Inhibition of nonsense-mediated mRNA decay reduces the tumorigenicity of human fibrosarcoma cells.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic RNA decay pathway with roles in cellular stress responses, differentiation, and viral defense. It functions in both quality control and post-transcriptional regulation of gene expression. NMD has also emerged as a modulator of cancer progression, although available evidence supports both a tumor suppressor and a pro-tumorigenic role, depending on the model. To further investigate the role of NMD in cancer, we knocked out the NMD factor SMG7 in the HT1080 human fibrosarcoma cell line, resulting in suppression of NMD function. We then compared the oncogenic properties of the parental cell line, the SMG7-knockout, and a rescue cell line in which we re-introduced both isoforms of SMG7. We also tested the effect of a drug inhibiting the NMD factor SMG1 to distinguish NMD-dependent effects from putative NMD-independent functions of SMG7. Using cell-based assays and a mouse xenograft tumor model, we showed that suppression of NMD function severely compromises the oncogenic phenotype. Molecular pathway analysis revealed that NMD suppression strongly reduces matrix metalloprotease 9 (MMP9) expression and that MMP9 re-expression partially rescues the oncogenic phenotype. Since MMP9 promotes cancer cell migration and invasion, metastasis and angiogenesis, its downregulation may contribute to the reduced tumorigenicity of NMD-suppressed cells. Collectively, our results highlight the potential value of NMD inhibition as a therapeutic approach

Enhancer turnover and conserved regulatory function in vertebrate evolution

Mutations in regulatory regions including enhancers are an important source of variation and innovation during evolution. Enhancers can evolve by changes in the sequence, arrangement and repertoire of transcription factor binding sites, but whole enhancers can also be lost or gained in certain lineages in a process of turnover. The proopiomelanocortin gene (Pomc), which encodes a prohormone, is expressed in the pituitary and hypothalamus of all jawed vertebrates. We have previously described that hypothalamic Pomc expression in mammals is controlled by two enhancers?nPE1 and nPE2?that are derived from transposable elements and that presumably replaced the ancestral neuronal Pomc regulatory regions. Here, we show that nPE1 and nPE2, even though they are mammalian novelties with no homologous counterpart in other vertebrates, nevertheless can drive gene expression specifically to POMC neurons in the hypothalamus of larval and adult transgenic zebrafish. This indicates that when neuronal Pomc enhancers originated de novo during early mammalian evolution, the newly created cis- and trans-codes were similar to the ancestral ones. We also identify the neuronal regulatory region of zebrafish pomca and confirm that it is not homologous to the mammalian enhancers. Our work sheds light on the process of gene regulatory evolution by showing how a locus can undergo enhancer turnover and nevertheless maintain the ancestral transcriptional output.Fil: Domene, Sabina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Bumaschny, Viviana Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Universidad de Buenos Aires. Facultad de Medicina; ArgentinaFil: Silva Junqueira de Souza, Flavio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Franchini, Lucia Florencia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Nasif, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Low, Malcolm J.. University of Michigan. Medical School. Department of Molecular and Integrative Physiology; Estados UnidosFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentin

Islet-1 specifies the identity of hypothalamic melanocortin neurons and is critical for normal food intake and adiposity in adulthood

