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Technologies to probe functions and mechanisms of long noncoding RNAs

Nature Structural & Molecular Biology volume 22, pages 29–35 (2015)Cite this article

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Abstract

Thousands of long noncoding RNAs (lncRNAs) have been discovered, but their functional characterization has been slowed by a limited set of research tools. Here we review emerging RNA-centric methods to interrogate the intrinsic structure of lncRNAs as well as their genomic localization and biochemical partners. Understanding these technologies, including their advantages and caveats, and developing them in the future will be essential to progress from description to comprehension of the myriad roles of lncRNAs.

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Figure 1: ChIRP sequencing (ChIRP-seq) and its applications.
Figure 2: Workflow of high-throughput RNA structure and RNA-protein analysis.
Figure 3: Knowledge gained by the use of various technologies can accelerate the virtuous cycle of developing biological insights and RNA engineering iteratively.

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References

  1. Yang, L., Froberg, J.E. & Lee, J.T. Long noncoding RNAs: fresh perspectives into the RNA world. Trends Biochem. Sci. 39, 35–43 (2014).

    Article  PubMed  CAS  Google Scholar 

  2. Batista, P.J. & Chang, H.Y. Long noncoding RNAs: cellular address codes in development and disease. Cell 152, 1298–1307 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Rinn, J.L. & Chang, H.Y. Genome regulation by long noncoding RNAs. Annu. Rev. Biochem. 81, 145–166 (2012).

    Article  CAS  PubMed  Google Scholar 

  4. Chu, C., Qu, K., Zhong, F.L., Artandi, S.E. & Chang, H.Y. Genomic maps of long noncoding RNA occupancy reveal principles of RNA-chromatin interactions. Mol. Cell 44, 667–678 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Simon, M.D. et al. The genomic binding sites of a noncoding RNA. Proc. Natl. Acad. Sci. USA 108, 20497–20502 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Engreitz, J.M. et al. The Xist lncRNA exploits three-dimensional genome architecture to spread across the X chromosome. Science 341, 1237973 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  7. Grabowski, P.J. & Sharp, P.A. Affinity chromatography of splicing complexes: U2, U5, and U4 + U6 small nuclear ribonucleoprotein particles in the spliceosome. Science 233, 1294–1299 (1986).

    Article  CAS  PubMed  Google Scholar 

  8. Baltz, A.G. et al. The mRNA-bound proteome and its global occupancy profile on protein-coding transcripts. Mol. Cell 46, 674–690 (2012).

    Article  CAS  PubMed  Google Scholar 

  9. Castello, A. et al. Insights into RNA biology from an atlas of mammalian mRNA-binding proteins. Cell 149, 1393–1406 (2012).

    Article  CAS  PubMed  Google Scholar 

  10. Franke, A. & Baker, B.S. The rox1 and rox2 RNAs are essential components of the compensasome, which mediates dosage compensation in Drosophila. Mol. Cell 4, 117–122 (1999).

    Article  CAS  PubMed  Google Scholar 

  11. Gendrel, A.V. & Heard, E. Fifty years of X-inactivation research. Development 138, 5049–5055 (2011).

    Article  CAS  PubMed  Google Scholar 

  12. Hacisuleyman, E. et al. Topological organization of multichromosomal regions by the long intergenic noncoding RNA Firre. Nat. Struct. Mol. Biol. 21, 198–206 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Simon, M.D. et al. High-resolution Xist binding maps reveal two-step spreading during X-chromosome inactivation. Nature 504, 465–469 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Quinn, J.J. et al. Revealing long noncoding RNA architecture and functions using domain-specific chromatin isolation by RNA purification. Nat. Biotechnol. 32, 933–940 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Liu, W. et al. Brd4 and JMJD6-associated anti-pause enhancers in regulation of transcriptional pause release. Cell 155, 1581–1595 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Li, W. et al. Functional roles of enhancer RNAs for oestrogen-dependent transcriptional activation. Nature 498, 516–520 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Rossetto, C.C. & Pari, G. KSHV PAN RNA associates with demethylases UTX and JMJD3 to activate lytic replication through a physical interaction with the virus genome. PLoS Pathog. 8, e1002680 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Powell, W.T. et al. A Prader-Willi locus lncRNA cloud modulates diurnal genes and energy expenditure. Hum. Mol. Genet. 22, 4318–4328 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Ng, S.Y., Bogu, G.K., Soh, B.S. & Stanton, L.W. The long noncoding RNA RMST interacts with SOX2 to regulate neurogenesis. Mol. Cell 51, 349–359 (2013).

