MicroRNA-Independent Modulation of DICER1 Expression by hAgo2

Abstract

Many proteins, including DICER1 and hAgo2, are involved in the biogenesis of microRNAs (miRNAs). Whether hAgo2 regulates DICER1 expression is unknown. Exogenously overexpressed hAgo2 suppressed DICER1 expression at the levels of both protein and mRNA, and the reduction in hAgo2 expression enhanced DICER1 expression. Precursor miRNA processing mediated by DICER1 was also modulated by hAgo2. However, hAgo2 protein did not suppress DICER1 promoter activity. Therefore, hAgo2 protein probably regulates DICER1 expression at the posttranscriptional level. Indeed, hAgo2 protein inhibited the reporter assay of the DICER1 mRNA 3' untranslated region (3'-UTR). Previous reports have demonstrated that miRNAs (e.g., let-7 and miR-103/107) inhibited DICER1 expression posttranscriptionally. However, hAgo2 still suppressed DICER1 expression in the cells depleted of these miRNAs. Moreover, the reporter activities of the DICER1 mRNA 3'-UTR without these miRNA binding sites were still suppressed by hAgo2. Therefore, in addition to an miRNA-dependent pathway, hAgo2 can also modulate DICER1 expression through an miRNA-independent mechanism. Downregulation of DICER1 expression was further proven to be dependent on both hAgo2 and AUF1 proteins. Interactions of hAgo2 and AUF1 proteins were demonstrated by the coimmunoprecipitation assay. As expected, hAgo2 could not suppress the DICER1 mRNA 3'-UTR reporter with a mutation in the potential AUF1-binding site. Thus, downregulation of DICER1 expression through the 3'-UTR requires both hAgo2 and AUF1.

Keywords: coimmunoprecipitation; knockdown; knockout; luciferase reporter assay; posttranscriptional level.

FIG 1

hAgo2 downregulated DICER1 expression. (A to D) Western blot of various proteins involved in the miRNA pathway, using samples from HeLa cells, HuH7, or A549 cells, transiently transfected with different amounts of either vector or the plasmid expressing hAgo2, as indicated (A), stably transfected with GFP or hAgo2 (two different infection experiments, 1 and 2) (B), transfected with different amounts of siRNA targeting hAgo2, as indicated (C), or stably transfected with shRNA targeting hAgo2 (two different shRNA clones, +6 and +8) (D). (E) Western blot of DICER1 and hAgo2 proteins using samples from HeLa cells (left) or A549 cells (right) transfected with sgRNA targeting hAgo2 (two different clones [2-5 and 1-5] were from HeLa cells, while the other two [2-19 and 1-14] were from A549 cells).

FIG 2

Real-time RT-PCR to detect the relative amounts of eight different precursor miRNAs using RNA samples from HeLa cells with shRNA targeting hAgo2 (two different clones, +6 and +8) (A) or overexpression of hAgo2 (two different clones, C1 and C2) (B). Results in panels A and B are the means (±SD) from three independent experiments.

FIG 3

hAgo2 downregulates DICER1 expression by affecting the 3′-UTR of its mRNA. (A) Western blot using protein samples from HeLa cells transiently transfected with expression plasmids with or without the MG132 treatment, as indicated. (B) Real-time RT-PCR to detect the DICER1 mRNA amount using RNA samples from HeLa cells transiently transfected with expression plasmids for hAgo2. (C) Real-time RT-PCR to detect the relative mRNA amount of hAgo2 and DICER1 using RNA samples from HeLa cells with shRNA targeting hAgo2 (two different clones, +6 and +8). (D) HeLa cells were transfected with the following plasmids: pGL3-DICER1-prom or pGL3-Basic, 0.1 μg; pRL-TK, 0.01 μg; pcDNA3-myc-hAgo2 (or pcDNA3 empty vector), different doses of 0.1, 0.2, and 0.4 μg. Cells were harvested, and luciferase activity was measured 48 h after transfection. (E) HeLa cells were transfected with the following plasmids: pIS1-DICER1-long UTR or pIS1, 0.1 μg; PIS0, 0.01 μg; pcDNA3-myc-hAgo2 (or pcDNA3 empty vector), different doses of 0.1, 0.2, and 0.4 μg. The cells were harvested, and luciferase activity was measured 48 h after transfection. (F) HeLa cells were transfected with the following plasmids: pIS1-DICER1-long UTR and pcDNA3-myc-hAgo2 (or pcDNA3 empty vector) with different doses of 0, 0.5, 1, and 2 μg. The cells were harvested, and the luciferase mRNA level was determined 48 h after transfection using real-time RT-PCR. Results in panels B, C, D, E, and F are the means (±SD) from three independent experiments.