Food intake and body weight regulation depend on proper expression of the proopiomelanocortin gene (Pomc) in a group of neurons located in the mediobasal hypothalamus of all vertebrates. These neurons release POMC-encoded melanocortins, which are potent anorexigenic neuropeptides, and their absence from mice or humans leads to hyperphagia and severe obesity. Although the pathophysiology of hypothalamic POMC neurons is well understood, the genetic program that establishes the neuronal melanocortinergic phenotype and maintains a fully functional neuronal POMC phenotype throughout adulthood remains unknown. Here, we report that the early expression of the LIM-homeodomain transcription factor Islet 1 (ISL1) in the developing hypothalamus promotes the terminal differentiation of melanocortinergic neurons and is essential for hypothalamic Pomc expression since its initial onset and throughout the entire lifetime. We detected ISL1 in the prospective hypothalamus just before the onset of Pomc expression and, from then on, Pomc and Isl1 coexpress. ISL1 binds in vitro and in vivo to critical homeodomain binding DNA motifs present in the neuronal Pomc enhancers nPE1 and nPE2, and mutations of these sites completely disrupt the ability of these enhancers to drive reporter gene expression to hypothalamic POMC neurons in transgenic mice and zebrafish. ISL1 is necessary for hypothalamic Pomc expression during mouse and zebrafish embryogenesis. Furthermore, conditional Isl1 inactivation from POMC neurons impairs Pomc expression, leading to hyperphagia and obesity. Our results demonstrate that ISL1 specifies the identity of hypothalamic melanocortin neurons and is required for melanocortin-induced satiety and normal adiposity throughout the entire lifespanFil: Nasif, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Silva Junqueira de Souza, Flavio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Gonzalez, Laura Elisabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Yamashita, Miho. University of Michigan. Department of Molecular and Integrative Physiology; Estados UnidosFil: Orquera, Daniela Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Low, Malcolm J.. University of Michigan. Department of Molecular and Integrative Physiology; Estados UnidosFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina. University of Michigan. Department of Molecular and Integrative Physiology; Estados Unido

Production of human cell lines with mutations and deletions in endogenous NMD factors using CRISPR/Cas genome editing

Nonsense-mediated mRNA decay (NMD) is a eukaryotic RNA degradation pathway that modulates the abundance of 3-10% of all cellular mRNAs in mammalian cells. NMD constitutes a cellular quality control system that prevents the production of potentially harmful truncated proteins by targeting for degradation mRNAs with premature termination codons. Additionally, NMD targets many physiological mRNAs that encode full-length functional proteins and has therefore been implicated in the regulation of many different biological processes like stress response and cellular differentiation. Mammalian NMD relies on, at least, two different pathways for degrading its target mRNAs: the endonucleolytic pathway that depends on SMG6 endonuclease activity and the deadenylation-coupled exonucleolytic pathway for which the SMG5-SMG7 heterodimer is required. Available data suggests that these two degradation pathways act redundantly in human cells, but the accurate quantitative assessment of the relative contribution of each pathway to overall NMD has been elusive until now, mainly because of the use of knockdown strategies that never result in complete inactivation of the respective pathway. We therefore plan to investigate this by generating genome-edited human cell lines in which we mutate or delete SMG6 and SMG7 to inactivate, respectively, the endo- or exo- nucleolytic degradation pathways. The experimental approaches to generate the mutant cell lines by CRISPR/Cas genome editing will be presented. Characterization of these mutant cell lines will enable us to quantitatively assess the contribution of each decay pathway to NMD and to determine if different transcripts are preferentially targeted by one or the other pathway, providing further insight into the different roles of NMD as a general post-transcriptional gene regulatory mechanism

Nonsense-mediated mRNA decay: novel mechanistic insights and biological impact

Nonsense-mediated mRNA decay (NMD) was originally coined to define a quality control mechanism that targets mRNAs with truncated open reading frames due to the presence of a premature termination codon. Meanwhile, it became clear that NMD has a much broader impact on gene expression and additional biological functions beyond quality control are continuously being discovered. We review here the current views regarding the molecular mechanisms of NMD, according to which NMD ensues on mRNAs that fail to terminate translation properly, and point out the gaps in our understanding. We further summarize the recent literature on an ever-rising spectrum of biological processes in which NMD appears to be involved, including homeostatic control of gene expression, development and differentiation, as well as viral defense

Beyond quality control: The role of nonsense-mediated mRNA decay (NMD) in regulating gene expression

Nonsense-mediated mRNA decay (NMD) has traditionally been described as a quality control system that rids cells of aberrant mRNAs with crippled protein coding potential. However, transcriptome-wide profiling of NMD deficient cells identified a plethora of seemingly intact mRNAs coding for functional proteins as NMD targets. This led to the view that NMD constitutes an additional post-transcriptional layer of gene expression control involved in the regulation of many different biological pathways. Here, we review our current knowledge about the role of NMD in embryonic development and tissue-specific cell differentiation. We further summarize how NMD contributes to balancing of the integrated stress response and to cellular homeostasis of splicing regulators and NMD factors through auto-regulatory feedback loops. In addition, we discuss recent evidence that suggests a role for NMD as an innate immune response against several viruses. Altogether, NMD appears to play an important role in a broad spectrum of biological pathways, many of which still remain to be discovered

