A G-Rich Motif in the lncRNA Braveheart Interacts with a Zinc-Finger Transcription Factor to Specify the Cardiovascular Lineage

Mol Cell. 2016 Oct 6;64(1):37-50. doi: 10.1016/j.molcel.2016.08.010. Epub 2016 Sep 8.


Long non-coding RNAs (lncRNAs) are an emerging class of transcripts that can modulate gene expression; however, their mechanisms of action remain poorly understood. Here, we experimentally determine the secondary structure of Braveheart (Bvht) using chemical probing methods and show that this ∼590 nt transcript has a modular fold. Using CRISPR/Cas9-mediated editing of mouse embryonic stem cells, we find that deletion of 11 nt in a 5' asymmetric G-rich internal loop (AGIL) of Bvht (bvhtdAGIL) dramatically impairs cardiomyocyte differentiation. We demonstrate a specific interaction between AGIL and cellular nucleic acid binding protein (CNBP/ZNF9), a zinc-finger protein known to bind single-stranded G-rich sequences. We further show that CNBP deletion partially rescues the bvhtdAGIL mutant phenotype by restoring differentiation capacity. Together, our work shows that Bvht functions with CNBP through a well-defined RNA motif to regulate cardiovascular lineage commitment, opening the door for exploring broader roles of RNA structure in development and disease.

Keywords: Braveheart; CNBP; SHAPE; cardiac; long non-coding RNA.

MeSH terms

  • Animals
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Base Composition
  • Base Sequence
  • Binding Sites
  • CRISPR-Associated Protein 9
  • Cell Differentiation
  • Cell Lineage / genetics
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Endonucleases / genetics
  • Endonucleases / metabolism
  • Gene Deletion
  • Gene Editing
  • Gene Expression Regulation
  • Humans
  • Mice
  • Mouse Embryonic Stem Cells / cytology
  • Mouse Embryonic Stem Cells / metabolism*
  • Myocytes, Cardiac / cytology
  • Myocytes, Cardiac / metabolism*
  • Nucleic Acid Conformation
  • Phenotype
  • Protein Binding
  • RNA, Long Noncoding / chemistry
  • RNA, Long Noncoding / genetics*
  • RNA, Long Noncoding / metabolism
  • RNA-Binding Proteins / genetics*
  • RNA-Binding Proteins / metabolism
  • Signal Transduction


  • Bacterial Proteins
  • Cnbp1 protein, mouse
  • RNA, Long Noncoding
  • RNA-Binding Proteins
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases