Ana Eulalio

P bodies: at the crossroads of post-transcriptional pathways

Post-transcriptional processes have a central role in the regulation of eukaryotic gene expression. Although it has been known for a long time that these processes are functionally linked, often by the use of common protein factors, it has only recently become apparent that many of these processes are also physically connected. Indeed, proteins that are involved in mRNA degradation, translational repression, mRNA surveillance and RNA-mediated gene silencing, together with their mRNA targets, colocalize within discrete cytoplasmic domains known as P bodies. The available evidence indicates that P bodies are sites where mRNAs that are not being translated accumulate, the information carried by associated proteins and regulatory RNAs is integrated, and their fate — either translation, silencing or decay — is decided.

P-Body Formation Is a Consequence, Not the Cause, of RNA-Mediated Gene Silencing

ABSTRACT P bodies are cytoplasmic domains that contain proteins involved in diverse posttranscriptional processes, such as mRNA degradation, nonsense-mediated mRNA decay (NMD), translational repression, and RNA-mediated gene silencing. The localization of these proteins and their targets in P bodies raises the question of whether their spatial concentration in discrete cytoplasmic domains is required for posttranscriptional gene regulation. We show that processes such as mRNA decay, NMD, and RNA-mediated gene silencing are functional in cells lacking detectable microscopic P bodies. Although P bodies are not required for silencing, blocking small interfering RNA or microRNA silencing pathways at any step prevents P-body formation, indicating that P bodies arise as a consequence of silencing. Consistently, we show that releasing mRNAs from polysomes is insufficient to trigger P-body assembly: polysome-free mRNAs must enter silencing and/or decapping pathways to nucleate P bodies. Thus, even though P-body components play crucial roles in mRNA silencing and decay, aggregation into P bodies is not required for function but is instead a consequence of their activity.

Functional screening identifies miRNAs inducing cardiac regeneration

In mammals, enlargement of the heart during embryonic development is primarily dependent on the increase in cardiomyocyte numbers. Shortly after birth, however, cardiomyocytes stop proliferating and further growth of the myocardium occurs through hypertrophic enlargement of the existing myocytes. As a consequence of the minimal renewal of cardiomyocytes during adult life, repair of cardiac damage through myocardial regeneration is very limited. Here we show that the exogenous administration of selected microRNAs (miRNAs) markedly stimulates cardiomyocyte proliferation and promotes cardiac repair. We performed a high-content microscopy, high-throughput functional screening for human miRNAs that promoted neonatal cardiomyocyte proliferation using a whole-genome miRNA library. Forty miRNAs strongly increased both DNA synthesis and cytokinesis in neonatal mouse and rat cardiomyocytes. Two of these miRNAs (hsa-miR-590 and hsa-miR-199a) were further selected for testing and were shown to promote cell cycle re-entry of adult cardiomyocytes ex vivo and to promote cardiomyocyte proliferation in both neonatal and adult animals. After myocardial infarction in mice, these miRNAs stimulated marked cardiac regeneration and almost complete recovery of cardiac functional parameters. The miRNAs identified hold great promise for the treatment of cardiac pathologies consequent to cardiomyocyte loss.

GW182 interaction with Argonaute is essential for miRNA-mediated translational repression and mRNA decay

MicroRNAs (miRNAs) silence gene expression by binding 3′ untranslated regions of target mRNAs. Recent studies suggested silencing is achieved through either recruitment of eIF6, which prevents ribosome assembly, or displacement of eIF4E from the mRNA 5′ cap structure. Using Drosophila melanogaster cells, we show that eIF6 is not required for silencing. In contrast, silencing is abolished by mutating Argonaute 1 (AGO1) at two conserved phenylalanine residues predicted to mediate binding to the cap structure. Notably, we found these mutations also prevented AGO1 from interacting with GW182 and miRNAs, indicating that the essential role of these residues is unrelated to cap binding. Consistently, depleting GW182 or overexpressing its AGO1 binding domain relieved silencing of all reporters tested, including those lacking a poly(A) tail. Together, our findings show that miRNA function is effected by AGO1–GW182 complexes and the role of GW182 in silencing goes beyond promoting deadenylation.

Analysis of the host microRNA response to Salmonella uncovers the control of major cytokines by the let-7 family

MicroRNAs have well‐established roles in eukaryotic host responses to viruses and extracellular bacterial pathogens. In contrast, microRNA responses to invasive bacteria have remained unknown. Here, we report cell type‐dependent microRNA regulations upon infection of mammalian cells with the enteroinvasive pathogen, Salmonella Typhimurium. Murine macrophages strongly upregulate NF‐κB associated microRNAs; strikingly, these regulations which are induced by bacterial lipopolysaccharide (LPS) occur and persist regardless of successful host invasion and/or replication, or whether an inflammatory response is mounted, suggesting that microRNAs belong to the first line of anti‐bacterial defence. However, a suppression of the global immune regulator miR‐155 in endotoxin‐tolerant macrophages revealed that microRNA responses also depend on the status of infected cells. This study identifies the let‐7 family as the common denominator of Salmonella‐regulated microRNAs in macrophages and epithelial cells, and suggests that repression of let‐7 relieves cytokine IL‐6 and IL‐10 mRNAs from negative post‐transcriptional control. Our results establish a paradigm of microRNA‐mediated feed‐forward activation of inflammatory factors when mammalian cells are targeted by bacterial pathogens.

