RESUMEN
Many of the most highly conserved elements in the human genome are "poison exons," alternatively spliced exons that contain premature termination codons and permit post-transcriptional regulation of mRNA abundance through induction of nonsense-mediated mRNA decay (NMD). Poison exons are widely assumed to be highly conserved due to their presumed importance for organismal fitness, but this functional importance has never been tested in the context of a whole organism. Here, we report that a poison exon in Smndc1 is conserved across mammals and plants and plays a molecular autoregulatory function in both kingdoms. We generated mouse and A. thaliana models lacking this poison exon to find its loss leads to deregulation of SMNDC1 protein levels, pervasive alterations in mRNA processing, and organismal size restriction. Together, these models demonstrate the importance of poison exons for both molecular and organismal phenotypes that likely explain their extraordinary conservation.
Asunto(s)
Empalme Alternativo , Arabidopsis , Exones , Degradación de ARNm Mediada por Codón sin Sentido , Animales , Exones/genética , Ratones , Humanos , Degradación de ARNm Mediada por Codón sin Sentido/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Empalme Alternativo/genética , Codón sin Sentido/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secuencia ConservadaRESUMEN
Alternative polyadenylation (APA) is strikingly dysregulated in many cancers. Although global APA dysregulation is frequently associated with poor prognosis, the importance of most individual APA events is controversial simply because few have been functionally studied. Here, we address this gap by developing a CRISPR-Cas9-based screen to manipulate endogenous polyadenylation and systematically quantify how APA events contribute to tumor growth in vivo. Our screen reveals individual APA events that control mouse melanoma growth in an immunocompetent host, with concordant associations in clinical human cancer. For example, forced Atg7 3' UTR lengthening in mouse melanoma suppresses ATG7 protein levels, slows tumor growth, and improves host survival; similarly, in clinical human melanoma, a long ATG7 3' UTR is associated with significantly prolonged patient survival. Overall, our study provides an easily adaptable means to functionally dissect APA in physiological systems and directly quantifies the contributions of recurrent APA events to tumorigenic phenotypes.
Asunto(s)
Melanoma , Poliadenilación , Animales , Ratones , Humanos , Regiones no Traducidas 3'/genética , Melanoma/genética , Detección Precoz del CáncerRESUMEN
Lack of oxygen (hypoxia and anoxia) is detrimental to cell function and survival and underlies many disease conditions. Hence, metazoans have evolved mechanisms to adapt to low oxygen. One such mechanism, metabolic suppression, decreases the cellular demand for oxygen by downregulating ATP-demanding processes. However, the molecular mechanisms underlying this adaptation are poorly understood. Here, we report on the role of ndrg1a in hypoxia adaptation of the anoxia-tolerant zebrafish embryo. ndrg1a is expressed in the kidney and ionocytes, cell types that use large amounts of ATP to maintain ion homeostasis. ndrg1a mutants are viable and develop normally when raised under normal oxygen. However, their survival and kidney function is reduced relative to WT embryos following exposure to prolonged anoxia. We further demonstrate that Ndrg1a binds to the energy-demanding sodium-potassium ATPase (NKA) pump under anoxia and is required for its degradation, which may preserve ATP in the kidney and ionocytes and contribute to energy homeostasis. Lastly, we show that sodium azide treatment, which increases lactate levels under normoxia, is sufficient to trigger NKA degradation in an Ndrg1a-dependent manner. These findings support a model whereby Ndrg1a is essential for hypoxia adaptation and functions downstream of lactate signaling to induce NKA degradation, a process known to conserve cellular energy.
Asunto(s)
Hipoxia , Pez Cebra , Adenosina Trifosfato/metabolismo , Animales , Hipoxia/genética , Lactatos , Oxígeno/metabolismo , Potasio/metabolismo , Sodio/metabolismo , Azida Sódica/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Pez Cebra/metabolismoRESUMEN
Many cancers carry recurrent, change-of-function mutations affecting RNA splicing factors. Here, we describe a method to harness this abnormal splicing activity to drive splicing factor mutation-dependent gene expression to selectively eliminate tumor cells. We engineered synthetic introns that were efficiently spliced in cancer cells bearing SF3B1 mutations, but unspliced in otherwise isogenic wild-type cells, to yield mutation-dependent protein production. A massively parallel screen of 8,878 introns delineated ideal intronic size and mapped elements underlying mutation-dependent splicing. Synthetic introns enabled mutation-dependent expression of herpes simplex virus-thymidine kinase (HSV-TK) and subsequent ganciclovir (GCV)-mediated killing of SF3B1-mutant leukemia, breast cancer, uveal melanoma and pancreatic cancer cells in vitro, while leaving wild-type cells unaffected. Delivery of synthetic intron-containing HSV-TK constructs to leukemia, breast cancer and uveal melanoma cells and GCV treatment in vivo significantly suppressed the growth of these otherwise lethal xenografts and improved mouse host survival. Synthetic introns provide a means to exploit tumor-specific changes in RNA splicing for cancer gene therapy.
