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Reading the RNA Code Ralph E. Kleiner* Department of Chemistry, Princeton University, Princeton, New Jersey 08540, United States
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which they recognize through an aromatic cage,3 analogous to how epigenetic reader proteins recognize methylated lysine residues in proteins. In addition, HNRNPA2B1 has been shown to bind m6A-containing RNAs and regulate their splicing.4 So far, additional m6A readers have not been identified; however, given the abundance of this modification, and its involvement in diverse cellular signaling pathways, it is likely that more may exist. In addition, the m6A modification may preclude certain protein−RNA interactions. RNA−protein interactions mediate many aspects of mRNA biology, including regulating stability, trafficking, localization, and protein translation. Our current understanding of the molecular mechanisms underlying epitranscriptomic modifications suggests that these marks may function by regulating specific RNA−protein binding events. Of course, these modifications could also affect mRNA structure or alter base pairing interactions during ribosomal decoding; such models are attractive for modifications such as m1A and inosine but less applicable to modifications that do not have major effects on nucleobase hydrogen bonding. Therefore, to understand the function of epitranscriptomic modifications, it will be necessary to characterize their effect on protein−RNA interactions in the cell. Yang et al.5 report such a study with 5-methylcytidine (m5C), a modification that is well-established on tRNA and rRNA but only recently confirmed on mRNA. m 5 C modifications in RNA can be sequenced using bisulfite- or immunoprecipitation-based approaches. Multiple studies of m5C in mammalian cells have revealed its presence in mRNA, although a clear consensus sequence has not emerged. Using an optimized bisulfite sequencing-based approach, Yang et al. find ∼5000 m5C modifications in ∼2000 HeLa cell mRNAs. These modifications are enriched in CG-rich motifs as well as downstream of translation initiation sites. In addition, they profile m5C modifications in various murine tissues and find it to have a tissue-specific distribution and exhibit dynamic behavior. To characterize the interactions of m5C RNA with cellular proteins, the authors perform side-by-side pull downs with m5C and unmodified RNA oligos and find that the mRNA export protein ALYREF binds specifically to m5C RNA. This is the first report of an mRNA binding protein that is specific for m5C mRNA, although m5C has been reported to regulate protein− RNA interactions in the lncRNA XIST, and methyl binding domains (MBDs) specific for 5-methyldeoxycytidine in DNA have been well-characterized. The authors characterize the ALYREF−m5C RNA interaction biochemically and identify a point mutation that abolishes binding. As ALYREF shuttles
hemical modifications regulate the function of biological macromolecules. Postsynthesis modifications on proteins and DNA are key players in biological signaling and have attracted considerable attention because their dysregulation can be associated with disease states. While post-transcriptional modifications on RNA have been studied for at least as long, these modifications have received less attention than their protein and DNA counterparts have. Perhaps this is due to the sheer number of modificationsmore than 100 structurally distinct modifications have been characterized on cellular RNAor perhaps it is due to the long-standing view of RNA as a passive intermediate between DNA and protein. Post-transcriptional modifications have been identified on all classes of RNA; however, most of these modifications reside on tRNA where they have important structural roles or modulate condon−anticodon interactions during protein translation. Enzymes that remove modifications on tRNA have only recently been described, and therefore, RNA modifications were long considered to be static and part of the maturation process of RNA molecules, rather than serving a regulatory role. The discovery that the oxidative demethylase FTO could remove N6-methyladenosine (m6A) from mRNA1 ushered in a new era of RNA modification biology. Subsequent sequencing and biological investigation of m6A have shown that this dynamic modification is ubiquitous in eukaryotic mRNA and plays a central role in RNA metabolism, splicing, and protein translation. M6A has emerged as the founding member of a new class of RNA modifications, so-called “epitranscriptomic” modifications2 that occur within the mRNA sequence (distinct from the cap modifications