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. 2012 Jan;40(1):438-48.
doi: 10.1093/nar/gkr722. Epub 2011 Sep 13.

The biogenesis and characterization of mammalian microRNAs of mirtron origin

Christopher R Sibley  1 , Yiqi Seow, Sheena Saayman, Krijn K Dijkstra, Samir El Andaloussi, Marc S Weinberg, Matthew J A Wood
Affiliations

The biogenesis and characterization of mammalian microRNAs of mirtron origin

Christopher R Sibley et al. Nucleic Acids Res. 2012 Jan.

Abstract

Mirtrons, short hairpin pre-microRNA (miRNA) mimics directly produced by intronic splicing, have recently been identified and experimentally confirmed in invertebrates. While there is evidence to suggest several mammalian miRNAs have mirtron origins, this has yet to be experimentally demonstrated. Here, we characterize the biogenesis of mammalian mirtrons by ectopic expression of splicing-dependent mirtron precursors. The putative mirtrons hsa-miR-877, hsa-miR-1226 and mmu-miR-1224 were designed as introns within eGFP. Correct splicing and function of these sequences as introns was shown through eGFP fluorescence and RT-PCR, while all mirtrons suppressed perfectly complementary luciferase reporter targets to levels similar to that of corresponding independently expressed pre-miRNA controls. Splicing-deficient mutants and disruption of key steps in miRNA biogenesis demonstrated that mirtron-mediated gene knockdown was splicing-dependent, Drosha-independent and had variable dependence on RNAi pathway elements following pre-miRNA formation. The silencing effect of hsa-miR-877 was further demonstrated to be mediated by the generation of short anti-sense RNA species expressed with low abundance. Finally, the mammalian mirtron hsa-miR-877 was shown to reduce mRNA levels of an endogenous transcript containing hsa-miR-877 target sites in neuronal SH-SY5Y cells. This work confirms the mirtron origins of three mammalian miRNAs and suggests that they are a functional class of splicing-dependent miRNAs which are physiologically active.

