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Area of Science:

  • Neuroscience
  • Molecular Biology
  • Genetics

Background:

  • Circular RNAs (circRNAs) are abundant in the brain, with expression rising during neuronal differentiation.
  • The regulatory mechanisms governing circRNA biogenesis in the developing brain remain largely unknown.
  • NOVA1 and NOVA2 are neural-enriched RNA-binding proteins involved in alternative splicing.

Purpose of the Study:

  • To identify factors regulating circRNA biogenesis in the developing mouse brain.
  • To investigate the role of NOVA1 and NOVA2 in circRNA production.
  • To elucidate the cis-regulatory elements and binding sites involved in NOVA2-mediated circRNA biogenesis.

Main Methods:

  • RNA sequencing (RNA-seq) to profile circRNAs in Nova knockout mouse models.
  • Isolation of inhibitory and excitatory cortical neurons for detailed circRNA analysis.
  • Development of a backsplicing reporter assay using the Efnb2 gene.
  • Cross-linking and immunoprecipitation (CLIP) to identify NOVA2 binding sites.

Main Results:

  • Global circRNA levels were significantly reduced in Nova2 knockout mice, but not Nova1 knockout mice.
  • Loss of NOVA2 led to a more pronounced reduction of circRNAs in isolated cortical neurons.
  • Mutagenesis of YCAY clusters in flanking introns impaired NOVA2-mediated backsplicing of circEfnb2.
  • CLIP and reporter assays confirmed the importance of NOVA2 binding sites in intronic regions flanking circRNA loci.

Conclusions:

  • NOVA2 plays a critical, widespread role in promoting circRNA biogenesis in the developing brain.
  • NOVA2 directly enhances circRNA formation by binding to flanking intronic sequences.
  • This study identifies NOVA2 as the first RNA-binding protein with a global function in promoting circRNA biogenesis in the developing brain.