Essential function of the transcription factor Rax in the early patterning of the mammalian hypothalamus

The hypothalamus is a region of the anterior forebrain that controls basic aspects of vertebrate physiology, but the genes involved in its development are still poorly understood. Here, we investigate the function of the homeobox gene Rax/Rx in early hypothalamic development using a conditional targeted inactivation strategy in the mouse. We found that lack of Rax expression prior to embryonic day 8.5 (E8.5) caused a general underdevelopment of the hypothalamic neuroepithelium, while inactivation at later timepoints had little effect. The early absence of Rax impaired neurogenesis and prevented the expression of molecular markers of the dorsomedial hypothalamus, including neuropeptides Proopiomelanocortin and Somatostatin. Interestingly, the expression domains of genes expressed in the ventromedial hypothalamus and infundibulum invaded dorsal hypothalamic territory, showing that Rax is needed for the proper dorsoventral patterning of the developing medial hypothalamus. The phenotypes caused by the early loss of Rax are similar to those of eliminating the expression of the morphogen Sonic hedgehog (Shh) specifically from the hypothalamus. Consistent with this similarity in phenotypes, we observed that Shh and Rax are coexpressed in the rostral forebrain at late head fold stages and that loss of Rax caused a downregulation of Shh expression in the dorsomedial portion of the hypothalamus.Fil: Orquera, Daniela Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; ArgentinaFil: Nasif, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; ArgentinaFil: Low, Malcolm J.. Department Of Molecular And Integrative Physiology; Estados UnidosFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina. University of Michigan; Estados UnidosFil: Silva Junqueira de Souza, Flavio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular ; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentin

The homeodomain transcription factor NKX2.1 is essential for the early specification of melanocortin neuron identity and activates Pomc expression in the developing hypothalamus

Food intake is tightly regulated by a group of neurons present in the arcuate nucleus of the hypothalamus, which release Pomc-encoded melanocortins, the absence of which induces marked hyperphagia and early-onset obesity. Although the relevance of hypothalamic POMCneurons in the regulation of body weight and energy balance is well appreciated, little is known about the transcription factors that establish the melanocortin neuron identity during brain development and its phenotypic maintenance in postnatal life. Here, we report that the transcription factor NKX2.1 is present in mouse hypothalamic POMC neurons from early development to adulthood. Electromobility shift assays showed that NKX2.1 binds in vitro to NKX binding motifs present in the neuronal Pomc enhancers nPE1 and nPE2 and chromatin immunoprecipitation assays detected in vivo binding of NKX2.1 to nPE1 and nPE2 in mouse hypothalamic extracts. Transgenic and mutant studies performed in mouse embryos of either sex and adult males showed that the NKX motifs present in nPE1 and nPE2 are essential for their transcriptional enhancer activity. The conditional early inactivation of Nkx2.1 in the ventral hypothalamus prevented the onset of Pomc expression. Selective Nkx2.1 ablation from POMC neurons decreased Pomc expression in adult males and mildly increased their body weight and adiposity. Our results demonstrate that NKX2.1 is necessary to activate Pomc expression by binding to conserved canonical NKX motifs present in nPE1 and nPE2. Therefore, NKX2.1 plays a critical role in the early establishment of hypothalamic melanocortin neuron identity and participates in the maintenance of Pomc expression levels during adulthood.Fil: Orquera, Daniela Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Tavella, Maria Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Silva Junqueira de Souza, Flavio. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; ArgentinaFil: Nasif, Sofia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; ArgentinaFil: Low, Malcolm J.. Michigan State University; Estados UnidosFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Fisiología, Biología Molecular y Celular; Argentina. Michigan State University; Estados Unido

CRISPR-Trap:a clean approach for the generation of gene knockouts and gene replacements in human cells

Conventional nonhomologous end joining–based gene knockouts can lead to the production of C-terminally truncated proteins with potentially residual or dominant negative functions. Combining CRISPR/Cas9 with gene traps targeting the first intron (CRISPR-Trap), however, completely prevents the expression of the open reading frame, resulting in clean gene knockouts.</jats:p