miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA

Abstract

MicroRNAs (miRNAs) are ∼22 nt non-coding RNAs that typically bind to the 3' UTR of target mRNAs in the cytoplasm, resulting in mRNA destabilization and translational repression. Here, we report that miRNAs can also regulate gene expression by targeting non-coding antisense transcripts in human cells. Specifically, we show that miR-671 directs cleavage of a circular antisense transcript of the Cerebellar Degeneration-Related protein 1 (CDR1) locus in an Ago2-slicer-dependent manner. The resulting downregulation of circular antisense has a concomitant decrease in CDR1 mRNA levels, independently of heterochromatin formation. This study provides the first evidence for non-coding antisense transcripts as functional miRNA targets, and a novel regulatory mechanism involving a positive correlation between mRNA and antisense circular RNA levels.

Figure 1

miR-671 represses CDR1 expression. (A) Schematic representation of the CDR1 locus. CDR1 mRNA (chrX: 139,693,091–139,694,389, hg18) and the antisense RNA (chrX: 139,693,005–139,694,491, hg18) derived from EST accessions (for additional detail, see Supplementary Figure S3). The enlargement shows the miR-671 antisense target region along with vertebrate conservation from UCSC Genome Browser (17-way conservation). (B) CDR1 expression in HEK293-eGFP-671 cells at indicated time points from 0 to 40 days of tetracycline induction. Expression was determined by qRT–PCR on random hexamer primed total RNA using the primer set CDR1(4)FW/CDR1(4)RE (normalized to 18S, relative to uninduced levels). (C) Northern blot showing subcellular localization of miR-671 in nuclear and cytoplasmic fractions of HEK293 cells transiently transfected with pJEBB-671. miR-15b is included as a localization control for an endogenously expressed miRNA and U6 snRNA and tRNA^Lys are nuclear- and cytoplasm-specific RNAs, respectively. (D) CDR1 expression in HEK293 cells transfected with anti-mir-671 or scrambled control determined by qPCR (as in B).

Figure 2

Characterization of the circular CDR1 antisense RNA. (A) Schematic illustration of the circular antisense RNA including the NAS splice donor (SD) and splice acceptor (SA) sites and the position of the NAS-specific primer set. (B) qRT–PCR for total or poly(A)-enriched RNA from HEK293 using primers specific for the NAS RNA. 7SK is included as a poly(A) tail-deficient control RNA. (C) NAS-specific qRT–PCR for RNA treated with TAP (Tobacco Acid Pyrophosphatase) and EXO (Terminator 5′ Phosphate-Dependent Exonuclease) normalized to untreated RNA. GAPDH mRNA and 7SK RNA are included as controls for TAP and EXO efficiencies. (D) Total HEK293 RNA was mixed with solidifying agarose and subjected to electrophoresis. In this system, circular RNA species will be physically trapped, whereas linear RNAs remain free to migrate. A no-electrophoresis control was included to determine gel-extraction efficiency, and −fold enrichment in CDR1 AS was quantified by NAS-specific qRT–PCR relative to total INPUT RNA and normalized to GAPDH. (E) Agarose northern blot with 2 μg RNA from HEK293 cells or human brain showing distinct AS migration using a CDR1 NAS-specific probe (top panel). Unspliced and spliced AS represent splicing of canonical intron II (see A). The approximate RNA sizes were deduced based on ribosomal RNA migration. 18S (bottom panel) serves as loading control.

Figure 3

Strand-specific quantification of CDR1 mRNA and the antisense RNA. (A) Schematic illustration of the CDR1 locus with primers used for strand-specific qRT–PCR of CDR1 mRNA (above) and antisense RNA (below), respectively. (B) Expression of the CDR1 mRNA (black) and NAS antisense RNA (grey) in uninduced, 3 and 40 days induced HEK293-eGFP-671 cells determined by strand-specific qRT–PCR. Left and right axes denote relative levels of CDR1 mRNA and CDR1 AS, respectively. (C, D) Northern blot showing CDR1 AS levels after 3 days miR-671 induction (C) or upon 100 nM anti-miR-671-5p treatment (D) compared with uninduced and anti-Control treatment, respectively. 18S serves as loading control.

Figure 4

Effect of CDR1 antisense-specific knockdown and overexpression. (A) Schematic illustration showing the target sites for strand-specific sisiRNAs. (B, C) HEK293 cells were transfected with 20 nM strand-specific sisiRNAs targeting the CDR1 antisense RNA (sisiRNA-AS) or mRNA (sisiRNA-S), or a mock control siRNA (Control). After 48 h, RNA levels were evaluated by qRT–PCR using mRNA-specific (B) or NAS-specific (C) primers, respectively. (D) Schematic illustration showing antisense vector design. (E) HEK293 cells were transfected with a vector expressing an antisense transcript harbouring the miR-671 target site (AS1), a vector expressing NAS-mimicking antisense RNA (AS2), a vector expressing a non-target site part of AS (AS3), or an empty vector (EV). The ASmt vectors carry a perfect miR-769 target site instead of the putative miR-671 target. RNA was harvested after 48 h, and the CDR1 mRNA level was quantified by strand-specific qRT–PCR. (F, G) NAS-specific qRT–PCR (F) and northern blot (G) with RNA extracted from cells transfected with a CDR1 mRNA expression vector or an empty vector (EV). Upper, middle, and lower panels show CDR1 mRNA, CDR1 AS, and 18S levels, respectively (G) (^*P<0.05; ^**P<0.01; ^***P<0.001).

Figure 5

Ago2-mediated cleavage of the CDR1 antisense transcript. (A) Firefly luciferase reporters (pISO) with a perfect miR-671 target (P target), the endogenous CDR1 antisense target (AS target), a mismatched target (MM target) or no target were co-transfected with a Renilla luciferase expression vector (pcDNA3-RL) and miR-671 (pJEBB-671) or miR-769 (pJEBB-769). Relative luminescence represents the Firefly/Renilla ratio for pJEBB-671 relative to pJEBB-769, normalized to the no target control. (B) Northern blot with RNA from HEK293 cells co-transfected with empty vector (EV), antisense wild-type (AS2) or antisense with an miR-769 target-site (AS2mt) and pJEBB-671 or pJEBB-769. Transient AS expression was determined with an AS-specific probe (upper panel). The NeoR probe reflects transfection efficiency of the AS expression vector (second panel), the eGFP probe shows ectopic gene expression from the transfected miRNA vector (third panel) and 18S serves as loading control (lower panel). (C) Set-up as in (A) but with transient co-transfection of Ago2-wild-type (Ago2-wt) or Ago2-slicer mutant (Ago2-D669A) expression vectors (Supplementary Figure S7A). (D) Antisense-specific qRT–PCR on RNA from HEK293 cells transiently transfected with miR-671 along with EV, Ago2 wild-type (Ago2-wt), or Ago2-slicer mutant (Ago2-D669A) expression vectors (Supplementary Figure S7B). (E) Northern blot on RNA from HEK293 cells transfected with either AS2 or AS2mt, after siRNA-mediated Xrn1 knockdown, showing AS2 full-length and 3′ cleavage fragment. (F) 5′ RACE on RNA from (E) showing 5′ ends of 3′ cleavage fragment and full-length AS2. (G) Clonal sequencing of cleavage fragments obtained from 5′ RACE (^***P<0.001).