RESUMEN
Small non-protein coding RNAs are involved in pathways that control the genome at the level of chromatin. In Schizosaccharomyces pombe, small interfering RNAs (siRNAs) are required for the faithful propagation of heterochromatin that is found at peri-centromeric repeats. In contrast to repetitive DNA, protein-coding genes are refractory to siRNA-mediated heterochromatin formation, unless siRNAs are expressed in mutant cells. Here we report the identification of 20 novel mutant alleles that enable de novo formation of heterochromatin at a euchromatic protein-coding gene by using trans-acting siRNAs as triggers. For example, a single amino acid substitution in the pre-mRNA cleavage factor Yth1 enables siRNAs to trigger silent chromatin formation with unparalleled efficiency. Our results are consistent with a kinetic nascent transcript processing model for the inhibition of small-RNA-directed de novo formation of heterochromatin and lay a foundation for further mechanistic dissection of cellular activities that counteract epigenetic gene silencing.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Silenciador del Gen , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Schizosaccharomyces/genética , Factores de Escisión y Poliadenilación de ARNm/genética , Alelos , Sustitución de Aminoácidos , Centrómero/química , Centrómero/metabolismo , Ensamble y Desensamble de Cromatina , Perfilación de la Expresión Génica , Heterocromatina/química , Heterocromatina/metabolismo , Cinética , Modelos Genéticos , Anotación de Secuencia Molecular , Mutación , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Schizosaccharomyces/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismoRESUMEN
In recent years it has become evident that RNA interference-related mechanisms can mediate the deposition and transgenerational inheritance of specific chromatin modifications in a truly epigenetic fashion. Rapid progress has been made in identifying the RNAi effector proteins and how they work together to confer long-lasting epigenetic responses, and initial studies hint at potential physiological relevance of such regulation. In this review, we highlight mechanistic studies in model organisms that advance our understanding of how small RNAs trigger long-lasting epigenetic changes in gene expression and we discuss observations that lend support for the idea that small RNAs might participate in mechanisms that trigger epigenetic gene expression changes in response to environmental cues and the effects these could have on population adaptation.
Asunto(s)
Epigénesis Genética/genética , Evolución Molecular , Interacción Gen-Ambiente , ARN/genética , Ensamble y Desensamble de Cromatina/genética , Regulación de la Expresión Génica/genética , Silenciador del Gen , Interferencia de ARN , Transducción de Señal/genéticaRESUMEN
Understanding the function of the thousands of cellular proteins is a central question in molecular cell biology. As proteins are typically part of multiple dynamic and often overlapping macromolecular complexes exerting distinct functions, the identification of protein-protein interactions (PPI) and their assignment to specific complexes is a crucial but challenging task. We present a protein fragments complementation assay integrated with the proximity-dependent biotinylation technique BioID. Activated on the interaction of two proteins, split-BioID is a conditional proteomics approach that allows in a single and simple assay to both experimentally validate binary PPI and to unbiasedly identify additional interacting factors. Applying our method to the miRNA-mediated silencing pathway, we can probe the proteomes of two distinct functional complexes containing the Ago2 protein and uncover the protein GIGYF2 as a regulator of miRNA-mediated translation repression. Hence, we provide a novel tool to study dynamic spatiotemporally defined protein complexes in their native cellular environment.