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. 2010 May;16(5):1032-9.
doi: 10.1261/rna.1851510. Epub 2010 Mar 26.

Differential regulation of microRNA stability

Sophie Bail  1 , Mavis Swerdel, Hudan Liu, Xinfu Jiao, Loyal A Goff, Ronald P Hart, Megerditch Kiledjian
Affiliations

Differential regulation of microRNA stability

Sophie Bail et al. RNA. 2010 May.

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 3' terminal seven nucleotides where mutations in this region increased the stability of the RNA. Moreover, the exosome 3'-5' 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.

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Figures

FIGURE 1.
FIGURE 1.
Differential stability of miRNAs in actinomycin D-treated HEK293 cells. (A) Total RNA from 8-h actinomycin D treated or untreated HEK293 cells were RT-PCR amplified with primers to detect the unstable c-myc proto-oncogene mRNA or stable GAPDH mRNA to confirm transcriptional inhibition. (B) Microarray results for a subset of miRNAs were verified by quantitative RT-PCR using TaqMan miRNA assay kit (Applied Biosystems). Data represent the ratio of miRNA at 8 h actinomycin D treatment relative to untreated cells and represent the mean of at least three independent experiments ± standard errors.
FIGURE 2.
FIGURE 2.
Differential stability of miRNAs in Dicer knockdown HEK293 cells. The level of miRNA remaining at the indicated times following Dicer-specific shRNA induction in HEK293 2b2 cells were determined by quantitative RT-PCR using the TaqMan miRNA assay kit (Applied Biosystems) and presented relative to the stable miR-378, which was set to 1. Data represent the mean of at least three independent experiments ± standard errors.
FIGURE 3.
FIGURE 3.
miR-382 levels are primarily responsive to the exosome complex. (A) HEK293T cells were transduced with lentiviruses expressing shRNAs directed either to Xrn1, Xrn2, or Rrp41. Levels of the indicated mRNAs in control and shRNA-expressing cells as determined by quantitative real time–PCR are graphed. Xrn1, Xrn2, and Rrp41 mRNA levels were reduced by 85%, 83%, and 78% in the respective knockdown cells. (B) miR-382 and miR-378 levels were determined using the TaqMan miRNA assay kit (Applied Biosystems) in each indicated knockdown cell line and presented relative to RNU43 snoRNA. Data represent the mean of at least three independent experiments ± standard errors.
FIGURE 4.
FIGURE 4.
Decay of mature miRNA in vitro. Incubation of mature miRNA in an uncoupled decay reaction does not recapitulate the differential miRNA stability of miR-382 and miR-378 observed in cells. Assays were carried out with 50 μg of HEK293T cytoplasmic extract at 15°C with the indicated 32P-labeled single-stranded miRNA. (A) Reactions were terminated with stop buffer spiked with 5′-end 32P-labeled loading control to enable normalization between the lanes. The miRNA and loading controls are indicated on the right. Quantitations of the decay reactions derived from mature single-stranded (B) or double-stranded (C) miR-382 and miR-378 are shown. Data represent the mean of at least three independent experiments ± standard errors.
FIGURE 5.
FIGURE 5.
Coupled pre-miRNA processing and miRNA decay in vitro. Coupled processing and decay assays were carried out with 5′-end 32P-labeled pre-miR-382 (A) and pre-miR-378 (B). 32P-labeled-pre-miRNAs were incubated with 10 μg of Dicer-overexpressing HEK293 cytoplasmic S15 extract at 37°C for 30 min to process the pre-miRNA into mature miRNAs. Decay of the mature miRNA was then followed by the addition of 50 μg of HEK293T cytoplasmic S15 at 18°C (lanes 2–5). Lanes 6–8 show the extent of background processing that occurs during the decay assay. The 32P-labeled pre-miRNA in lanes 6–8 were incubated directly at 18°C with a mixture of 10 μg of cytoplasmic S15 extract derived from Dicer-overexpressing cells and 50 μg of cytoplasmic S15 extract from HEK293 cells. Reactions were terminated as in the legend to Figure 4. The pre-miRNA, mature miRNA, and loading controls are indicated on the right and the size markers on the left. (C) Graphic representation of miR-382 and miR-378 decay relative to the level of miRNA present in the “0” time point are shown. 32P-signals were quantified using ImageQuant software. Data represent the mean of at least three independent experiments ± standard errors.
FIGURE 6.
FIGURE 6.
Requirement of miR-382 3′ terminus for its rapid decay. (A) Processing-coupled in vitro decay assays were carried out with pre-miR-382 containing substitutions within the first or second half of the miRNA or (B) at positions 13–15, 16–18, and 19–22 in the second half of the miRNA. Reaction conditions and labeling are as described in the legend to Figure 4. (C) The sequence of wild-type and mutants miR-382 are shown at the left, where lines demarcate the substituted sequences. Bars represent the ratio of wild-type and mutant miR-382 levels expressed as a ratio of miRNA remaining after an 80-min decay reaction relative to the initial level of miRNA prior to the decay. Data represent the mean of at least three independent experiments ± standard errors. Significance was determined by ANOVA using Graph Pad InStat software, *P < 0.05, **P < 0.01, ***P < 0.001.
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