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lncRNA Structure: Message to the Heart

2016; Elsevier BV; Volume: 64; Issue: 1 Linguagem: Inglês

10.1016/j.molcel.2016.09.030

1097-4164

Furqan M. Fazal, Howard Y. Chang,

RNA and protein synthesis mechanisms

In this issue, Xue et al., 2016Xue Z. Hennelly S. Doyle B. Gulati A.A. Novikova I.V. Sanbonmatsu K.Y. Boyer L.A. Mol. Cell. 2016; 64 (this issue): 37-50PubMed Google Scholar 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 this issue, Xue et al., 2016Xue Z. Hennelly S. Doyle B. Gulati A.A. Novikova I.V. Sanbonmatsu K.Y. Boyer L.A. Mol. Cell. 2016; 64 (this issue): 37-50PubMed Google Scholar 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 non-coding RNAs (lncRNAs), defined as RNAs greater than 200 nucleotides that do not code for proteins (Quinn and Chang, 2016Quinn J.J. Chang H.Y. Nat. Rev. Genet. 2016; 17: 47-62Crossref PubMed Scopus (2289) Google Scholar). These RNAs are 5′ capped and polyadenylated like most mRNAs. Many have been found to exhibit tissue-specific expression (Iyer et al., 2015Iyer M.K. Niknafs Y.S. Malik R. Singhal U. Sahu A. Hosono Y. Barrette T.R. Prensner J.R. Evans J.R. Zhao S. et al.Nat. Genet. 2015; 47: 199-208Crossref PubMed Scopus (1875) Google Scholar), 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, 2016Quinn J.J. Chang H.Y. Nat. Rev. Genet. 2016; 17: 47-62Crossref PubMed Scopus (2289) Google Scholar). 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 best-understand 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., 2016Bevilacqua P.C. Ritchey L.E. Su Z. Assmann S.M. Annu. Rev. Genet. 2016; (Published online September 14, 2016)https://doi.org/10.1146/annurev-genet-120215-035034Crossref PubMed Scopus (146) Google Scholar), 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., 2016Xue Z. Hennelly S. Doyle B. Gulati A.A. Novikova I.V. Sanbonmatsu K.Y. Boyer L.A. Mol. Cell. 2016; 64 (this issue): 37-50PubMed Google Scholar), 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 tissue-specific expression in heart tissue and found it to be essential for cardiac lineage commitment (Klattenhoff et al., 2013Klattenhoff C.A. Scheuermann J.C. Surface L.E. Bradley R.K. Fields P.A. Steinhauser M.L. Ding H. Butty V.L. Torrey L. Haas S. et al.Cell. 2013; 152: 570-583Abstract Full Text Full Text PDF PubMed Scopus (723) Google Scholar). 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., 2016Xue Z. Hennelly S. Doyle B. Gulati A.A. Novikova I.V. Sanbonmatsu K.Y. Boyer L.A. Mol. Cell. 2016; 64 (this issue): 37-50PubMed Google Scholar). Braveheart was found to have a modular structure comprising of helices, terminal loops, and internal loops, similar to roX RNAs (Ilik et al., 2013Ilik I.A. Quinn J.J. Georgiev P. Tavares-Cadete F. Maticzka D. Toscano S. Wan Y. Spitale R.C. Luscombe N. Backofen R. et al.Mol. Cell. 2013; 51: 156-173Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar) and HOTAIR (Somarowthu et al., 2015Somarowthu S. Legiewicz M. Chillón I. Marcia M. Liu F. Pyle A.M. Mol. Cell. 2015; 58: 353-361Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar). 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., 2001Liquori C.L. Ricker K. Moseley M.L. Jacobsen J.F. Kress W. Naylor S.L. Day J.W. Ranum L.P. Science. 2001; 293: 864-867Crossref PubMed Scopus (1003) Google Scholar). 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., 2013Ilik I.A. Quinn J.J. Georgiev P. Tavares-Cadete F. Maticzka D. Toscano S. Wan Y. Spitale R.C. Luscombe N. Backofen R. et al.Mol. Cell. 2013; 51: 156-173Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar, Somarowthu et al., 2015Somarowthu S. Legiewicz M. Chillón I. Marcia M. Liu F. Pyle A.M. Mol. Cell. 2015; 58: 353-361Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar) 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 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., 2016Quinn J.J. Zhang Q.C. Georgiev P. Ilik I.A. Akhtar A. Chang H.Y. Genes Dev. 2016; 30: 191-207Crossref PubMed Scopus (112) Google Scholar), 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., 2016Lee S. Kopp F. Chang T.C. Sataluri A. Chen B. Sivakumar S. Yu H. Xie Y. Mendell J.T. Cell. 2016; 164: 69-80Abstract Full Text Full Text PDF PubMed Scopus (542) Google Scholar), 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 (Klattenhoff et al., 2013Klattenhoff C.A. Scheuermann J.C. Surface L.E. Bradley R.K. Fields P.A. Steinhauser M.L. Ding H. Butty V.L. Torrey L. Haas S. et al.Cell. 2013; 152: 570-583Abstract Full Text Full Text PDF PubMed Scopus (723) Google Scholar) remains to be determined. A G-Rich Motif in the lncRNA Braveheart Interacts with a Zinc-Finger Transcription Factor to Specify the Cardiovascular LineageXue et al.Molecular CellSeptember 8, 2016In BriefXue et al. determine the secondary structure of lncRNA Bvht and show that 11 nt deletion of the AGIL motif (bvhtdAGIL) prevents the transition from the mesoderm to cardiac progenitor state. They found that zinc-finger protein CNBP interacts with AGIL and that cnbpKO partially rescues differentiation defect of bvhtdAGIL cells. Full-Text PDF Open Archive