Molecular Cell
Previews lncRNA Structure: Message to the Heart Furqan M. Fazal1 and Howard Y. Chang1,* 1Center for Personal Dynamic Regulomes and Program in Epithelial Biology, Stanford University School of Medicine, Stanford, CA 94305, USA *Correspondence:
[email protected] http://dx.doi.org/10.1016/j.molcel.2016.09.030
In this issue, Xue et al. (2016) describe the secondary structure of the heart-specific long non-coding RNA Braveheart, leading to the discovery of a short, asymmetric G-rich loop that controls cardiac lineage commitment by interacting with the transcription factor CNBP. In recent years, there has been an explosion of studies investigating long noncoding RNAs (lncRNAs), defined as RNAs greater than 200 nucleotides that do not code for proteins (Quinn and Chang, 2016). These RNAs are 50 capped and polyadenylated like most mRNAs. Many have been found to exhibit tissue-specific expression (Iyer et al., 2015), and some have important roles in regulating cell state, differentiation, development, and gene expression. lncRNAs have been proposed to function in a variety of ways, including serving as scaffolds, decoys, guides, or enhancers, and may act in cis or trans. Despite widespread interest, with the exception of a few well-studied candidates, we continue to know little about how these RNAs function at a molecular level (Quinn and Chang, 2016). However, it is clear that we cannot treat these RNAs as a single group, nor is it likely that there is a common modus operandus for the majority of lncRNAs. But in the cases of some of the bestunderstand lncRNAs—X-inactive specific transcript (XIST), sex-chromosome dosage compensating roX (RNA on X) RNAs, HOX transcript antisense RNA (HOTAIR), and telomerase RNA component (TERC)—knowledge of RNA structure has been crucial for understanding their mechanisms of action. With the development and improvement of RNA structure probing techniques, several insightful genome-wide studies have examined RNA structure in vivo and in vitro (Bevilacqua et al., 2016), though less abundant lncRNAs are often not sufficiently sampled in global studies. Now, by determining the structure of the low-abundance lncRNA Braveheart in vitro (Xue et al., 2016), the study of
the Boyer and Sanbonmatsu groups emphasizes just how critical RNA structural information can be to dissect lncRNA function in vivo (Figure 1). In 2013, the Boyer group identified the lncRNA Braveheart through its tissuespecific expression in heart tissue and found it to be essential for cardiac lineage commitment (Klattenhoff et al., 2013). That study also established that this RNA interacts with SUZ12, a component of the polycomb repressive complex 2 (PCR2), which mediates transcription repression. To further understand how this lncRNA functions, the authors determined the structure of Braveheart in vitro using structure-probing chemicals (DMS and SHAPE) that preferentially target unstructured RNA regions (Xue et al., 2016). Braveheart was found to have a modular structure comprising of helices, terminal loops, and internal loops, similar to roX RNAs (Ilik et al., 2013) and HOTAIR (Somarowthu et al., 2015). The authors went on to delete an 11-nt asymmetric internal G-rich loop (AGIL) of the RNA in embryonic stem cells (ESCs) using CRISPR/Cas9. This deletion (BraveheartdAGIL) does not substantially perturb the structure of the lncRNA, impact expression level, or affect the expression of embryonic transcription factors such as Oct4 and Nanog. However, this region of Braveheart was necessary for proper cardiomyocyte (CM) differentiation from ESCs through cardiac progenitor cells (CPCs). BraveheartdAGIL mouse embryonic stem cells (MESCs) produced fewer beating embryoid bodies and showed a reduction of CM markers during differentiation. Knocking out a different part of the RNA that is predicted to perturb the structure did not have such adverse effects.
To identify the proteins that recognize AGIL, the authors used a protein array comprised of over 9,400 recombinant proteins and tested binding in vitro against the wild-type (WT) and AGIL knockout lncRNA. Among the proteins identified that preferentially bound WT over the dAGIL lncRNA, the authors focused on CNBP/ZNF9, a zinc-finger transcription factor with an RNA-binding motif. This transcription factor is highly conserved and is particularly abundant in heart and skeletal muscle, and an inherited mutation in its gene has been linked to muscular dystrophy (Liquori et al., 2001). Further experiments revealed that CNBP acts as a negative regulator of the cardiac differentiation process, and overexpression of this protein in WT ESCs results in fewer cardiac cells upon differentiation. Knocking out CNBP partially restores the differentiation phenotype in the BraveheartdAGIL cells. Taken together, these experiments suggest that Braveheart influences the commitment of ESCs to CPCs by antagonizing CNBP, which in turn is a negative regulator of this process. The work of Xue et al. reinforces the notion that lncRNAs can have modular structures (Ilik et al., 2013; Somarowthu et al., 2015) analogous to the domain structure of proteins. Disrupting or deleting a critical module results in loss of function, similar to mutating the active site(s) of proteins. In addition, this study raises many interesting possibilities and questions about the mechanism of action of Braveheart, and future studies will undoubtedly focus on unraveling these mysteries. First, given the strong phenotype of the AGIL knockout and the important role Braveheart plays in mouse development, it is surprising that this lncRNA has not
Molecular Cell 64, October 6, 2016 ª 2016 Elsevier Inc. 1
Molecular Cell
Previews lncRNA Identification
lncRNA
lncRNA Locus
Figure 1. RNA Structure-Probing Experiments, lncRNA Characterization Studies, and Determination of RNA Interactions Together Inform How lncRNAs Function Some such well-studied lncRNAs include XIST, HOTAIR, TERC, and the roX RNAs. In Braveheart, an 11-nt asymmetric internal G-loop (AGIL) interacts with transcription factor CNBP to coordinate cardiac-lineage commitment.
been identified in rat or human. Focal conservation of structured motifs has allowed the identification of lncRNA orthologs with rapid-evolving flanking sequences (Quinn et al., 2016), and the AGIL motif may guide identification of lncRNAs that play similar roles to Braveheart in other species. Second, it is unclear mechanistically how Braveheart is able to antagonize CNBP. Previous arguments about how lncRNAs might function as molecular decoys to titrate away proteins, such as in the abundant lncRNA NORAD (Lee et al., 2016), do not apply here because Braveheart is expressed at low levels. Finally, how the CNBP story ties in with the previous report linking this RNA function to transcription repression via PRC2 recruitment (Klat-
2 Molecular Cell 64, October 6, 2016
tenhoff et al., 2013) remains to be determined.
M.L., Ding, H., Butty, V.L., Torrey, L., Haas, S., et al. (2013). Cell 152, 570–583.
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