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 , Humanos , Ratones , Empalme Alternativo/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Codón sin Sentido/genética , Secuencia Conservada , Exones/genética , Degradación de ARNm Mediada por Codón sin Sentido/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
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
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
Mis-expression of DUX4 in skeletal muscle causes facioscapulohumeral muscular dystrophy (FSHD). Human DUX4 and mouse Dux are retrogenes derived from retrotransposition of the mRNA from the parental DUXC gene. Primates and rodents have lost the parental DUXC gene, and it is unknown whether DUXC had a similar role in driving an early pluripotent transcriptional program. Dogs and other Laurasiatherians have retained DUXC, providing an opportunity to determine the functional similarity to the retrotransposed DUX4 and Dux. Here, we identify the expression of two isoforms of DUXC mRNA in canine testis tissues: one encoding the canonical double homeodomain protein (DUXC), similar to DUX4/Dux, and a second that includes an in-frame alternative exon that disrupts the conserved amino acid sequence of the first homeodomain (DUXC-ALT). The expression of DUXC in canine cells induces a pluripotent program similar to DUX4 and Dux and induces the expression of a similar set of retrotransposons of the ERV/MaLR and LINE-1 families, as well as pericentromeric satellite repeats; whereas DUXC-ALT did not robustly activate gene expression in these assays. Important for preclinical models of FSHD, human DUX4 and canine DUXC show higher conservation of their homeodomains and corresponding binding motifs compared with the conservation between human DUX4 and mouse Dux, and human DUX4 activates a highly similar transcriptional program in canine cells. Together, these findings show that retrotransposition resulted in the loss of an alternatively spliced isoform and that DUXC containing mammals might be good candidates for certain preclinical models ofFSHD.
Asunto(s)
Distrofia Muscular Facioescapulohumeral , Secuencia de Aminoácidos , Animales , Perros , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Masculino , Mamíferos/metabolismo , Ratones , Músculo Esquelético/metabolismo , Distrofia Muscular Facioescapulohumeral/genética , Distrofia Muscular Facioescapulohumeral/metabolismo , ARN Mensajero/metabolismo , Retroelementos/genéticaRESUMEN
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.
Asunto(s)
Adenocarcinoma del Pulmón/patología , Empalme Alternativo , Exones/genética , Genes Supresores de Tumor , Isoformas de ARN/genética , Factores de Empalme de ARN/genética , ARN Mensajero/genética , Proteínas del Complejo SMN/genética , Adenocarcinoma del Pulmón/genética , Animales , Proliferación Celular , Células HeLa , Ensayos Analíticos de Alto Rendimiento , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Ratones , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Facioscapulohumeral dystrophy (FSHD) is the third most prevalent muscular dystrophy. A progressive disease, it presents clinically as weakness and wasting of the face, shoulder and upper arm muscles, with later involvement of the trunk and lower extremities. FSHD develops through complex genetic and epigenetic events that converge on a common mechanism of toxicity with mis-expression of the transcription factor double homeobox 4 (DUX4). There is currently no treatment available for FSHD. However, the consensus that ectopic DUX4 expression in skeletal muscle is the root cause of FSHD pathophysiology has allowed research efforts to turn toward cultivating a deeper understanding of DUX4 biology and the pathways that underlie FSHD muscle pathology, and to translational studies aimed at developing targeted therapeutics using ever more sophisticated cell and animal-based models of FSHD. This review summarizes recent advances in our understanding of FSHD, including the regulation and activity of DUX4 in its normal developmental roles as well as its pathological contexts. We highlight how these advances raise new questions and challenges for the field as it moves into the next decade of FSHD research.