MicroRNAs are tightly associated with RNA-induced gene silencing complexes in vivo - PubMed (original) (raw)

MicroRNAs are tightly associated with RNA-induced gene silencing complexes in vivo

Fuchou Tang et al. Biochem Biophys Res Commun. 2008.

Abstract

Previous work has shown that synthesized siRNA/miRNA is tightly associated with RNA-induced Gene Silencing Complexes (RISCs) in vitro. However, it is unknown if the endogenous miRNAs are also stably bound to RISC complexes in vivo in cells under physiological conditions. Here we describe the use of the looped real-time PCR-based method to trace the location of endogenous miRNAs in intact cells. We found that most of the endogenous miRNAs are tightly bound to RISC complexes, and only a very small proportion of them are free in cells. Furthermore, synthesized single-stranded mature miRNA or hairpin miRNA precursor cannot replace endogenous miRNAs already present in RISC complexes. However, we found that modified 2-O-Methyl-ribonucleotides were able to dissociate the target miRNA specifically from the RISC complex. These findings have important implications for understanding the basis for the stability and metabolism of miRNAs in living cells.

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Figures

Figure 1

Figure 1. Most miRNA are tightly associated with RISCs.

(A-C) Measurement of miR-16 in ES cells, NIH/3T3 cells, and mouse embryonic fibroblasts (MEFs). (1) Three freeze-thaw cycles followed by 95°C for 5min; (2) Three freeze-thaw cycles only (3) Treatment at 95°C for 5min; (4) Treatment at 4°C only as a control. (D-F) Measurement of miR-20 level in ES cells, NIH/3T3 cells, and MEFs. Treatments 1, 2, 3 and 4 are as specified in A-C. (G) RNase I treatment of ES cell lysates. (1) ES cells after three freeze-thaw cycles, were treated with RNase I for 5min, and following exposure at 95°C for 5min to release all RISC-bond miRNAs. (2) Three freeze-thaw cycles followed by incubation in the buffer for 5min, and at 95°C for 5min to release all RISC-bond miRNAs (as a control for RNase I treatment). (3) Incubation at 95°C for 5min to release miRNAs from RISC complex, followed by RNase I treatment for 5min, and further incubation of the cell lysate at 95°C for 5min. (4) ES cells were incubated at 95°C for 5min to release miRNAs from RISC complex, followed by treatment with buffer treatment for 5min, and incubation at 95°C for 5min (as a control for RNase I treatment). (H) RNase I treatment of MEF (Mouse Embryonic Fibroblast) lysates. (1) MEFs after three freeze-thaw cycles, were treated with RNase I for 5min, and following exposure at 95°C for 5min to release all RISC-bond miRNAs. (2) Three freeze-thaw cycles followed by incubation in the buffer for 5min, and at 95°C for 5min to release all RISC-bond miRNAs (as a control for RNase I treatment). (3) Incubation at 95°C for 5min to release miRNAs from RISC complex, followed by RNase I treatment for 5min, and further incubation of the cell lysate at 95°C for 5min. (4) MEFs were incubated at 95°C for 5min to release miRNAs from RISC complex, followed by treatment with buffer treatment for 5min, and incubation at 95°C for 5min (as a control for RNase I treatment).

Figure 2

Figure 2. The influence of antagomirs, Ago2, and Dicer on miRNA.

(A) Excessive amounts of synthetic miRNA or its precursor cannot replace endogenous miRNAs-RISCs. (1) ES cells treated at 95°C for 5min to release miRNAs (positive control). (2) Treatment at 95°C for 5min followed by incubation with RNase I for 30min to degrade ‘free’ miRNAs, then 95°C for 5min to release RISC-bond miRNAs (as negative control). (3) ES cells subjected to freeze-thaw cycles, followed by incubation with 0.5uM synthesized let-7a for 30min, and then with RNase I for 30min to degrade ‘free’ miRNAs, and finally 95°C for 5min to release RISC-bond miRNAs. (4) ES cell lysate incubated with 0.5uM synthesized pre-let-7a for 30min, and then with RNase I for 30min to degrade ‘free’ miRNAs, and finally at 95°C for 5min to release RISC-bond miRNAs. (B-C) Antagomirs effect on complementary miRNAs. Cells (ES or MEFs) were lysed by 3 freeze-thaw cycles. Then resulting cell lysate was incubated with 0.5nM-50nM of antagomirs of miR-16 (antagomir-16) for 30min, or as a control, with 0.5nM-50nM antisense miR-16 RNA or an unrelated antagomirs of Let-7a. RNase I was added subsequently to the samples to degrade miRNAs that were released from RISC complexes. Finally, RNase I was inactivated and the remaining RISC-bound miRNAs were released by treatment of the cell lysate at 95°C for 5min. (D) Measurement of mir-16 expression in wildtype and Ago2-/- mouse embryonic fibroblasts (MEFs). (E) Measurement of mir-16 expression in wildtype and Dicer-/- mouse embryonic fibroblasts (MEFs).

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