The stringent regulation of cell cycle progression helps to maintain genetic stability in cells. MicroRNAs (miRNAs) are critical regulators of gene expression in diverse cellular pathways, including developmental patterning, hematopoietic differentiation and antiviral defense. Here, we show that two c-Myc-regulated miRNAs, miR-17 and miR-20a, govern the transition through G1 in normal diploid human cells. Inhibition of these miRNAs leads to a G1 checkpoint due to an accumulation of DNA double-strand breaks, resulting from premature temporal accumulation of the E2F1 transcription factor. Surprisingly, gross changes in E2F1 levels were not required to initiate the DNA damage response and checkpoint, as these responses could occur with a less than twofold change in E2F1 protein levels. Instead, our findings indicate that the precise timing of E2F1 expression dictates S-phase entry and that accurate timing of E2F1 accumulation requires converging signals from the Rb/E2F pathway and the c-Myc-regulated miR-17 and miR-20a miRNAs to circumvent a G1 checkpoint arising from the untimely accumulation of E2F1. These data provide a mechanistic view of miRNA-based regulation of E2F1 in the context of the emerging model that miRNAs coordinate the timing of cell cycle progression.

In the complex relationships of mammalian viruses with their hosts, it is currently unclear as to what role RNA silencing pathways play during the course of infection. RNA silencing-based immunity is the cornerstone of plant and invertebrate defense against viral pathogens, and examples of host defense mechanisms and numerous viral counterdefense mechanisms exist. Recent studies indicate that RNA silencing might also play an active role in the context of a mammalian virus infection. We show here that a mammalian virus, human adenovirus, interacts with RNA silencing pathways during infection, as the virus produces microRNAs (miRNAs) and regulates the expression of Dicer, a key component of RNA silencing mechanisms. Our work demonstrates that adenovirus encodes two miRNAs within the loci of the virus-associated RNA I (VA RNA I). We find that one of these miRNAs, miR-VA “g”, enters into a functional, Argonaute-2 (Ago-2)-containing silencing complex during infection. Currently, the cellular or viral target genes for these miRNAs remain unidentified. Inhibition of the function of the miRNAs during infection did not affect viral growth in a highly cytopathic cell culture model. However, studies from other viruses implicate viral miRNAs in the establishment of latent or chronic infections. Additionally, we find that adenovirus infection leads to the reduced expression of Dicer. This downregulation does not appear to be dependent on the presence of VA RNA or its associated miRNAs. Rather, Dicer levels appear to inversely correlate with the level of viral replication, indicating that another viral gene product is responsible for this activity. Misregulation of Dicer expression does not appear to influence viral growth in a cell culture model of infection, and also does not lead to gross changes in the pool of cellular miRNAs. Taken together, our results demonstrate that RNA silencing pathways are active participants in the process of infection with human adenovirus. The production of viral miRNAs and the regulation of cellular Dicer levels during infection implicate RNA silencing mechanisms in both viral fitness as well as potential host defense strategies.