Functional sequestration of microRNA-122 from Hepatitis C Virus by circular RNA sponges

Figure 1.. Characterization of miRNA-122 binding sites. (A) Representation of the miRNA-122 wildtype binding sites (wt, left) used in this study as well as the corresponding seed mutants (mut, right). (B) Dual luciferase reporter assay using four copies of the binding sites shown in (A) inserted into the 3′-UTR of a firefly luciferase reporter. LAPTM4A, HNRNPK and KPNB1 3′-UTR fragments of similar length without miRNA-122 binding sites served as controls. Firefly luciferase activity was normalized to a renilla luciferase reporter encoded by the same plasmid and to LAPTM4A control. Error bars represent s.d. (n = 3). *P < 0.05; ***P < 0.001. (C) RT-PCR of the reporter RNAs from (B). Firefly and renilla luciferase RNA were detected, as well as cellular GAPDH mRNA. As a control for the absence of plasmid DNA, an experiment without reverse transcriptase was used (firefly luc -RT). (D) Neutravidin-biotin pulldown from HuH-7 cytoplasmic extracts using in vitro transcribed and biotinylated RNA carrying four copies of the binding sites displayed in (A) or KPNB1 3′-UTR fragment of the same length. Precipitated miRNA-122 was detected by northern blot. Input and bound biotinylated RNA were analyzed by…

Figure 2.. Preparation and analysis on circular RNA in vitro. (A) Schematic of in vitro circularization constructs. Transcripts to be circularized consist of a terminal 10 nt open loop structure (black) and a reverse-complementary repeat sequence of 11 nt, which forms an intramolecular stem (red). This structure is followed by a 63 nt constant region for detection by northern blot or PCR (blue), followed by the miRNA-122 sponge (bulge; perfect) or a scrambled control sequence (shuffle) in grey. (B) Schematic of the in vitro ligation reaction. 4-fold excess of GMP over GTP results in ∼80% of the transcripts containing a 5′-monophosphate, enabling efficient in vitro ligation by T4 RNA ligase. Ligation products are circular RNAs (intramolecular ligation) or linear dimers (intermolecular ligation). (C) In vitro ligation reactions described in (B) were analyzed on 5%, 6% or 7% polyacrylamide-urea gels by ethidium bromide staining. While mobility of linear RNAs remains unchanged compared to RNA marker, the apparent mobility of circular RNA is lower in higher percentage gels (indicated by dash/double dash or circle). (D) Purified linear or circular RNAs from (C) were transfected in HuH-7.5 cells…

Figure 3.. Circular miRNA-122 RNA sponges affect HCV translation. (A) Schematic of the HCV/luciferase reporter construct. The HCV polyprotein open reading frame (ORF) was replaced by a firefly luciferase gene, leaving the original start codon and part of the Core protein ORF intact due to IRES secondary structure elements present in the region. miRNA-122 binding sites 1 and 2 are depicted, with Ago/miRNA bound and indicate its function in protection from Xrn-1-mediated degradation and in translation stimulation. (B) Linear and circular RNAs shown in Fig. 2C or Miravirsen were transfected one day prior to the reporter construct shown in (A). Luciferase activity of the reporter was measured 4 h post-transfection and normalized to reporter-only transfection (“mock”). Error bars represent s.d. (n = 3) *P < 0.05; **P < 0.01; ***P < 0.001. (C) Ethidium bromide staining (left panel) of RNA prior to co-transfection into HuH-7.5 cells together with full-length HCV RNA (strain Jc1), and same RNAs detected via northern blot 5 days post-transfection (right panel). (D) Western blot of cell lysates from HuH-7.5 cells co-transfected with full-length HCV RNA and linear/circular RNAs from (C) or Miravirsen…