MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms

Abstract

MicroRNAs (miRNAs) are endogenously encoded small noncoding RNAs, derived by processing of short RNA hairpins, that can inhibit the translation of mRNAs bearing partially complementary target sequences. In contrast, small interfering RNAs (siRNAs), which are derived by processing of long double-stranded RNAs and are often of exogenous origin, degrade mRNAs bearing fully complementary sequences. Here, we demonstrate that an endogenously encoded human miRNA is able to cleave an mRNA bearing fully complementary target sites, whereas an exogenously supplied siRNA can inhibit the expression of an mRNA bearing partially complementary sequences without inducing detectable RNA cleavage. These data suggest that miRNAs and siRNAs can use similar mechanisms to repress mRNA expression and that the choice of mechanism may be largely or entirely determined by the degree of complementary of the RNA target.

Fig. 1.

Indicator construct design. (A) Sequences of the synthetic RNA targets used in this study and their predicted pairing with the miR-30, anti-miR-30, or miR-21 miRNA or the dNxt siRNA. Target sequences were either P complementary or were designed to form a central B. A random sequence, for which no complementary small RNA is known to exist, was used as a control. (B) Structure of the pCMV-luc-Target and pCMV-luc-Target-CAT indicator constructs. The Targets, represented by black boxes, are eight tandem repeats of one of the sequences shown in A. PA, polyadenylation signal.

Fig. 2.

Biological activity of the miR-30 and anti-miR-30 miRNAs. (A) The level of expression of miR-30, anti-miR-30, and of miR-21 in mock-transfected 293T cells, or in 293T cells transfected with the indicated miRNA expression plasmids, was determined by primer extension. (B) The luc enzyme activities detected in 293T cell cultures transfected with the listed indicator and effector plasmids, as well as the pBC12/CMV/β-gal control plasmid, were determined ≈40 h after transfection and then adjusted based on minor variations observed in the CAT internal control. These values are presented normalized to the culture transfected with pCMV-luc-random-CAT and pCMV-miR-21, which was arbitrarily set at 1. Average of three independent experiments with SD indicated. The number of nanograms of each miRNA expression plasmid transfected into each culture is indicated. (C) Parallel Northern analysis to detect the luc-reporter mRNA (Upper) and the control β-gal mRNA (Lower). Shown above Upper are the amounts of pCMV-miR-30 or pCMV-miR-21 transfected per culture. The level of luc enzyme activity detected for each indicator construct is given as a percentage of the level obtained on cotransfection with the pCMV-miR-21 control plasmid. Lane 1, RNA from mock-transfected 293T cells. The arrow indicates the position of the ≈1.8-kb luc mRNA cleavage product.

Fig. 3.

Effect of target complementarity on mRNA fate. (A) Predicted pairing of the artificial miR-30(B) miRNA with the miR-30(B) and miR-30(P) mRNA target sites. (B) Northern analysis demonstrating overexpression of the mature miR-21 and miR-30(B) miRNAs (M) in transfected 293T cells. The predicted ≈67-nt pre-miRNA precursors (P) are also detected. (C) Induced levels of luc enzyme activity in 293T cells transfected with the indicator effector and indicator plasmids. This assay was performed as described in Fig. 2B except that the parental pCMV plasmid, which is not predicted to express any miRNA, was used as an additional negative control. The presented values are normalized to the culture transfected with pCMV-luc-random-CAT and pCMV. (D) Parallel Northern analysis to detect the luc reporter mRNA, performed as described in Fig. 2C. The arrow indicates the position of the ≈1.8-kb luc mRNA cleavage product.

Fig. 4.

Biological activity of the human miR-21 miRNA. (A) This experiment was performed as described in Fig. 2B. Data shown are the average of four independent experiments. (B) Parallel Northern analysis of luc (Upper) and β-gal (Lower) mRNA expression. The level of luc enzyme activity detected with each indicator construct is given as a percentage of the level obtained on cotransfection with the pCMV-miR-30 control plasmid. The arrow indicates the position of the ≈1.8-kb luc mRNA cleavage product.

Fig. 5.

Inhibition of mRNA utilization by a synthetic siRNA. (A) Cultures were cotransfected with one of the three listed indicator plasmids together with the dNxt or dTap siRNA and the pRL-CMV and pBC12/CMV/β-gal internal control plasmids. The amount of each siRNA used is given in picomoles. Approximately 40 h after transfection, cultures were used for the dual luciferase assay or for RNA isolation. Firefly luc activities were adjusted for minor variations in the Renilla luc internal control and are presented normalized to the activity observed in the culture transfected with the pCMV-luc-random control plasmid and the dTap control siRNA, which was set at 1. Average of three independent experiments with SD indicated. (B) Northern analysis of firefly luc (Upper) and β-gal (Lower) mRNA expression. The level of firefly luc enzyme activity detected for each indicator construct is given as a percentage of the level obtained with the dTap control siRNA. The arrow indicates the position of the ≈1.8-kb luc mRNA cleavage product.