    Article  CAS  PubMed  Google Scholar 

  20. Li, Z. et al. The long noncoding RNA THRIL regulates TNFα expression through its interaction with hnRNPL. Proc. Natl. Acad. Sci. USA 111, 1002–1007 (2014).

    Article  CAS  PubMed  Google Scholar 

  21. Vance, K.W. et al. The long non-coding RNA Paupar regulates the expression of both local and distal genes. EMBO J. 33, 296–311 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Colak, D. et al. Promoter-bound trinucleotide repeat mRNA drives epigenetic silencing in fragile X syndrome. Science 343, 1002–1005 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Guttman, M. et al. Chromatin signature reveals over a thousand highly conserved large non-coding RNAs in mammals. Nature 458, 223–227 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Johnson, C.L. & Spence, A.M. Epigenetic licensing of germline gene expression by maternal RNA in C. elegans. Science 333, 1311–1314 (2011).

    Article  CAS  PubMed  Google Scholar 

  25. Riley, K.J., Yario, T.A. & Steitz, J.A. Association of Argonaute proteins and microRNAs can occur after cell lysis. RNA 18, 1581–1585 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Carey, J., Cameron, V., de Haseth, P.L. & Uhlenbeck, O.C. Sequence-specific interaction of R17 coat protein with its ribonucleic acid binding site. Biochemistry 22, 2601–2610 (1983).

    Article  CAS  PubMed  Google Scholar 

  27. Hogg, J.R. & Collins, K. RNA-based affinity purification reveals 7SK RNPs with distinct composition and regulation. RNA 13, 868–880 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Schnapp, G., Rodi, H.P., Rettig, W.J., Schnapp, A. & Damm, K. One-step affinity purification protocol for human telomerase. Nucleic Acids Res. 26, 3311–3313 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Hutvágner, G., Simard, M.J., Mello, C.C. & Zamore, P.D. Sequence-specific inhibition of small RNA function. PLoS Biol. 2, E98 (2004).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Déjardin, J. & Kingston, R.E. Purification of proteins associated with specific genomic loci. Cell 136, 175–186 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  31. Clemson, C.M., McNeil, J.A., Willard, H.F. & Lawrence, J.B. XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure. J. Cell Biol. 132, 259–275 (1996).

    Article  CAS  PubMed  Google Scholar 

  32. Julka, S. & Regnier, F. Quantification in proteomics through stable isotope coding: a review. J. Proteome Res. 3, 350–363 (2004).

    Article  CAS  PubMed  Google Scholar 

  33. Tsvetanova, N.G., Riordan, D.P. & Brown, P.O. The yeast Rab GTPase Ypt1 modulates unfolded protein response dynamics by regulating the stability of HAC1 RNA. PLoS Genet. 8, e1002862 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Hu, S. et al. Profiling the human protein-DNA interactome reveals ERK2 as a transcriptional repressor of interferon signaling. Cell 139, 610–622 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Rapicavoli, N.A., Poth, E.M., Zhu, H. & Blackshaw, S. The long noncoding RNA Six3OS acts in trans to regulate retinal development by modulating Six3 activity. Neural Dev. 6, 32 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Kretz, M. et al. Control of somatic tissue differentiation by the long non-coding RNA TINCR. Nature 493, 231–235 (2013).

    Article  CAS  PubMed  Google Scholar 

  37. Hasegawa, Y. et al. The matrix protein hnRNP U is required for chromosomal localization of Xist RNA. Dev. Cell 19, 469–476 (2010).