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Figures

Figure 1.
Figure 1.
miR-877 and miR-1224 are functional mammalian introns. (A) Putative mirtrons were studied by inserting appropriate sequences as introns within an eGFP transcript. (B) Predicted mirtron alignments for miR-877 and miR-1224 sequences incorporated into the pMirt vector. Variants with modifications made to splicing regulatory sequences are indicated by dashed boxes. US = unspliceable, DUS = double unspliceable, BP = Branch-point, Scr = Scrambled. Blue and red nucleotides represent the guide sequence and modified nucleotides, respectively. (C) Representative fluorescent microscopy images of different mirt-miR-877 and mirt-miR-1224 variants 48 h after transfection in HEK-293 cells. NAD represents a control intron of comparable length to miR-877 and miR-1224 with no hairpin-forming potential. (D) Upper panel—Quantification of eGFP fluorescence following expression of mirt-miR-877 and mirt-miR-1224 variants in HEK-293 cells. Values represent mean fluorescent ±SD from n = 3. *P < 0.05 relative to non-transfected cells. Lower panel—RT–PCR with intron-spanning primers following expression of miR-877 and miR-1224 variants in HEK-293 cells.
Figure 2.
Figure 2.
miR-877 and miR-1224 are splicing-dependent mammalian mirtrons processed into small RNA species. (A) Dual-luciferase reporter assays showing knockdown of a complementary miR-877 target or miR-1224 target following co-transfection with indicated mirtron (dark bars) and pre-miRNA control (light bars) variants. Values represent mean ratios of Renilla: Firefly luciferase ±SD from n = 6. Mirtron variants are normalized to cells transfected with the NAD variant. Pre-miRNA variants are normalized to cells transfected with a non-specific U6 pre-miRNA hairpin. *P < 0.05 relative to respective normalizing control. (B) Upper panel—3′ RACE RT–PCR of total RNA from HEK-293 cells transfected with NAD construct or mirtron/pre-miRNA variants of miR-877 using a miR-877 specific forward primer. Lower panel—Sequencing of 3′ RACE RT-PCR products identifies Dicer cleavage sites. (C) 18–25 nt small RNAs were extracted by polyacrylamide gel isolation from total RNA of HEK cells transfected with equimolar amounts of miR-877 and miR-1224 each as introns within separate eGFP mRNAs. Linkers were added to both ends of the RNA for RT–PCR amplification before deep sequencing using an Illumina Genome Analyzer. Sequences highly matched to any part of miR-877 or miR-1224 hairpin were identified and the incidence of each nucleotide in the sequenced small RNAs was plotted against miR-877 or miR-1224 sequences.
Figure 3.
Figure 3.
Mammalian mirtrons are Drosha independent. (A) Western blotting of protein lysates following transduction of HEK-293 cells with lentivirus expressing a non-specific control shRNA or Drosha targeting shRNA. (B) Dual-luciferase reporter assay showing knockdown of a HBV target sequence following co-transfection with an HBV-targeting pri-miR-122 mimic in the presence of a non-specific shRNA or Drosha-targeting shRNA. Values represent mean ratios of Renilla: Firefly luciferase ±SD from n = 6. Values are normalized to cells transfected with a non-specific pri-miR-122 mimic in the presence of the respective shRNA construct.*P < 0.05 relative to respective normalizing control. (C) Dual-luciferase reporter assay showing knockdown of a miR-877 target sequence following co-transfection with indicated mirt-miR-877, pre-miR-877 or miR-877 pri-miR-30 mimic in the presence of a non-specific shRNA or Drosha-targeting shRNA. Values represent mean ratios of Renilla: Firefly luciferase ±SD from n = 6. Mirtron variants are normalized to cells transfected with the NAD variant. Pre-miRNA variants are normalized to cells transfected with a non-specific U6 pre-miRNA hairpin. Pri-miRNA-30 mimics are normalized to cells transfected with a non-specific pri-miR-30 mimic. *P < 0.05 relative to respective normalizing control.
Figure 4.
Figure 4.
Mammalian mirtrons have variable dependence on the canonical RNAi pathway following pre-miRNA formation. (A and B) Dual-luciferase reporter assay showing knockdown of a miR-877 target sequence following co-transfection with indicated pre-miR-877 variants (A) or mirt-miR-877 variants (B) in the presence of increasing concentrations of VA1 expressed from the pAdvantage vector. Values represent mean ratios of Renilla: Firefly luciferase ±SD from n = 6. Pre-miRNA variants (A) are normalized to cells transfected with a non-specific U6 pre-miRNA hairpin and no pAdvantage vector. Mirtron variants (B) are normalized to cells transfected with mirt-miR-877DUS and no pAdvantage vector. (C and D) Dual-luciferase reporter assay showing knockdown of a miR-1224 target sequence following co-transfection with indicated pre-miR-1224 variants (C) or mirt-miR-1224 variants (D) in the presence of increasing concentrations of VA1 expressed from the pAdvantage vector. Values represent mean ratios of Renilla: Firefly luciferase ±SD from n = 6. Pre-miRNA variants (C) are normalized to cells transfected with a non-specific U6 pre-miRNA hairpin and no pAdVantage vector. Mirtron variants (D) are normalized to cells transfected with mirt-miR-1224DUS and no pAdVantage vector.
Figure 5.
Figure 5.
miR-877 silences the predicted seed-matched target FXR2 in neuronal SH-SY5Y cells. (A) Dual-luciferase reporter assay showing knockdown of predicted miR-877 target sequences following co-transfection with indicated mirt-miR-877 or pre-miR-877 variants. Values represent mean ratios of Renilla: Firefly luciferase ±SD from n = 3. For each unique target, mirtron variants are normalized to cells transfected the NAD variant and pre-miRNA variants are normalized to cells transfected with a non-specific U6 pre-miRNA hairpin. *P < 0.05 relative to respective normalizing control. (B) qPCR detection of miR-877 in SH-SY5Y cells in the presence or absence of mirt-miR-877. Values represent relative miR-877 expression normalized to RNU-24 expression using the 2−ΔCt method and n = 6. (C) Relative expression of putative miR-877 target transcripts determined with RT–qPCR following transfection of SH-SY5Y cells with the NAD variant or mirt-miR-877. Values represent mean expression relative to β-actin expression ±SD using relative standard curve method and n = 6. Transcript levels in presence of mirt-mir-877 are normalized to cells transfected with the NAD variant.
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