RESUMEN
We report the discovery of a class of abundant circular noncoding RNAs that are produced during metazoan tRNA splicing. These transcripts, termed tRNA intronic circular (tric)RNAs, are conserved features of animal transcriptomes. Biogenesis of tricRNAs requires anciently conserved tRNA sequence motifs and processing enzymes, and their expression is regulated in an age-dependent and tissue-specific manner. Furthermore, we exploited this biogenesis pathway to develop an in vivo expression system for generating "designer" circular RNAs in human cells. Reporter constructs expressing RNA aptamers such as Spinach and Broccoli can be used to follow the transcription and subcellular localization of tricRNAs in living cells. Owing to the superior stability of circular vs. linear RNA isoforms, this expression system has a wide range of potential applications, from basic research to pharmaceutical science.
Asunto(s)
Drosophila/genética , ARN de Transferencia/química , ARN de Transferencia/genética , ARN/química , ARN/metabolismo , Animales , Femenino , Genes Reporteros , Células HEK293 , Humanos , Intrones , Masculino , Modelos Moleculares , Conformación de Ácido Nucleico , Estabilidad del ARN , ARN Circular , TranscriptomaRESUMEN
Circular (circ)RNAs have recently become a subject of great biologic interest. It is now clear that they represent a diverse and abundant class of RNAs with regulated expression and evolutionarily conserved functions. There are several mechanisms by which RNA circularization can occur in vivo. Here, we focus on the biogenesis of tRNA intronic circular RNAs (tricRNAs) in archaea and animals, and we detail their use as research tools for orthogonal, directed circRNA expression in vivo.
Asunto(s)
Aptámeros de Nucleótidos/genética , Ingeniería Genética/métodos , Precursores del ARN/genética , Sitios de Empalme de ARN , Empalme del ARN , ARN de Transferencia/genética , ARN/genética , Animales , Aptámeros de Nucleótidos/metabolismo , Archaea/genética , Archaea/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Exones , Células HEK293 , Células HeLa , Humanos , Intrones , Ratones , ARN/metabolismo , Precursores del ARN/metabolismo , ARN Circular , ARN de Transferencia/metabolismo , Empalmosomas/metabolismo , Empalmosomas/ultraestructuraRESUMEN
Mutations in the human survival motor neuron 1 (SMN) gene are the primary cause of spinal muscular atrophy (SMA), a devastating neuromuscular disorder. SMN protein has a well-characterized role in the biogenesis of small nuclear ribonucleoproteins (snRNPs), core components of the spliceosome. Additional tissue-specific and global functions have been ascribed to SMN; however, their relevance to SMA pathology is poorly understood and controversial. Using Drosophila as a model system, we created an allelic series of twelve Smn missense mutations, originally identified in human SMA patients. We show that animals expressing these SMA-causing mutations display a broad range of phenotypic severities, similar to the human disease. Furthermore, specific interactions with other proteins known to be important for SMN's role in RNP assembly are conserved. Intragenic complementation analyses revealed that the three most severe mutations, all of which map to the YG box self-oligomerization domain of SMN, display a stronger phenotype than the null allele and behave in a dominant fashion. In support of this finding, the severe YG box mutants are defective in self-interaction assays, yet maintain their ability to heterodimerize with wild-type SMN. When expressed at high levels, wild-type SMN is able to suppress the activity of the mutant protein. These results suggest that certain SMN mutants can sequester the wild-type protein into inactive complexes. Molecular modeling of the SMN YG box dimer provides a structural basis for this dominant phenotype. These data demonstrate that important structural and functional features of the SMN YG box are conserved between vertebrates and invertebrates, emphasizing the importance of self-interaction to the proper functioning of SMN.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila/genética , Atrofia Muscular Espinal/genética , Proteínas de Unión al ARN/genética , Proteínas del Complejo SMN/genética , Animales , Modelos Animales de Enfermedad , Proteínas de Drosophila/química , Humanos , Neuronas Motoras/patología , Atrofia Muscular Espinal/patología , Mutación Missense/genética , Fenotipo , Multimerización de Proteína/genética , Proteínas de Unión al ARN/química , Ribonucleoproteínas Nucleares Pequeñas/genética , Proteínas del Complejo SMN/química , Relación Estructura-ActividadRESUMEN
Here we report a human intellectual disability disease locus on chromosome 14q31.3 corresponding to mutation of the ZC3H14 gene that encodes a conserved polyadenosine RNA binding protein. We identify ZC3H14 mRNA transcripts in the human central nervous system, and we find that rodent ZC3H14 protein is expressed in hippocampal neurons and colocalizes with poly(A) RNA in neuronal cell bodies. A Drosophila melanogaster model of this disease created by mutation of the gene encoding the ZC3H14 ortholog dNab2, which also binds polyadenosine RNA, reveals that dNab2 is essential for development and required in neurons for normal locomotion and flight. Biochemical and genetic data indicate that dNab2 restricts bulk poly(A) tail length in vivo, suggesting that this function may underlie its role in development and disease. These studies reveal a conserved requirement for ZC3H14/dNab2 in the metazoan nervous system and identify a poly(A) RNA binding protein associated with a human brain disorder.