The GW182 protein family in animal cells: New insights into domains required for miRNA-mediated gene silencing

GW182 family proteins interact directly with Argonaute proteins and are required for miRNA-mediated gene silencing in animal cells. The domains of the GW182 proteins have recently been studied to determine their role in silencing. These studies revealed that the middle and C-terminal regions function as an autonomous domain with a repressive function that is independent of both the interaction with Argonaute proteins and of P-body localization. Such findings reinforce the idea that GW182 proteins are key components of miRNA repressor complexes in metazoa.

The mammalian microRNA response to bacterial infections

MicroRNAs are small RNAs that post-transcriptionally regulate eukaryotic gene expression. In addition to their involvement in a wide range of physiological and pathological processes, including viral infections, microRNAs are increasingly implicated in the eukaryotic response to bacterial pathogens. Recent studies have characterized changes in host microRNA expression following infection with exclusively extracellular (Helicobacter pylori) or intracellular (Salmonella enterica) Gram-negative bacteria, as well as in the response to Gram-positive (Listeria monocytogenes) and other pathogens (Mycobacterium and Francisella species). In this review, we discuss the emerging roles of microRNAs in mammalian host signaling and defense against bacterial pathogens.

A C-terminal silencing domain in GW182 is essential for miRNA function

Proteins of the GW182 family are essential for miRNA-mediated gene silencing in animal cells; they interact with Argonaute proteins (AGOs) and are required for both the translational repression and mRNA degradation mediated by miRNAs. To gain insight into the role of the GW182-AGO1 interaction in silencing, we generated protein mutants that do not interact and tested them in complementation assays. We show that silencing of miRNA targets requires the N-terminal domain of GW182, which interacts with AGO1 through multiple glycine-tryptophan (GW)-repeats. Indeed, a GW182 mutant that does not interact with AGO1 cannot rescue silencing in cells depleted of endogenous GW182. Conversely, silencing is impaired by mutations in AGO1 that strongly reduce the interaction with GW182 but not with miRNAs. We further show that a GW182 mutant that does not localize to P-bodies but interacts with AGO1 rescues silencing in GW182-depleted cells, even though in these cells, AGO1 also fails to localize to P-bodies. Finally, we show that in addition to the N-terminal AGO1-binding domain, the middle and C-terminal regions of GW182 (referred to as the bipartite silencing domain) are essential for silencing. Together our results indicate that miRNA silencing in animal cells is mediated by AGO1 in complex with GW182, and that P-body localization is not required for silencing.

Structural Basis for the Mutually Exclusive Anchoring of P Body Components Edc3 and Tral to the Dead Box Protein Ddx6/Me31B.

The DEAD box helicase DDX6/Me31B functions in translational repression and mRNA decapping. How particular RNA helicases are recruited specifically to distinct functional complexes is poorly understood. We present the crystal structure of the DDX6 C-terminal RecA-like domain bound to a highly conserved FDF sequence motif in the decapping activator EDC3. The FDF peptide adopts an alpha-helical conformation upon binding to DDX6, occupying a shallow groove opposite to the DDX6 surface involved in RNA binding and ATP hydrolysis. Mutagenesis of Me31B shows the relevance of the FDF interaction surface both for Me31Bs accumulation in P bodies and for its ability to repress the expression of bound mRNAs. The translational repressor Tral contains a similar FDF motif. Together with mutational and competition studies, the structure reveals why the interactions of Me31B with EDC3 and Tral are mutually exclusive and how the respective decapping and translational repressor complexes might hook onto an mRNA substrate.

The interactions of GW182 proteins with PABP and deadenylases are required for both translational repression and degradation of miRNA targets

Animal miRNAs silence the expression of mRNA targets through translational repression, deadenylation and subsequent mRNA degradation. Silencing requires association of miRNAs with an Argonaute protein and a GW182 family protein. In turn, GW182 proteins interact with poly(A)-binding protein (PABP) and the PAN2-PAN3 and CCR4-NOT deadenylase complexes. These interactions are required for the deadenylation and decay of miRNA targets. Recent studies have indicated that miRNAs repress translation before inducing target deadenylation and decay; however, whether translational repression and deadenylation are coupled or represent independent repressive mechanisms is unclear. Another remaining question is whether translational repression also requires GW182 proteins to interact with both PABP and deadenylases. To address these questions, we characterized the interaction of Drosophila melanogaster GW182 with deadenylases and defined the minimal requirements for a functional GW182 protein. Functional assays in D. melanogaster and human cells indicate that miRNA-mediated translational repression and degradation are mechanistically linked and are triggered through the interactions of GW182 proteins with PABP and deadenylases.