Asunto(s)
Neoplasias de la Mama , Leucemia , Melanoma , Animales , Antivirales , Neoplasias de la Mama/genética , Femenino , Ganciclovir/metabolismo , Ganciclovir/farmacología , Terapia Genética/métodos , Humanos , Intrones/genética , Leucemia/genética , Melanoma/genética , Melanoma/terapia , Ratones , Mutación/genética , Factores de Empalme de ARN/genética , Timidina Quinasa/genética , Timidina Quinasa/metabolismo , Neoplasias de la ÚveaRESUMEN
CRISPR screens have accelerated the discovery of important cancer vulnerabilities. However, single-gene knockout phenotypes can be masked by redundancy among related genes. Paralogs constitute two-thirds of the human protein-coding genome, so existing methods are likely inadequate for assaying a large portion of gene function. Here, we develop paired guide RNAs for paralog genetic interaction mapping (pgPEN), a pooled CRISPR-Cas9 single- and double-knockout approach targeting more than 2,000 human paralogs. We apply pgPEN to two cell types and discover that 12% of human paralogs exhibit synthetic lethality in at least one context. We recover known synthetic lethal paralogs MEK1/MEK2, important drug targets CDK4/CDK6, and other synthetic lethal pairs including CCNL1/CCNL2. Additionally, we identify ten tumor suppressor paralog pairs whose compound loss promotes cell proliferation. These findings nominate drug targets and suggest that paralog genetic interactions could shape the landscape of positive and negative selection in cancer.
Asunto(s)
Duplicación de Gen , Genes Letales , Genes Sintéticos , Genes Supresores de Tumor , Genoma Humano , Neoplasias/genética , Adulto , Antineoplásicos/farmacología , Proteína 9 Asociada a CRISPR/genética , Sistemas CRISPR-Cas , Proliferación Celular , Femenino , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Humanos , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Neoplasias/tratamiento farmacológico , Neoplasias/patología , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismoRESUMEN
CRISPR-based cancer dependency maps are accelerating advances in cancer precision medicine, but adequate functional maps are limited to the most common oncogenes. To identify opportunities for therapeutic intervention in other rarer subsets of cancer, we investigate the oncogene-specific dependencies conferred by the lung cancer oncogene, RIT1. Here, genome-wide CRISPR screening in KRAS, EGFR, and RIT1-mutant isogenic lung cancer cells identifies shared and unique vulnerabilities of each oncogene. Combining this genetic data with small-molecule sensitivity profiling, we identify a unique vulnerability of RIT1-mutant cells to loss of spindle assembly checkpoint regulators. Oncogenic RIT1M90I weakens the spindle assembly checkpoint and perturbs mitotic timing, resulting in sensitivity to Aurora A inhibition. In addition, we observe synergy between mutant RIT1 and activation of YAP1 in multiple models and frequent nuclear overexpression of YAP1 in human primary RIT1-mutant lung tumors. These results provide a genome-wide atlas of oncogenic RIT1 functional interactions and identify components of the RAS pathway, spindle assembly checkpoint, and Hippo/YAP1 network as candidate therapeutic targets in RIT1-mutant lung cancer.
Asunto(s)
Neoplasias Pulmonares/genética , Oncogenes/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Ciclo Celular/genética , Línea Celular Tumoral , Receptores ErbB/genética , Femenino , Técnicas de Inactivación de Genes , Ensayos Analíticos de Alto Rendimiento , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Masculino , Ratones , Terapia Molecular Dirigida , Mutación , Células 3T3 NIH , Proteínas Proto-Oncogénicas p21(ras)/genética , Factores de Transcripción/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Señalizadoras YAP , Proteínas rasRESUMEN
Although mutations in DNA are the best-studied source of neoantigens that determine response to immune checkpoint blockade, alterations in RNA splicing within cancer cells could similarly result in neoepitope production. However, the endogenous antigenicity and clinical potential of such splicing-derived epitopes have not been tested. Here, we demonstrate that pharmacologic modulation of splicing via specific drug classes generates bona fide neoantigens and elicits anti-tumor immunity, augmenting checkpoint immunotherapy. Splicing modulation inhibited tumor growth and enhanced checkpoint blockade in a manner dependent on host T cells and peptides presented on tumor MHC class I. Splicing modulation induced stereotyped splicing changes across tumor types, altering the MHC I-bound immunopeptidome to yield splicing-derived neoepitopes that trigger an anti-tumor T cell response in vivo. These data definitively identify splicing modulation as an untapped source of immunogenic peptides and provide a means to enhance response to checkpoint blockade that is readily translatable to the clinic.
Asunto(s)
Neoplasias/genética , Neoplasias/inmunología , Empalme del ARN/genética , Animales , Presentación de Antígeno/efectos de los fármacos , Presentación de Antígeno/inmunología , Antígenos de Neoplasias/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Epítopos/inmunología , Etilenodiaminas/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Hematopoyesis/efectos de los fármacos , Hematopoyesis/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inhibidores de Puntos de Control Inmunológico/farmacología , Inmunoterapia , Inflamación/patología , Ratones Endogámicos C57BL , Péptidos/metabolismo , Isoformas de Proteínas/metabolismo , Pirroles/farmacología , Empalme del ARN/efectos de los fármacos , Sulfonamidas/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/inmunologíaRESUMEN
While RNA-seq has enabled comprehensive quantification of alternative splicing, no correspondingly high-throughput assay exists for functionally interrogating individual isoforms. We describe pgFARM (paired guide RNAs for alternative exon removal), a CRISPR-Cas9-based method to manipulate isoforms independent of gene inactivation. This approach enabled rapid suppression of exon recognition in polyclonal settings to identify functional roles for individual exons, such as an SMNDC1 cassette exon that regulates pan-cancer intron retention. We generalized this method to a pooled screen to measure the functional relevance of 'poison' cassette exons, which disrupt their host genes' reading frames yet are frequently ultraconserved. Many poison exons were essential for the growth of both cultured cells and lung adenocarcinoma xenografts, while a subset had clinically relevant tumor-suppressor activity. The essentiality and cancer relevance of poison exons are likely to contribute to their unusually high conservation and contrast with the dispensability of other ultraconserved elements for viability.