts from synthetic sites in the nucleus to the cytoplasm has been under scrutiny for years, but details of the pathway remain obscure. A new high-resolution method for mapping the pathway is described; HeLa cells are grown in Br-U so that the analogue is incorporated into RNA and exported to the cytoplasm, before Br-RNA is localized by immuno-electron microscopy. After exposure to low concentrations of Br-U for short periods, cells grow normally.
BACKGROUND. The homeostasis of the hematopoietic stem/progenitor cell pool relies on a fine-tuned... more BACKGROUND. The homeostasis of the hematopoietic stem/progenitor cell pool relies on a fine-tuned balance between self-renewal, differentiation and proliferation. Recent studies have proposed that mitochondria regulate these processes. Although recent work has contributed to understand the role of mitochondria during stem cell differentiation, it remains elusive whether the mitochondrial content/function impacts human hematopoietic stem versus progenitor function. DESIGN AND METHODS.
Alternative splicing enables higher eukaryotes to increase their repertoire of proteins derived f... more Alternative splicing enables higher eukaryotes to increase their repertoire of proteins derived from a restricted number of genes. However, the possibility that functional diversity may also be augmented by splicing between adjacent genes has been largely neglected. Here, we show that the human melanocortin 1 receptor (MC1R) gene, a critical component of the facultative skin pigmentation system, has a highly complex and inefficient poly(A) site which is instrumental in allowing intergenic splicing between this locus and its immediate downstream neighbour tubulin-β-III (TUBB3). These transcripts, which produce two distinct protein isoforms localizing to the plasma membrane and the endoplasmic reticulum, seem to be restricted to humans as no detectable chimeric mRNA could be found in MC1R expressing mouse melanocytes. Significantly, treatment with the MC1R agonist α-MSH or activation of the stress response kinase p38-MAPK, both key molecules associated with ultraviolet radiation dermal insult and subsequent skin tanning, result in a shift in expression from MC1R in favour of chimeric MC1R-TUBB3 isoforms in cultured melanocytes. We propose that these chimeric proteins serve to equip melanocytes with novel cellular phenotypes required as part of the pigmentation response.
The nuclear membrane is the defining feature of eukaryotes. It divides the cell into two function... more The nuclear membrane is the defining feature of eukaryotes. It divides the cell into two functionally specialized compartments, and it is widely assumed that translation is restricted to only one: the cytoplasm. However, recent results suggest that some translation takes place in nuclei closely coupled to transcription. Various labeling techniques are described that enable nascent peptides to be labeled and then localized wherever they might be in the cell.
Calreticulin (CALR) is a multifaceted protein primarily involved in intracellular protein control... more Calreticulin (CALR) is a multifaceted protein primarily involved in intracellular protein control processes. The identification of CALR mutations in essential thrombocythemia (ET) and primary myelofibrosis that are mutually exclusive with the JAK2 V617F mutation has stirred an intensive research interest about the molecular functions of CALR and its mutants in myeloproliferative neoplasms (MPNs) and its diagnostic/prognostic value. The recently characterized protein–protein interaction of CALR mutants and MPL receptor has advanced our knowledge on the functional role of CALR mutants in thrombocythemia but it has also uncovered limitations of the current established research models. Human cell lines and mouse models provide useful information but they lack the advantages provided by ex vivo primary cultures of physiologically relevant to the disease cell types [i.e., megakaryocytes (MKs), platelets]. The results from gene expression and chromatin occupancy analysis have focused on the JAK-STAT pathway activated in both JAK2 V617F-and CALR-mutated MPN patient groups, although a more complete analysis is needed to be performed in MKs. Stress related processes seem to be affected in CALR mutant ET-MKs, but the precise mechanism is not known yet. Herein, we describe a culture method for human MKs from peripheral blood progenitors, which could help further toward an unbiased characterization of the role of CALR in ET and MK differentiation.
Gene expression activity is heterogeneous in a population of isogenic cells. Identifying the mole... more Gene expression activity is heterogeneous in a population of isogenic cells. Identifying the molecular basis of this variability will improve our understanding of phenomena like tumor resistance to drugs, virus infection, or cell fate choice. The complexity of the molecular steps and machines involved in transcription and translation could introduce sources of randomness at many levels, but a common constraint to most of these processes is its energy dependence. In eukaryotic cells, most of this energy is provided by mitochondria. A clonal population of cells may show a large variability in the number and functionality of mitochondria. Here, we discuss how differences in the mitochondrial content of each cell contribute to heterogeneity in gene products. Changes in the amount of mitochondria can also entail drastic alterations of a cell's gene expression program, which ultimately leads to phenotypic diversity. Also watch the Video Abstract.
Noise in gene expression is a main determinant of phenotypic variability. Increasing experimental... more Noise in gene expression is a main determinant of phenotypic variability. Increasing experimental evidence suggests that genome-wide cellular constraints largely contribute to the heterogeneity observed in gene products. It is still unclear, however, which global factors mostly affect gene expression noise, and to which extent. Since eukaryotic gene expression is an energy demanding process, differences in the energy budget of each cell could determine gene expression differences. Here, we quantify the contribution of mitochondrial variability (a natural source of ATP variation) to global variability in gene expression. We find that changes in mitochondrial content can account for ~50% of the variability observed in protein levels. This is the combined result of the effect of mitochondria dosage on transcription and translation apparatus content and activities. Moreover, we find that mitochondrial levels have a large impact on alternative splicing, thus modulating both the abundance...
