RESUMEN
MicroRNAs (miRNAs) guide Argonaute (AGO) proteins to bind mRNA targets. Although most targets are destabilized by miRNA-AGO binding, some targets induce degradation of the miRNA instead. These special targets are also referred to as trigger RNAs. All triggers identified thus far have binding sites with greater complementarity to the miRNA than typical target sites. Target-directed miRNA degradation (TDMD) occurs when trigger RNAs bind the miRNA-AGO complex and recruit the ZSWIM8 E3 ubiquitin ligase, leading to AGO ubiquitination and proteolysis and subsequent miRNA destruction. More than 100 different miRNAs are regulated by ZSWIM8 in bilaterian animals, and hundreds of trigger RNAs have been predicted computationally. Disruption of individual trigger RNAs or ZSWIM8 has uncovered important developmental and physiologic roles for TDMD across a variety of model organisms and cell types. In this review, we highlight recent progress in understanding the mechanistic basis and functions of TDMD, describe common features of trigger RNAs, outline best practices for validating trigger RNAs, and discuss outstanding questions in the field.
Asunto(s)
MicroARNs , Estabilidad del ARN , Animales , Proteínas Argonautas/metabolismo , Sitios de Unión , MicroARNs/genética , MicroARNs/metabolismo , Proteolisis , UbiquitinaciónRESUMEN
MicroRNAs (miRNAs) guide Argonaute (AGO) proteins to bind and repress target RNAs. However, some unusual targets trigger destruction of the miRNA, a phenomenon known as target-directed miRNA degradation (TDMD). This Snapshot depicts our current understanding of how TDMD occurs and highlights established functions of TDMD in viruses and model organisms. To view this SnapShot, open or download the PDF.
Asunto(s)
MicroARNs , MicroARNs/genética , MicroARNs/metabolismo , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Estabilidad del ARNRESUMEN
MicroRNAs (miRNAs) pair to sites in mRNAs to direct the degradation of these RNA transcripts. Conversely, certain RNA transcripts can direct the degradation of particular miRNAs. This target-directed miRNA degradation (TDMD) requires the ZSWIM8 E3 ubiquitin ligase. Here, we report the function of ZSWIM8 in the mouse embryo. Zswim8 -/- embryos were smaller than their littermates and died near the time of birth. This highly penetrant perinatal lethality was apparently caused by a lung sacculation defect attributed to failed maturation of alveolar epithelial cells. Some mutant individuals also had heart ventricular septal defects. These developmental abnormalities were accompanied by aberrant accumulation of more than 50 miRNAs observed across 12 tissues, which often led to enhanced repression of their mRNA targets. These ZSWIM8-sensitive miRNAs were preferentially produced from genomic miRNA clusters, and in some cases, ZSWIM8 caused a switch in the dominant strand or isoform that accumulated from a miRNA hairpin-observations suggesting that TDMD provides a mechanism to uncouple coproduced miRNAs from each other. Overall, our findings indicate that the regulatory influence of ZSWIM8, and presumably TDMD, in mammalian biology is widespread and consequential, and posit the existence of many yet-unidentified transcripts that trigger miRNA degradation.
Asunto(s)
MicroARNs , Animales , Ratones , Embrión de Mamíferos/metabolismo , Genoma , Crecimiento y Desarrollo , Mamíferos/genética , MicroARNs/genética , MicroARNs/metabolismoRESUMEN
MicroRNAs (miRNAs) associate with Argonaute (AGO) proteins to direct widespread posttranscriptional gene repression. Although association with AGO typically protects miRNAs from nucleases, extensive pairing to some unusual target RNAs can trigger miRNA degradation. We found that this target-directed miRNA degradation (TDMD) required the ZSWIM8 Cullin-RING E3 ubiquitin ligase. This and other findings support a mechanistic model of TDMD in which target-directed proteolysis of AGO by the ubiquitin-proteasome pathway exposes the miRNA for degradation. Moreover, loss-of-function studies indicated that the ZSWIM8 Cullin-RING ligase accelerates degradation of numerous miRNAs in cells of mammals, flies, and nematodes, thereby specifying the half-lives of most short-lived miRNAs. These results elucidate the mechanism of TDMD and expand its inferred role in shaping miRNA levels in bilaterian animals.
