RESUMEN
Autophagy is a highly conserved degradation pathway that ensures nutrient recycling and removal of unwanted substrates. This process has a fundamental role in stress adaptation and maintenance of cellular homeostasis. Here, we discuss emerging aspects of the autophagy-RNA interplay, including autophagy-mediated degradation of RNA, RNA-binding proteins (RBPs), and ribonucleoprotein (RNP) complexes. Beyond degradation, we review new roles for autophagy players in the secretion and intracellular transport of RNA and related complexes. We discuss the physiological importance of these events for RNA homeostasis and gene expression programs, as well as their implications for disease, including cancer and neurodegeneration. Lastly, we examine how post-transcriptional regulation of autophagy, through specialized processing and selective translation of key transcripts, challenges and updates our current view of autophagy complexity.
Asunto(s)
Autofagia , ARN/metabolismo , Transporte Biológico , Homeostasis , Hidrólisis , Lisosomas/metabolismo , Ribonucleoproteínas/metabolismoRESUMEN
Phosphorylation of histone H3 at serine 10 (H3S10ph) by Aurora kinases plays an important role in mitosis; however, H3S10ph also marks regulatory regions of inducible genes in interphase mammalian cells, implicating mitosis-independent functions. Using the fluorescent ubiquitin-mediated cell cycle indicator (FUCCI), we found that 30% of the genome in interphase mouse embryonic stem cells (ESCs) is marked with H3S10ph. H3S10ph broadly demarcates gene-rich regions in G1 and is positively correlated with domains of early DNA replication timing (RT) but negatively correlated with H3K9me2 and lamin-associated domains (LADs). Consistent with mitosis-independent kinase activity, this pattern was preserved in ESCs treated with Hesperadin, a potent inhibitor of Aurora B/C kinases. Disruption of H3S10ph by expression of nonphosphorylatable H3.3S10A results in ectopic spreading of H3K9me2 into adjacent euchromatic regions, mimicking the phenotype observed in Drosophila JIL-1 kinase mutants. Conversely, interphase H3S10ph domains expand in Ehmt1 (also known as Glp) null ESCs, revealing that H3S10ph deposition is restricted by H3K9me2. Strikingly, spreading of H3S10ph at RT transition regions (TTRs) is accompanied by aberrant transcription initiation of genes co-oriented with the replication fork in Ehmt1-/- and Ehmt2-/- ESCs, indicating that establishment of repressive chromatin on the leading strand following DNA synthesis may depend upon these lysine methyltransferases. H3S10ph is also anti-correlated with H3K9me2 in interphase murine embryonic fibroblasts (MEFs) and is restricted to intragenic regions of actively transcribing genes by EHMT2. Taken together, these observations reveal that H3S10ph may play a general role in restricting the spreading of repressive chromatin in interphase mammalian cells.
Asunto(s)
Cromatina/metabolismo , Replicación del ADN/fisiología , Fibroblastos/metabolismo , Histonas/metabolismo , Interfase/fisiología , Células Madre Embrionarias de Ratones/metabolismo , Animales , Cromatina/genética , Drosophila melanogaster , Fibroblastos/citología , Histonas/genética , Ratones , Ratones Noqueados , Células Madre Embrionarias de Ratones/citologíaRESUMEN
In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.
Asunto(s)
Autofagia , Animales , Autofagosomas , Autofagia/fisiología , Proteínas Relacionadas con la Autofagia/metabolismo , Bioensayo/normas , Biomarcadores , Humanos , LisosomasRESUMEN
Autophagy is a conserved degradation process that occurs in all eukaryotic cells and its dysfunction has been associated with various diseases including cancer. While a number of large-scale attempts have recently identified new molecular players in autophagy regulation, including proteins and microRNAs, little is known regarding the function of long non-coding RNAs (lncRNAs) in the regulation of this process. To identify new long non-coding RNAs with functional implications in autophagy, we performed a high-throughput RNAi screen targeting more than 600 lncRNA transcripts and monitored their effects on autophagy in MCF-7 cells. We identified 63 lncRNAs that affected GFP-LC3B puncta numbers significantly. We validated the strongest hit, the lncRNA DRAIC previously shown to impact cell proliferation, and revealed a novel role for this lncRNA in the regulation of autophagic flux. Interestingly, we find DRAIC's pro-proliferative effects to be autophagy-independent. This study serves as a valuable resource for researchers from both the lncRNA and autophagy fields as it advances the current understanding of autophagy regulation by non-coding RNAs.