Differential regulation of microRNA stability

Abstract

MicroRNAs (miRNAs) are endogenous single-stranded RNA molecules of about 21 nucleotides in length that are fundamental post-transcriptional regulators of gene expression. Although the transcriptional and processing events involved in the generation of miRNAs have been extensively studied, very little is known pertaining to components that regulate the stability of individual miRNAs. All RNAs have distinct inherent half-lives that dictate their level of accumulation and miRNAs would be expected to follow a similar principle. Here we demonstrate that although most miRNA appear to be stable, like mRNAs, miRNAs possess differential stability in human cells. In particular, we found that miR-382, a miRNA that contributes to HIV-1 provirus latency, is unstable in cells. To determine the region of miR-382 responsible for its rapid decay, we developed a cell-free system that recapitulated the observed cell-based-regulated miR-382 turnover. The system utilizes in vitro-processed mature miRNA derived from pre-miRNA and follows the decay of the processed miRNA. Using this system, we demonstrate that instability of miR-382 is driven by sequences outside its seed region and required the 39 terminal seven nucleotides where mutations in this region increased the stability of the RNA. Moreover, the exosome 39-59 exoribonuclease complex was identified as the primary nuclease involved in miR-382 decay with a more modest contribution by the Xrn1 and no detectable contribution by Xrn2. These studies provide evidence for an miRNA element essential for rapid miRNA decay and implicate the exosome in this process. The development of a biochemically amendable system to analyze the mechanism of differential miRNA stability provides an important step in efforts to regulate gene expression by modulating miRNA stability.

DcpS is a transcript-specific modulator of RNA in mammalian cells

“…HEK293T cells were transfected by pLKO.1-shXRN1 (Sigma #TRCN0000049676) or pLKO.1-shRRp41 (Sigma #TRCN0000051365), and pCMV-VSVG and psPAX2 with Lipofectamin2000 (Life Technologies) to generate viral particles. Culture medium was harvested two days post-transfection and frozen at −80°C for storage (Bail et al 2010). …”

Degradation dynamics of microRNAs revealed by a novel pulse-chase approach

“…A target-dependent mechanism has also been identified, in which turnover is mediated by the interaction of miRNAs with their mRNA targets, promoting miRNA unloading from AGO and degradation (Baccarini et al 2011;Ruegger and Grosshans 2012;De et al 2013;de la Mata et al 2015). One main reason for this lack of knowledge is the lack of experimental approaches that allow the analysis of miRNA half-lives at the global level without perturbing gene transcription or miRNA biogenesis (Bail et al 2010;Gantier et al 2011;Guo et al 2015). To overcome such issues, we exploited metabolic pulse labeling, the gold standard method used to determine RNA and protein halflives, and developed a "pulse-chase" approach optimized for the analysis of miRNAs.…”

RNA-mediated degradation of microRNAs: A widespread viral strategy?

“…In 2010, we reported that miR-27 is rapidly down-regulated in mouse cells infected with murine cytomegalovirus (MCMV), resulting in >90% reduction by 24 hours post-infection. 17 This observation was intriguing because the half-lives of most miRNAs are reported to be in the range of days 18 . We and others subsequently identified an MCMV transcript that directly associates with mature miR-27 and mediates its degradation.…”