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.

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.

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 RNAmediated 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.

MicroRNAs are approximately 22 nucleotide-long RNAs that silence gene expression posttranscriptionally by binding to the 3' untranslated regions of target mRNAs. Although much is known about their biogenesis and biological functions, the mechanisms allowing miRNAs to silence gene expression in animal cells are still under debate. Here, we discuss current models for miRNA-mediated gene silencing and formulate a hypothesis to reconcile differences.

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.