RESUMEN Objetivos Describimos un nuevo método para visualizar y cuantificar la actividad transcri... more RESUMEN Objetivos Describimos un nuevo método para visualizar y cuantificar la actividad transcripcional en la retina, como alternativa a los métodos autorradiográficos hasta ahora existentes y que nos ha permitido detectar" in vivo" la síntesis y transporte del ácido ribonucleico (ARN) con una alta resolución (Iborra y cols. J. Cell Science'98).
Este trabajo ha sido subvencionado por un proyecto de investigación FIS-FEDER (P1020191) del Inst... more Este trabajo ha sido subvencionado por un proyecto de investigación FIS-FEDER (P1020191) del Instituto de Salud Carlos III (Investigador Principal Dra. Pinazo-Durán) y beca de formación en investigación oftalmológica asociada a este proyecto otorgada a Sheila Pons Vázquez, y por un proyecto de investigación Fundaçao para a ciencia e tecnologia, Portugal (praxis XXI/BD/3395, praxis P/SAU/12287/1998) (Investigador Principal Prof.
La vitreorretinopatía proliferante (VRP) es la causa más frecuente de fracaso en la cirugía del d... more La vitreorretinopatía proliferante (VRP) es la causa más frecuente de fracaso en la cirugía del desprendimiento de retina (1-3). La VRP está causada por una proliferación fibrocelular que constituye membranas con capacidad contráctil, en la superficie y debajo de la retina (4).
Genes on different chromosomes can be spatially associated in the nucleus in several transcriptio... more Genes on different chromosomes can be spatially associated in the nucleus in several transcriptional and regulatory situations; however, the functional significance of such associations remains unclear. Using human erythropoiesis as a model, we show that five cotranscribed genes, which are found on four different chromosomes, associate with each other at significant but variable frequencies. Those genes most frequently in association lie in decondensed stretches of chromatin.
Summary Transcription factories are nuclear domains where gene transcription takes place although... more Summary Transcription factories are nuclear domains where gene transcription takes place although the molecular basis for their formation and maintenance are unknown. In this study, we explored how the properties of chromatin as a polymer may contribute to the structure of transcription factories. We found that transcriptional active chromatin contains modifications like histone H4 acetylated at Lysine 16 (H4K16ac). Single fibre analysis showed that this modification spans the entire body of the gene.
Summary The route taken by transcripts from synthetic sites in the nucleus to the cytoplasm has b... more Summary The route taken by transcripts from synthetic sites in the nucleus to the cytoplasm has been under scrutiny for years, but details of the pathway remain obscure. A new high-resolution method for mapping the pathway is described; HeLa cells are grown in Br-U so that the analogue is incorporated into RNA and exported to the cytoplasm, before Br-RNA is localized by immuno-electron microscopy. After exposure to low concentrations of Br-U for short periods, cells grow normally.
BACKGROUND. The homeostasis of the hematopoietic stem/progenitor cell pool relies on a fine-tuned... more BACKGROUND. The homeostasis of the hematopoietic stem/progenitor cell pool relies on a fine-tuned balance between self-renewal, differentiation and proliferation. Recent studies have proposed that mitochondria regulate these processes. Although recent work has contributed to understand the role of mitochondria during stem cell differentiation, it remains elusive whether the mitochondrial content/function impacts human hematopoietic stem versus progenitor function. DESIGN AND METHODS.
Alternative splicing enables higher eukaryotes to increase their repertoire of proteins derived f... more Alternative splicing enables higher eukaryotes to increase their repertoire of proteins derived from a restricted number of genes. However, the possibility that functional diversity may also be augmented by splicing between adjacent genes has been largely neglected. Here, we show that the human melanocortin 1 receptor (MC1R) gene, a critical component of the facultative skin pigmentation system, has a highly complex and inefficient poly(A) site which is instrumental in allowing intergenic splicing between this locus and its immediate downstream neighbour tubulin-β-III (TUBB3). These transcripts, which produce two distinct protein isoforms localizing to the plasma membrane and the endoplasmic reticulum, seem to be restricted to humans as no detectable chimeric mRNA could be found in MC1R expressing mouse melanocytes. Significantly, treatment with the MC1R agonist α-MSH or activation of the stress response kinase p38-MAPK, both key molecules associated with ultraviolet radiation dermal insult and subsequent skin tanning, result in a shift in expression from MC1R in favour of chimeric MC1R-TUBB3 isoforms in cultured melanocytes. We propose that these chimeric proteins serve to equip melanocytes with novel cellular phenotypes required as part of the pigmentation response.
The nuclear membrane is the defining feature of eukaryotes. It divides the cell into two function... more The nuclear membrane is the defining feature of eukaryotes. It divides the cell into two functionally specialized compartments, and it is widely assumed that translation is restricted to only one: the cytoplasm. However, recent results suggest that some translation takes place in nuclei closely coupled to transcription. Various labeling techniques are described that enable nascent peptides to be labeled and then localized wherever they might be in the cell.
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Papers by Francisco Iborra