Asunto(s)
Proteínas Argonautas/metabolismo , MicroARNs/metabolismo , Estabilidad del ARN , ARN Largo no Codificante/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Elonguina/genética , Elonguina/metabolismo , Técnicas de Silenciamiento del Gen , Humanos , Células K562 , Ratones , Células 3T3 NIH , Proteolisis , ARN Largo no Codificante/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Noncoding RNAs (ncRNAs) play increasingly appreciated gene-regulatory roles. Here, we describe a regulatory network centered on four ncRNAs-a long ncRNA, a circular RNA, and two microRNAs-using gene editing in mice to probe the molecular consequences of disrupting key components of this network. The long ncRNA Cyrano uses an extensively paired site to miR-7 to trigger destruction of this microRNA. Cyrano-directed miR-7 degradation is much more effective than previously described examples of target-directed microRNA degradation, which come primarily from studies of artificial and viral RNAs. By reducing miR-7 levels, Cyrano prevents repression of miR-7-targeted mRNAs and enables accumulation of Cdr1as, a circular RNA known to regulate neuronal activity. Without Cyrano, excess miR-7 causes cytoplasmic destruction of Cdr1as in neurons, in part through enhanced slicing of Cdr1as by a second miRNA, miR-671. Thus, several types of ncRNAs can collaborate to establish a sophisticated regulatory network.
Asunto(s)
Encéfalo/metabolismo , Redes Reguladoras de Genes , ARN no Traducido/metabolismo , Animales , Citoplasma/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , MicroARNs/genética , MicroARNs/metabolismo , Neuronas/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
BACKGROUND AND PURPOSE: The Heart of Glass (HEG) receptor binds KRIT1 and functions with KRIT1, CCM2, and PDCD10 in a common signaling pathway required for heart and vascular development. Mutations in KRIT1, CCM2, and PDCD10 also underlie human cerebral cavernous malformation (CCM) and postnatal loss of these genes in the mouse endothelium results in rapid CCM formation. Here, we test the role of HEG in CCM formation in mice and in humans. METHODS: We constitutively or conditionally deleted Heg and Ccm2 genes in genetically modified mice. Mouse embryos, brain, and retina tissues were analyzed to assess CCM lesion formation. RESULTS: In postnatal mice, CCMs form with Ccm2-/- but not with Heg-/- or Heg-/-;Ccm2+/- endothelial cells. Consistent with these findings, human patients with CCM who lack exonic mutations in KRIT1, CCM2, or PDCD10 do not have mutations in HEG. CONCLUSIONS: These findings suggest that the HEG-CCM signaling functions during cardiovascular development and growth, whereas CCMs arise because of loss of HEG-independent CCM signaling in the endothelium of the central nervous system after birth.
Asunto(s)
Endotelio/patología , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Proteínas de la Membrana/fisiología , Animales , Proteínas Reguladoras de la Apoptosis/genética , Encéfalo/patología , Proteínas Portadoras/genética , Feto/patología , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Humanos , Proteína KRIT1 , Proteínas de la Membrana/genética , Ratones , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Proteínas Asociadas a Microtúbulos/genética , Proteínas Proto-Oncogénicas/genética , Retina/patologíaRESUMEN
Cardiovascular growth must balance stabilizing signals required to maintain endothelial connections and network integrity with destabilizing signals that enable individual endothelial cells to migrate and proliferate. The cerebral cavernous malformation (CCM) signaling pathway utilizes the adaptor protein CCM2 to strengthen endothelial cell junctions and stabilize vessels. Here we identify a CCM2 paralog, CCM2L, that is expressed selectively in endothelial cells during periods of active cardiovascular growth. CCM2L competitively blocks CCM2-mediated stabilizing signals biochemically, in cultured endothelial cells, and in developing mice. Loss of CCM2L reduces endocardial growth factor expression and impairs tumor growth and wound healing. Our studies identify CCM2L as a molecular mechanism by which endothelial cells coordinately regulate vessel stability and growth during cardiovascular development, as well as postnatal vessel growth.