that have been studied extensively). In addition to m6A, at least six other epitranscriptomic modifications have been identified, including N6,2′-O-dimethyladenosine (m6Am), N1-methyladenosine (m1A), pseudouridine (ψ), inosine (I), 5-methylcytidine (m5C), and 5hydroxymethylcytidine (hm5C). These modifications are postulated to serve as an epigenetic code regulating mRNA behavior and gene expression. If m6A is excluded, our current understanding of the functional significance of these modifications is extremely limited. How does the addition of a single methyl group on adenosine, in a manner that does not impede Watson−Crick base pairing, have profound effects on mRNA function and homeostasis? Epigenetic modifications on histone proteins regulate gene expression in part by serving as docking platforms for effector proteins (“readers”) that specifically bind to these modifications. Indeed, m6A appears to function in much the same way, by recruiting effector proteins to modified mRNA molecules. YTH domain-containing proteins, of which there are five family members in mammals, have been identified as m6A reader proteins and mediate much of m6A biology. Structural, biochemical, and transcriptomic data have shown that these proteins bind tightly and specifically to m6A-modified RNA, © XXXX American Chemical Society
Special Issue: Future of Biochemistry Received: September 1, 2017
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DOI: 10.1021/acs.biochem.7b00866 Biochemistry XXXX, XXX, XXX−XXX
Biochemistry
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between the nucleus and cytoplasm, the authors postulate that m5C binding may be involved in nuclear export of m5Ccontaining mRNAs. Indeed, knockdown of ALYREF or of the m5C methyltransferase NSUN2 results in the accumulation of m5C-containing RNA in the nucleus. Together, their findings demonstrate a new function for m5C in mRNA and implicate a protein reader in this process. Their work is an important contribution to our understanding of this abundant epitranscriptomic modification and lays the groundwork for further investigation of the role of m5C in mRNA function. RNA modifications play an important role in regulating mRNA function in the cell. Given the diversity of known RNA chemistry, it is likely that additional mRNA modifications await discovery, together with their corresponding reader proteins (Figure 1). Identifying and elucidating the functional significance of these molecular interactions represents one of the major challenges at the forefront of RNA biology.
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REFERENCES
(1) Jia, G., Fu, Y., Zhao, X., Dai, Q., Zheng, G., Yang, Y., Yi, C., Lindahl, T., Pan, T., Yang, Y. G., and He, C. (2011) N6methyladenosine in nuclear RNA is a major substrate of the obesityassociated FTO. Nat. Chem. Biol. 7, 885−887. (2) Roundtree, I. A., Evans, M. E., Pan, T., and He, C. (2017) Dynamic RNA Modifications in Gene Expression Regulation. Cell 169, 1187−1200. (3) Xu, C., Wang, X., Liu, K., Roundtree, I. A., Tempel, W., Li, Y., Lu, Z., He, C., and Min, J. (2014) Structural basis for selective binding of m6A RNA by the YTHDC1 YTH domain. Nat. Chem. Biol. 10, 927− 929. (4) Alarcon, C. R., Goodarzi, H., Lee, H., Liu, X., Tavazoie, S., and Tavazoie, S. F. (2015) HNRNPA2B1 Is a Mediator of m(6)ADependent Nuclear RNA Processing Events. Cell 162, 1299−1308. (5) Yang, X., Yang, Y., Sun, B. F., Chen, Y. S., Xu, J. W., Lai, W. Y., Li, A., Wang, X., Bhattarai, D. P., Xiao, W., Sun, H. Y., Zhu, Q., Ma, H. L., Adhikari, S., Sun, M., Hao, Y. J., Zhang, B., Huang, C. M., Huang, N., Jiang, G. B., Zhao, Y. L., Wang, H. L., Sun, Y. P., and Yang, Y. G. (2017) 5-methylcytosine promotes mRNA export - NSUN2 as the methyltransferase and ALYREF as an m5C reader. Cell Res. 27, 606− 625.
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NOTE ADDED IN PROOF After this article was received, a proteomic study characterizing the m6A interactome in mammalian cells and identifying new readers and proteins repelled by this modification was published online (Nature Structural and Molecular Biology (2017) doi:10.1038/nsmb.3462).
Figure 1. Protein readers of epitranscriptomic RNA modifications. (a) YTH proteins bind to m6A-modified mRNA and regulate stability, splicing, and translation. (b) ALYREF is a reader of m5C-modified mRNA and is involved in mRNA export. (c) Epitranscriptomic modifications for which no protein reader is currently known.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Ralph E. Kleiner: 0000-0003-0508-9975 Notes
The author declares no competing financial interest. B
DOI: 10.1021/acs.biochem.7b00866 Biochemistry XXXX, XXX, XXX−XXX
