Cell-cell contact globally activates microRNA biogenesis

Abstract

MicroRNAs (miRNAs) are 18- to 24-nt RNA molecules that regulate messenger RNAs (mRNAs). Posttranscriptional mechanisms regulate miRNA abundance during development as well as in cancer cells where miRNAs frequently exhibit dysregulated expression. The molecular mechanisms that govern the global efficiency of miRNA biogenesis in these settings remain incompletely understood, and experimental systems for the biochemical dissection of these pathways are currently lacking. Here, we demonstrate that miRNAs are subject to dynamic posttranscriptional regulation in widely used cell culture systems. As diverse mammalian and Drosophila cell lines are grown to increasing density, miRNA biogenesis is globally activated, leading to elevated mature miRNA levels and stronger repression of target constructs. This broad increase in miRNA abundance is associated with enhanced processing of miRNAs by Drosha and more efficient formation of RNA-induced silencing complexes. These findings uncover a critical parameter necessary for accurate analysis of miRNAs in cell culture settings, establish a tractable system for the study of regulated miRNA biogenesis, and may provide insight into mechanisms that influence miRNA expression in physiologic and pathophysiologic states.

Fig. 1.

Widespread up-regulation of miRNA expression in confluent cells. (A) Scatter plots showing miRNA expression in confluent versus subconfluent primary fibroblast, NIH 3T3, and HeLa cell lines. miRNAs that were detectable in both conditions are plotted. (B) Northern blots demonstrating miRNA abundance in HeLa cells at increasing levels of confluency. Representative images of U6 snRNA and ethidium bromide staining are shown. For this and subsequent figures, numbers below blots represent relative abundance of each miRNA normalized to U6 expression. (C) miRNA expression 24 h after plating HeLa and NIH 3T3 cells at increasing density. (D) Relative firefly luciferase activity derived from 3′ UTR reporter constructs containing sites of complete (perfect) or partial (bulged) complementarity to miR-15a or miR-143 after transfection into subconfluent or confluent HeLa cells. Values were normalized to luciferase activity produced from a reporter plasmid lacking miRNA-binding sites (pGL3-control) under each condition. Renilla luciferase activity produced from a cotransfected plasmid controlled for differences in transfection efficiency. Error bars represent standard deviations from 3 independent measurements. Representative data from 3 independent experimental trials, all of which yielded similar results, are shown.

Fig. 2.

Globally increased miRNA abundance in confluent cultures of diverse animal cell lines. (A) Northern blots demonstrating miRNA abundance in subconfluent (S) and confluent (C) human and mouse cell lines. (B) Limited miRNA accumulation in confluent HEK293 cells. (C) Up-regulation of both precursor and mature miRNAs in Drosophila S2R⁺ cells at increasing confluency. 2S rRNA served as a loading control.

Fig. 3.

Cell–cell contact likely triggers the global increase in miRNA abundance in confluent cultures. (A) miRNA abundance, cell number, and confluency were monitored daily during prolonged culture of HeLa cells. miRNA levels correlated with confluency despite no measurable reduction in cellular proliferation rate. (B) Increased miRNA abundance is not induced by cellular quiescence as demonstrated by unchanged or reduced expression of miRNAs in serum-starved subconfluent primary fibroblasts and NIH 3T3 cells (Upper). Propidium iodide staining and flow cytometry confirms efficient cell-cycle arrest after serum starvation (Lower). (C) Increased miRNA abundance is not triggered by diffusible factors, as demonstrated by unchanged or reduced expression of miRNAs in HeLa cells grown in media conditioned by subconfluent or confluent cells. (D) miRNAs do not accumulate significantly in high-density suspension cultures of Ramos or K562 cells.

Fig. 4.

Increased Drosha processing activity and enhanced RISC formation contribute to the global increase in miRNA abundance in confluent cells. (A) Quantitative PCR demonstrating unchanged or decreased abundance of most miRNA primary transcripts in HeLa cells at increasing confluency. (B) In vitro Drosha processing assay demonstrating increased activity in extracts from confluent HeLa cells (M, mock; 5–30, length of reaction in minutes). Plots show the amount of pre-miRNA produced at each time point relative to the first time point in the subconfluent group. (C) Transfection with synthetic pre-miR-1 or si-Luc demonstrates enhanced RISC formation in confluent cells. HeLa cells were transfected with pre-miR-1 or si-Luc alone (Mock) or in combination with HA-tagged Ago1 or Ago2 expression plasmids and then plated at subconfluent (S) or confluent (C) densities. Northern blot analysis was used to assess mature miR-1 or si-Luc abundance in total lysate (Input) or anti-HA immunoprecipitates (IP). Representative ethidium bromide (EtBr)-stained gel demonstrates equal loading of total input. Western blot analysis using anti-HA antibody demonstrated approximately equal recovery of Ago proteins from subconfluent and confluent cells.