Asunto(s)
Malformaciones Vasculares del Sistema Nervioso Central/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Neovascularización Patológica , Secuencia de Aminoácidos , Animales , Malformaciones Vasculares del Sistema Nervioso Central/embriología , Malformaciones Vasculares del Sistema Nervioso Central/genética , Embrión de Mamíferos/irrigación sanguínea , Embrión de Mamíferos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Uniones Intercelulares/metabolismo , Proteína KRIT1 , Ratones , Ratones Noqueados , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/deficiencia , Datos de Secuencia Molecular , Unión Proteica , Proteínas Proto-Oncogénicas/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de SeñalRESUMEN
Cerebral cavernous malformation is a common human vascular disease that arises due to loss-of-function mutations in genes encoding three intracellular adaptor proteins, cerebral cavernous malformations 1 protein (CCM1), CCM2, and CCM3. CCM1, CCM2, and CCM3 interact biochemically in a pathway required in endothelial cells during cardiovascular development in mice and zebrafish. The downstream effectors by which this signaling pathway regulates endothelial function have not yet been identified. Here we have shown in zebrafish that expression of mutant ccm3 proteins (ccm3Delta) known to cause cerebral cavernous malformation in humans confers cardiovascular phenotypes identical to those associated with loss of ccm1 and ccm2. CCM3Delta proteins interacted with CCM1 and CCM2, but not with other proteins known to bind wild-type CCM3, serine/threonine protein kinase MST4 (MST4), sterile 20-like serine/threonine kinase 24 (STK24), and STK25, all of which have poorly defined biological functions. Cardiovascular phenotypes characteristic of CCM deficiency arose due to stk deficiency and combined low-level deficiency of stks and ccm3 in zebrafish embryos. In cultured human endothelial cells, CCM3 and STK25 regulated barrier function in a manner similar to CCM2, and STKs negatively regulated Rho by directly activating moesin. These studies identify STKs as essential downstream effectors of CCM signaling in development and disease that may regulate both endothelial and epithelial cell junctions.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/fisiología , Sistema Cardiovascular/embriología , Hemangioma Cavernoso del Sistema Nervioso Central/etiología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas de la Membrana/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Transducción de Señal/fisiología , Proteínas de Pez Cebra/fisiología , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/genética , Secuencia Conservada , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Proteínas de Microfilamentos/metabolismo , Proteínas Asociadas a Microtúbulos/fisiología , Datos de Secuencia Molecular , Proteínas Musculares , Fosforilación , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Alineación de Secuencia , Pez Cebra , Proteínas de Pez Cebra/química , Proteínas de Pez Cebra/genéticaRESUMEN
Cerebral cavernous malformations (CCMs) are human vascular malformations caused by mutations in three genes of unknown function: KRIT1, CCM2 and PDCD10. Here we show that the heart of glass (HEG1) receptor, which in zebrafish has been linked to ccm gene function, is selectively expressed in endothelial cells. Heg1(-/-) mice showed defective integrity of the heart, blood vessels and lymphatic vessels. Heg1(-/-); Ccm2(lacZ/+) and Ccm2(lacZ/lacZ) mice had more severe cardiovascular defects and died early in development owing to a failure of nascent endothelial cells to associate into patent vessels. This endothelial cell phenotype was shared by zebrafish embryos deficient in heg, krit1 or ccm2 and reproduced in CCM2-deficient human endothelial cells in vitro. Defects in the hearts of zebrafish lacking heg or ccm2, in the aortas of early mouse embryos lacking CCM2 and in the lymphatic vessels of neonatal mice lacking HEG1 were associated with abnormal endothelial cell junctions like those observed in human CCMs. Biochemical and cellular imaging analyses identified a cell-autonomous pathway in which the HEG1 receptor couples to KRIT1 at these cell junctions. This study identifies HEG1-CCM protein signaling as a crucial regulator of heart and vessel formation and integrity.
Asunto(s)
Sistema Cardiovascular/embriología , Proteínas Portadoras/fisiología , Hemangioma Cavernoso del Sistema Nervioso Central/fisiopatología , Glicoproteínas de Membrana/fisiología , Proteínas de Pez Cebra/fisiología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Hemorragia/genética , Humanos , Proteína KRIT1 , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
We have defined inactive alpha and omega fragments of beta-lactamase that can complement to form a functional enzyme in both bacteria and mammalian cells, serving as a readout for the interaction of proteins fused to the fragments. Critical to this advance was the identification of a tripeptide, Asn-Gly-Arg, which when juxtaposed at the carboxyl terminus of the alpha fragment increased complemented enzyme activity by up to 4 orders of magnitude. beta-Lactamase is well suited to monitoring constitutive and inducible protein interactions because it is small (29 kDa), monomeric, and assayable with a fluorescent cell-permeable substrate. The negligible background, the magnitude of induced signal caused by enzymatic amplification, and detection of signal within minutes are unparalleled in mammalian protein interaction detection systems published to date.