FIG 4

Downregulation of DICER1 by hAgo2 is not through miRNAs. (A) Western blot of various proteins involved in the miRNA pathway using samples from HeLa cells transfected with miRNA Let-7a. (B, left) Western blot of DICER1 and hAgo2 proteins using samples from HeLa cells stably transfected with control sponge (labeled as CXCR4) or sponges to remove Let-7a or Let-7b. (Right) Western blotting of DICER1 protein using samples from HeLa cells stably transfected with different sponges and transiently transfected with the plasmid expressing hAgo2. (C) Renilla luciferase activity was measured using samples from HeLa cells with different amounts of pIS1-DICER1 long-mut-let7 (pIS0 empty firefly luciferase plasmid as control). (D, left) Western blotting of DICER1 and hAgo2 proteins using samples from HeLa cells stably transfected with sponges to remove miR103/107. (Right) Western blotting of DICER1 protein using samples from HeLa cells stably transfected with miR103/107 sponges and transiently transfected with the plasmid expressing hAgo2. (E) Luciferase activity was measured using samples from HeLa cells with pIS1-DICER1 long-mut-miR103/107. (F) Renilla luciferase activity was measured using samples from HeLa cells with the pIS1-DICER1 long ALL mutant. (G) Western blotting of DICER1 protein using samples from HeLa cells stably transfected with shRNA targeting GW182 (two different shRNA clones) and transiently transfected with expressing plasmids for hAgo2. Results in panels C, E, and F are the means (±SD) from three independent experiments.

FIG 5

Downregulation of DICER1 by AUF1(s) requires hAgo2. (A) Western blot of various proteins involved in the miRNA pathway using samples from HeLa cells stably transfected with shRNA targeting AUF1 (two different shRNA clones, I and II). (B) Western blotting of DICER1 protein using samples from HeLa cells transiently transfected with the expression plasmids for one of the four different isoforms of AUF1 (p37, p40, p42, or p45). The expected proteins were marked by arrows, while the smaller proteins were probably the degraded products. (C) Western blot of DICER1 and hAgo2 proteins using samples from HeLa cells transiently transfected with different amounts of either vector or the plasmids expressing four different forms of AUF1, as indicated. (D) Western blot of various proteins involved in the miRNA pathway using samples from HeLa cells transiently transfected with the expression plasmids for the four isoforms of AUF1. (E) Western blot of DICER1 protein using samples from A549 cells stably transfected with sgRNA targeting hAgo2 (two different clones [2-19 and 1-14] from A549 cells) and transiently transfected with expression plasmids for four different forms of AUF1. (F) Western blotting of DICER1 protein using samples from HeLa cells stably transfected with shRNA targeting hAgo2 (two different shRNA clones, +6 and +8) and transiently transfected with expression plasmids for four different forms of AUF1.

FIG 6

Both hAgo2 and AUF1(s) are required to downregulate DICER1 expression. (A) Western blotting of DICER1 protein using samples from HeLa cells stably transfected with shRNA targeting AUF1 (two different shRNA clones, I and II) and transiently transfected with an expression plasmid for hAgo2. (B) Luciferase activity was measured using samples from HeLa cells with pIS1-DICER1 long AREM. (C) Coimmunoprecipitation of hAgo2 and AUF1 proteins. Forty-eight hours after transfection of HEK293T cells with different plasmids, as indicated, cell lysates from these cells were directly analyzed by Western blotting (input) for expression control or immunoprecipitated with the anti-Myc antibody prior to Western blotting (bottom). AUF1 p45 protein was marked by an arrow. Results in panel B are the means (±SD) from three independent experiments.

FIG 7

Western blot of DICER1 protein using samples from HeLa cells transiently transfected with an expression plasmid for hAgo1 (A, upper), hAgo3 (A, bottom), the N-terminal 550 aa of hAgo2 (B, left), the C terminus of hAgo2 (B, right), or hAgo2 (1 to 830 aa) (C).

FIG 8

Proposed model for this study. DICER1 expression could be downregulated by hAgo2 and different miRNAs (e.g., let-7a or miRNA 103/107). In addition to this mechanism, hAgo2, in collaboration with AUF1s, could also suppress DICER1 expression through an miRNA-independent pathway. In the AUF1 complex, the circle with a solid line represents p45, while the three circles with dotted lines represent p37, p40, and p42.