A G-Rich Motif in the lncRNA Braveheart Interacts with a Zinc-Finger Transcription Factor to Specify the Cardiovascular Lineage.
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 bvhtdAGILmutant 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.
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Zhihong
Z
Xue
Los Alamos National Laboratory, Theoretical Biology and Biophysics Group, Los Alamos, NM 87545, USA; New Mexico Consortium, Los Alamos, NM 87544, USA.
Scott
S
Hennelly
Undergraduate Research Opportunities Program, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Physics, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Boryana
B
Doyle
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Arune A
AA
Gulati
Los Alamos National Laboratory, Theoretical Biology and Biophysics Group, Los Alamos, NM 87545, USA; Pacific Northwest National Laboratory, Environmental Molecular Sciences Laboratory, Richmond, WA 99354, USA.
Irina V
IV
Novikova
Los Alamos National Laboratory, Theoretical Biology and Biophysics Group, Los Alamos, NM 87545, USA; New Mexico Consortium, Los Alamos, NM 87544, USA.
Karissa Y
KY
Sanbonmatsu
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA; Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. Electronic address: lboyer@mit.edu.
Laurie A
LA
Boyer
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D000076987:CRISPR-Associated Protein 9; D002454:Cell Differentiation; D019070:Cell Lineage / Q000235:genetics; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D000072669:Gene Editing; D005786:Gene Expression Regulation; D006801:Humans; D051379:Mice; D000066450:Mouse Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D010641:Phenotype; D011485:Protein Binding; D062085:RNA, Long Noncoding / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D015398:Signal Transduction
D001426:Bacterial Proteins; D000076987:CRISPR-Associated Protein 9; D004720:Endonucleases; D062085:RNA, Long Noncoding; D016601:RNA-Binding Proteins; C499012:Cnbp protein, mouse; C000606107:Cas9 endonuclease Streptococcus pyogenes
Braveheart; CNBP; SHAPE; cardiac; long non-coding RNA