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Peer-reviewed journal article

A Network of Noncoding Regulatory RNAs Acts in the Mammalian Brain

Benjamin KleavelandiD, Charlie Y. ShiiD, Joanna Stefano, David P. BarteliD

Abstract

Noncoding RNAs (ncRNAs) play increasingly appreciated gene-regulatory roles. Here, we describe a regulatory network centered on four ncRNAs--a long ncRNA, a circular RNA, and two microRNAs--using gene editing in mice to probe the molecular consequences of disrupting key components of this network. The long ncRNA Cyrano uses an extensively paired site to miR-7 to trigger destruction of this microRNA. Cyrano-directed miR-7 degradation is much more efficient than previously described examples of target-directed microRNA degradation, which come from studies of artificial and viral RNAs. By reducing miR-7 levels, Cyrano prevents repression of miR-7-targeted mRNAs and enables the accumulation of Cdr1as, a circular RNA known to regulate neuronal activity. Without Cyrano, excess miR-7 causes cytoplasmic destruction of Cdr1as, in part through enhanced slicing of Cdr1as by a second miRNA, miR-671. Thus, several types of ncRNAs can collaborate to establish a sophisticated regulatory network.\n\nHIGHLIGHTSA long noncoding RNA, a circular RNA, and two microRNAs form a regulatory network\n\nThe Cyrano long noncoding RNA directs potent, multiple-turnover destruction of miR-7\n\nUnchecked miR-7 prevents accumulation of Cdr1as circular RNA in cytoplasm of neurons miR-7 prevents this accumulation by enhancing the miR-671-directed slicing of Cdr1as

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In-depth summary

✦ Multi-agent verified full-text synthesis

Claude Sonnet 4.6 · xhigh effort · 8 Nov 2026 · from the full text of version 1 (9 Mar 2018) · CC BY 4.0

The lncRNA Cyrano drives multiple-turnover degradation of miR-7 in mouse brain, which protects the circRNA Cdr1as from miR-7-dependent, partly miR-671-sliced destruction.

How target RNA-directed miRNA degradation works on endogenous transcripts, and how circRNAs such as Cdr1as are turned over, were poorly understood. This work shows an endogenous lncRNA is a potent miR-7 degrader and uses a mouse knockout panel to link it to Cdr1as stability.

Key results

  1. ShownLoss of Cyrano raises mature miR-7 strongly in brain without changing precursors or the passenger strand, which points to degradation of Argonaute-loaded miR-7. The effect is conserved in human cells.

    Small-RNA-seq (n = 3 per genotype): miR-7a and miR-7b up 40- and 47-fold in cerebellum, 6- and 7-fold in hippocampus; miR-7* strands unchanged. RNA blots in other tissues: 3-38 fold. CRISPRi of CYRANO in K562 cells: ~3-fold. · Cyrano is a cytoplasmic lncRNA that promotes…

  2. ShownCyrano-directed miR-7 destruction is multiple-turnover and far more efficient than earlier TDMD examples, based on absolute molecule counts in cultured cerebellar granule neurons.

    Cyrano 102 ± 16 and miR-7 40 ± 7 molecules per cell in wild-type CGNs; miR-7 rose 45 fold to 1800 molecules per cell in Cyrano-deficient CGNs; ~17.6 miR-7 destroyed per Cyrano; 18-180-times more efficient than prior examples. · The miR-7 site in Cyrano directs…; Insights into target RNA-directed miRNA…

  3. ShownThe extensively paired miR-7 site in Cyrano is required, and degradation is very sensitive to Watson-Crick pairing. Every Cas9-made site mutant failed to induce miR-7 degradation.

    All six alleles M1-M6 (n = 4 per genotype, RNA blots) lost activity. The three seed-disrupting alleles resembled Cyrano-deficient mice. M1, a 2-nt deletion removing one seed pair, was also largely inactive. · The miR-7 site in Cyrano directs…

  4. ShownCyrano-induced oligoadenylylation of miR-7 is not on the degradation pathway. Removing the cytoplasmic polyA polymerase Gld2, or knocking down candidate exonucleases, does not change miR-7 levels.

    Gld2-/- hippocampus lost oligoadenylylated miR-7 reads yet showed no miR-7 change. In K562, CRISPRi knockdown of PAPD4 (98%), PARN (99%) and DIS3L2 (98%) left miR-7 unchanged. · Uncoupling tailing from trimming does not block…

  5. ShownCyrano indirectly sustains Cdr1as. Without Cyrano, raised miR-7 destroys Cdr1as, partly by boosting miR-671-directed slicing and partly by another miR-7-dependent mechanism.

    Cdr1as fell ~10-fold in Cyrano-/- and Cyrano M3/M3 cerebellum but was insensitive to Cyrano loss in Cyrano-/-; Mir7a1-/-; Mir7b-/- triple knockouts. In Cyrano-/- mice, deleting the miR-671 site raised Cdr1as 7.5 fold in cerebellum, implying 5 fold extra slicing and a presumed 3.4-fold effect from another mechanism. · Cyrano enables the Cdr1as circRNA to accumulate…; Cdr1as is regulated by miR-7 and miR-671

Methods, data and reproducibility

Mouse knockouts and Cas9 site mutants (Cyrano, Mir7a1/Mir7b, Cdr1as miR-671 site) were profiled by small-RNA-seq, RNA-seq, RNA blots, RT-qPCR and single-molecule FISH. CRISPRi knockdowns in human cell lines tested candidate factors.

  • Small-RNA-seq of wild-type vs Cyrano-/- cerebellum and hippocampus (1-month-old males) · Mouse · n = n = 3 biological replicates per genotype
  • PolyA RNA-seq of wild-type vs Cyrano-/- tissues for miR-7 target analysis (ten tissues) · Mouse · n = n = 4-6 samples per genotype per tissue
  • Cyrano miR-7 site Cas9 alleles M1-M6, RNA blot of cerebellum · Mouse · n = 6 alleles; n = 4 biological replicates
  • Mir7a1-/-; Mir7b-/- double knockout (one founder from F0 screen), triple knockout with Cyrano, and Cdr1as miR-671 site deletions (18 and 54 nt) · Mouse · n = 102 F0 mice screened for the Mir7 knockout
  • Single-molecule FISH for Cyrano and Cdr1as in cultured cerebellar granule neurons · Mouse · n = 40 fields per probe and genotype
  • CRISPRi cell lines targeting CYRANO to test miR-7 regulation · Human and mouse · n = 35 cell lines
  • Gld2-/- hippocampus small-RNA-seq reanalysed from Mansur et al. (2016) · Mouse · n = n = 6 biological replicates per genotype
Methods & tools
STAR v2.4, htseq-count v0.6.1p1, DESeq2, TargetScanMouse v7.1, cutadapt, FastX Toolkit, miRBase v20 and v21, CRISPRi (dCas9-KRAB), Single-molecule FISH, ImageM (MATLAB), IGV v2.3.34, GraphPad Prism 7
Reagents & environment
STAR v2.4, htseq-count v0.6.1p1, TargetScanMouse v7.1, Ensembl GRCm38.73 annotation, mm10, miRBase v20 (mouse) and v21 (human), IGV v2.3.34, ImageQuant TL v8.1.0.0, GraphPad Prism 7.
Reproducibility gaps
Sequencing data are stated as being deposited in GEO, available upon publication, with no accession printed. No code repository is given; custom tailing scripts are not linked.
Limitations (5)
  • The protein factors that execute Cyrano-directed miR-7 degradation remain unidentified. A 197-nt Cyrano region gave only ~3-fold miR-7 reduction in HEK293T vs 45-fold in neurons, so efficiency determinants are unresolved. · stated by the authors
  • A second miR-7-dependent mechanism that degrades Cdr1as (about 3.4-fold in cerebellum and 2.3-fold in cortex) is not identified; only miR-671 slicing is partly explained. · stated by the authors
  • Single-molecule FISH quantification uses z-projected images, so nuclear dot counts are likely overestimated. · stated by the authors
  • The 18-180-fold efficiency comparison combines molecule counts from cultured neurons with values calculated from other published systems, which differ in cell type and construct design. · noted in this summary
  • Only male mice were used (except one blot). Cyrano-deficient mice showed no overt abnormalities and unremarkable basic neurobehavioral testing, and miR-7 target repression was modest, with strong evidence in only three of ten tissues. · noted in this summary

Who should read:Read this if you study miRNA turnover, target RNA-directed miRNA degradation, or circRNA regulation and want in vivo knockout evidence for a lncRNA, circRNA and miRNA network.

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