    Article  CAS  PubMed  Google Scholar 

  38. Helwak, A. & Tollervey, D. Mapping the miRNA interactome by cross-linking ligation and sequencing of hybrids (CLASH). Nat. Protoc. 9, 711–728 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Kudla, G., Granneman, S., Hahn, D., Beggs, J.D. & Tollervey, D. Cross-linking, ligation, and sequencing of hybrids reveals RNA-RNA interactions in yeast. Proc. Natl. Acad. Sci. USA 108, 10010–10015 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Martin, R. et al. A pre-ribosomal RNA interaction network involving snoRNAs and the Rok1 helicase. RNA 20, 1173–1182 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Trang, P., Hsu, A.W. & Liu, F. Nuclease footprint analyses of the interactions between RNase P ribozyme and a model mRNA substrate. Nucleic Acids Res. 27, 4590–4597 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Tijerina, P., Mohr, S. & Russell, R. DMS footprinting of structured RNAs and RNA-protein complexes. Nat. Protoc. 2, 2608–2623 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Latham, J.A. & Cech, T.R. Defining the inside and outside of a catalytic RNA molecule. Science 245, 276–282 (1989).

    Article  CAS  PubMed  Google Scholar 

  44. Pan, T. Probing RNA structure by lead cleavage. Curr. Protoc. Nucleic Acid Chem. 00, 6.3 (2001).

    Google Scholar 

  45. Spitale, R.C. et al. RNA SHAPE analysis in living cells. Nat. Chem. Biol. 9, 18–20 (2013).

    Article  CAS  PubMed  Google Scholar 

  46. Ilik, I.A. et al. Tandem stem-loops in roX RNAs act together to mediate X chromosome dosage compensation in Drosophila. Mol. Cell 51, 156–173 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Maenner, S., Muller, M., Frohlich, J., Langer, D. & Becker, P.B. ATP-dependent roX RNA remodeling by the helicase maleless enables specific association of MSL proteins. Mol. Cell 51, 174–184 (2013).

    Article  CAS  PubMed  Google Scholar 

  48. Novikova, I.V., Hennelly, S.P. & Sanbonmatsu, K.Y. Structural architecture of the human long non-coding RNA, steroid receptor RNA activator. Nucleic Acids Res. 40, 5034–5051 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Tsai, M.C. et al. Long noncoding RNA as modular scaffold of histone modification complexes. Science 329, 689–693 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Weeks, K.M. & Mauger, D.M. Exploring RNA structural codes with SHAPE chemistry. Acc. Chem. Res. 44, 1280–1291 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Wan, Y. et al. Landscape and variation of RNA secondary structure across the human transcriptome. Nature 505, 706–709 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Wan, Y., Qu, K., Ouyang, Z. & Chang, H.Y. Genome-wide mapping of RNA structure using nuclease digestion and high-throughput sequencing. Nat. Protoc. 8, 849–869 (2013).

    Article  CAS  PubMed  Google Scholar 

  53. Wan, Y. et al. Genome-wide measurement of RNA folding energies. Mol. Cell 48, 169–181 (2012).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  54. Kertesz, M. et al. Genome-wide measurement of RNA secondary structure in yeast. Nature 467, 103–107 (2010).

    Article  CAS  PubMed  Google Scholar 

  55. Rouskin, S., Zubradt, M., Washietl, S., Kellis, M. & Weissman, J.S. Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo. Nature 505, 701–705 (2014).

    Article  CAS  PubMed  Google Scholar 

  56. Ding, Y. et al. In vivo genome-wide profiling of RNA secondary structure reveals novel regulatory features. Nature 505, 696–700 (2014).

    Article  CAS  PubMed  Google Scholar 

  57. Lucks, J.B. et al. Multiplexed RNA structure characterization with selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). Proc. Natl. Acad. Sci. USA 108, 11063–11068 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Siegfried, N.A., Busan, S., Rice, G.M., Nelson, J.A. & Weeks, K.M. RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP). Nat. Methods 11, 959–965 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Murigneux, V., Sauliere, J., Roest Crollius, H. & Le Hir, H. Transcriptome-wide identification of RNA binding sites by CLIP-seq. Methods 63, 32–40 (2013).

