Research Article

Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function

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Science  22 Sep 2017:
Vol. 357, Issue 6357, eaam8526
DOI: 10.1126/science.aam8526
  • Cdr1as is a brain-enriched circular RNA, expressed in hundreds of copies within neurons and essential for maintaining normal brain function.

    Genetic ablation of the Cdr1as locus in mice led to deregulation of miR-7 and miR-671 in the brain, up-regulation of immediate early genes, synaptic malfunctions, and a deficit in prepulse inhibition of the startle reflex, a behavioral phenotype associated with neuropsychiatric disorders.

  • Fig. 1 The circRNA Cdr1as is bound by miR-7 and miR-671 and highly expressed in excitatory neurons.

    (A) Cdr1as is densely bound by AGO:miRNA complexes containing miR-7 and miR-671. Bars on the circle represent circRNA:miRNA chimeric reads from AGO2 HITS-CLIP data from mouse brains. (B) Cdr1as is predominantly expressed in excitatory as opposed to inhibitory neurons and is not detected in glial cells. Markers: GFAP, astrocytes; NeuN, neurons, OLIG2, oligodendrocytes; VGLUT1, excitatory neurons; GAD67, inhibitory neurons. Arrows mark Cdr1as expression overlap with inhibitory neurons. Cdr1as, VGLUT1, and GAD67 mRNAs were detected by in situ hybridization (ISH) and GFAP, NeuN, and OLIG2 by immunostainings. (C) Cdr1as is broadly distributed in neuronal somas and neurites. Leftmost panel, single-molecule fluorescent in situ hybridization (FISH) for Cdr1as in cultured primary cortical neurons [in vitro day 14; DAPI (4′,6-diamidino-2-phenylindole) nuclear staining]. Right three panels, single excitatory pyramidal neuron at lamina II. (D) Using CRISPR-Cas9, the Cdr1as locus was deleted. The sequences given (left) denote protospacer adjacent motifs. kb, kilobases. RNA ISH (right) confirmed successful genetic ablation of Cdr1as.

  • Fig. 2 miRNA expression changes in Cdr1as knockout (KO) brain regions.

    Small RNAs were sequenced from mouse (A) cerebellum, (B) cortex, (C) hippocampus, and (D) olfactory bulb, each in biological replicates of n = 3, except Cdr1as KO hippocampus (n = 2). Shades of green indicate miRNAs of the same family. Gray, miRNAs with no significant expression change. WT, wild type.

  • Fig. 3 miR-7a is down-regulated in Cdr1as KO brains.

    miRNA-7a expression in Cdr1as KO and WT mouse brains detected using (A) Northern blotting, (B) FISH, and (C) chromogenic ISH. nCx, neocortex; St, striatum; Sp, septum; pCx, piriform cortex; L1 to L6, cortical layers; WM, white matter; U6 and miR-124, control RNAs.

  • Fig. 4 Gene expression changes in Cdr1as KO brains.

    Polyadenylated RNAs were sequenced from mouse (A) cerebellum, (B) cortex, (C) hippocampus, and (D) olfactory bulb, each in biological replicates of n = 3. DE, significantly differentially expressed; gray, no significant expression change. (E) Western Blot analysis of differentially expressed immediate early genes in cortical lysates; GAPDH serves as a loading control. (F) Top, c-Fos immunohistochemistry combined with ISH for VGLUT1 mRNA in the somatosensory cortex. Bottom, c-Fos signal intensity quantification across images (n = 60 per genotype). Boxes are defined by the first and third quartiles; horizontal bars, medians; whiskers span 1.5 times the interquartile range; gray circles, outliers; AU, arbitrary units.

  • Fig. 5 Loss of Cdr1as locus contributes to dysfunctional synaptic neurotransmission and abnormal brain function associated with neuropsychiatric disorders.

    (A and B) Cdr1as KO neurons showed increased spontaneous vesicle release and normal calcium-evoked excitatory postsynaptic currents (EPSCs). The left panel of (A) shows miniature EPSC (mEPSC) frequencies of WT (n = 34) and Cdr1as KO (n = 34) autaptic neurons. Right panel of (A), representative traces from WT (green) and Cdr1as KO (blue) in standard extracellular solution and in AMPA receptor–blocking NBQX (2,3-dihydroxy-6-nitro-7-sulfamoylbenzo[f]quinoxaline) solution. The left panel of (B) shows EPSC amplitudes of WT (n = 35) and Cdr1as KO (n = 34) neurons. Right panel of (B), 25-ms interstimulus interval paired-pulse ratio for WT (n = 30) and Cdr1as KO (n = 30) neurons. Representative traces of evoked EPSCs are shown. Time of action potentials (AP) are indicated by arrows, and currents associated with AP induction were blanked to enhance visibility of the synaptic current. Mann-Whitney U test; *P < 0.05. All data are represented as means ± SEM. (C) Cdr1as KO mice showed deficits in prepulse inhibition (PPI) of the startle reflex. PPI was measured as the percentage of the basal startle response. WT females, n = 13; Cdr1as KO females, n = 13; WT males, n = 10; Cdr1as KO males, n = 10. Boxes are defined by the first and third quartiles; medians are indicated as horizontal bars; whiskers span 1.5 times the interquartile range. Three-way analysis of variance with Bonferroni-corrected Welch t test; **P < 0.01, ***P < 0.001. Because there was no significant effect of gender, the male and female mice were pooled in post hoc tests.

Supplementary Materials

  • Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function

    Monika Piwecka, Petar Glažar, Luis R. Hernandez-Miranda, Sebastian Memczak, Susanne A. Wolf, Agnieszka Rybak-Wolf, Andrei Filipchyk, Filippos Klironomos, Cledi Alicia Cerda Jara, Pascal Fenske, Thorsten Trimbuch, Vera Zywitza, Mireya Plass, Luisa Schreyer, Salah Ayoub, Christine Kocks, Ralf Kühn, Christian Rosenmund, Carmen Birchmeier, Nikolaus Rajewsky

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

    Download Supplement
    • Materials and Methods
    • Figs. S1 to S18
    • Table S6
    • Captions for Tables S1 to S5 and S7
    • References
    Table S1
    miRNA:RNA Chimeric read data.
    Table S2
    miRNA differential expression analysis, DESeq2 output.
    Table S3
    polyA-selected RNA-seq differential expression analysis, DESeq2 output.
    Table S4
    rRNA-depleted RNA-seq differential expression analysis, DESeq2 output.
    Table S5
    Nanostring normalized counts.
    Table S7
    DNA oligo list.

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