A short loop at the heart of a long RNA

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Sci. Signal.  18 Oct 2016:
Vol. 9, Issue 450, pp. ec240
DOI: 10.1126/scisignal.aal2098

Long noncoding RNAs (lncRNAs), defined as non–protein-coding RNAs longer than 200 base pairs, have diverse, tissue-specific roles. The lncRNA encoded by Braveheart (Bvht) is essential for cardiac lineage commitment. Xue et al. identified an lncRNA–transcription factor interaction that is necessary for specifying cell fate. Using chemical probing with dimethyl sulfate (DMS) and selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) of purified Braveheart transcripts in vitro, the authors defined the secondary structure, which included a 5' asymmetric G-rich internal loop motif (AGIL). Targeted 11-nt deletion in this motif (bvhtdAGIL) using CRISPR/Cas9 homology-directed repair in mouse embryonic stem cells (mESCs) did not affect transcription of Bvht or the pluripotency markers Oct4 or Nanog and did not alter cell morphology or division. Under appropriate conditions, wild-type mESCs form spontaneously beating embryoid bodies (EBs), an indication of cardiomyocyte differentiation, or can be induced toward neuronal or endodermal fates. However, bvhtdAGIL cells showed a marked reduction in the percent of beating EBs, as well as a reduction in expression of cardiac marker genes encoding cardiac troponin T (cTnT) and myosin heavy chains, without compromised induction of neuronal or endodermal markers under appropriate differentiation conditions. In contrast, a deletion in the Braveheart five-way helical junction did not affect formation of beating EBs. These results suggested that the Braveheart AGIL motif is specifically involved in the specification of cardiac cell fate. Of the proteins that exhibited an AGIL-dependent interaction with Bvht in a microarray screen, four were conserved between human and mouse. One of these was CNBP (also known as ZNF9), a zinc finger transcription factor that has been linked to heart defects in humans and mice and has an affinity for G-rich single-stranded nucleic acids. CRISPR/Cas9 deletion of two different small regions of cnbp (cnbpKO) in wild-type mESCs resulted in a higher percentage of cTnT+ cells, and overexpression of Flag-tagged CNBP reduced the fraction of cTnT+ cells, suggesting that CNBP negatively regulates cardiac differentiation. Indeed, cnbpKO;bvhtdAGIL double mutants had increased percentages of cTnT+ cells compared with bvhtdAGIL single mutants. Fazal and Chang note that this mechanism of transcriptional repression appears to be distinct from a previously reported interaction of Bvht with polycomb repressive complex 2, which was not affected by the AGIL deletion. By determining the structural basis of a specific regulatory function in an lncRNA, this study adds to the growing list of protein-like functions carried out by RNA molecules.

Z. Xue, S. Hennelly, B. Doyle, A. A. Gulati, I. V. Novikova, K. Y. Sanbonmatsu, L. A. Boyer, A G-rich motif in the lncRNA Braveheart interacts with a zinc-finger transcription factor to specify the cardiovascular lineage. Mol. Cell 64, 37–50 (2016). [PubMed]

F. M. Fazal, H. Y. Chang, lncRNA structure: Message to the heart. Mol. Cell 64, 1–2 (2016). [PubMed]