    Article  CAS  PubMed  Google Scholar 

  60. Martin, L. et al. Systematic reconstruction of RNA functional motifs with high-throughput microfluidics. Nat. Methods 9, 1192–1194 (2012).

    Article  CAS  PubMed  Google Scholar 

  61. Buenrostro, J.D. et al. Quantitative analysis of RNA-protein interactions on a massively parallel array reveals biophysical and evolutionary landscapes. Nat. Biotechnol. 32, 562–568 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Szeto, K. et al. High-throughput binding characterization of RNA aptamer selections using a microplate-based multiplex microcolumn device. Anal. Bioanal. Chem. 406, 2727–2732 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Lambert, N. et al. RNA Bind-n-Seq: quantitative assessment of the sequence and structural binding specificity of RNA binding proteins. Mol. Cell 54, 887–900 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Buenrostro, J.D. et al. Quantitative analysis of RNA-protein interactions on a massively parallel array reveals biophysical and evolutionary landscapes. Nat. Biotechnol. 32, 562–568 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Raj, A., van den Bogaard, P., Rifkin, S.A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877–879 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Jung, J., Lifland, A.W., Zurla, C., Alonas, E.J. & Santangelo, P.J. Quantifying RNA-protein interactions in situ using modified-MTRIPs and proximity ligation. Nucleic Acids Res. 41, e12 (2013).

    Article  CAS  PubMed  Google Scholar 

  67. Lubeck, E., Coskun, A.F., Zhiyentayev, T., Ahmad, M. & Cai, L. Single-cell in situ RNA profiling by sequential hybridization. Nat. Methods 11, 360–361 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Hsu, P.D., Lander, E.S. & Zhang, F. Development and applications of CRISPR-Cas9 for genome engineering. Cell 157, 1262–1278 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  69. Ouyang, Z., Snyder, M.P. & Chang, H.Y. SeqFold: genome-scale reconstruction of RNA secondary structure integrating high-throughput sequencing data. Genome Res. 23, 377–387 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Jiang, J. et al. Translating dosage compensation to trisomy 21. Nature 500, 296–300 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Takahashi, M.K. & Lucks, J.B. A modular strategy for engineering orthogonal chimeric RNA transcription regulators. Nucleic Acids Res. 41, 7577–7588 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Lucks, J.B., Qi, L., Mutalik, V.K., Wang, D. & Arkin, A.P. Versatile RNA-sensing transcriptional regulators for engineering genetic networks. Proc. Natl. Acad. Sci. USA 108, 8617–8622 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Ran, F.A. et al. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell 154, 1380–1389 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  74. West, J.A. et al. The long noncoding RNAs NEAT1 and MALAT1 bind active chromatin sites. Mol. Cell 55, 791–802 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank members of our laboratory for thoughtful discussions. This work was supported by the Singapore Agency for Science, Technology, and Research (A*STAR to C.C.), the A.P. Giannini Foundation (R.C.S.) and the US National Institutes of Health (H.Y.C.). H.Y.C. is supported as an Early Career Scientist of the Howard Hughes Medical Institute.

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Authors and Affiliations

  1. Program in Epithelial Biology, Stanford University School of Medicine, Stanford, California, USA

    Ci Chu, Robert C Spitale & Howard Y Chang

  2. Department of Pharmaceutical Sciences, University of California at Irvine, Irvine, California, USA

    Robert C Spitale

  3. Howard Hughes Medical Institute, Stanford University, Stanford, California, USA

    Howard Y Chang

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  1. Ci Chu
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  2. Robert C Spitale
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Correspondence to Robert C Spitale or Howard Y Chang.

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Competing interests

C.C. and H.Y.C. are inventors of a patent on ChIRP technology held by Stanford University. R.C.S. and H.Y.C. are inventors of a patent on SHAPE probes held by Stanford University.

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Chu, C., Spitale, R. & Chang, H. Technologies to probe functions and mechanisms of long noncoding RNAs. Nat Struct Mol Biol 22, 29–35 (2015). https://doi.org/10.1038/nsmb.2921

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