Protocol | Published:

Identification of RNA-binding protein targets with HyperTRIBE

Nature Protocolsvolume 13pages18291849 (2018) | Download Citation

Abstract

RNA-binding proteins (RBPs) accompany RNA from birth to death, affecting RNA biogenesis and functions. Identifying RBP–RNA interactions is essential to understanding their complex roles in different cellular processes. However, detecting in vivo RNA targets of RBPs, especially in a small number of discrete cells, has been a technically challenging task. We previously developed a novel technique called TRIBE (targets of RNA-binding proteins identified by editing) to overcome this problem. TRIBE expresses a fusion protein consisting of a queried RBP and the catalytic domain of the RNA-editing enzyme ADAR (adenosine deaminase acting on RNA) (ADARcd), which marks target RNA transcripts by converting adenosine to inosine near the RBP binding sites. These marks can be subsequently identified via high-throughput sequencing. In spite of its usefulness, TRIBE is constrained by a low editing efficiency and editing-sequence bias from the ADARcd. Therefore, we developed HyperTRIBE by incorporating a previously characterized hyperactive mutation, E488Q, into the ADARcd. This strategy increases the editing efficiency and reduces sequence bias, which markedly increases the sensitivity of this technique without sacrificing specificity. HyperTRIBE provides a more powerful strategy for identifying RNA targets of RBPs with an easy experimental and computational protocol at low cost, that can be performed not only in flies, but also in mammals. The HyperTRIBE experimental protocol described below can be carried out in cultured Drosophila S2 cells in 1 week, using tools available in a common molecular biology laboratory; the computational analysis requires 3 more days.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

1. TRIBE: hijacking an RNA-editing enzyme to identify cell-specific targets of RNA-binding proteins: https://doi.org/10.1016/j.cell.2016.03.007

2. Mechanistic implications of enhanced editing by a HyperTRIBE RNA-binding protein: https://doi.org/10.1261/rna.064691.117

References

  1. 1.

    Gerstberger, S., Hafner, M. & Tuschl, T. A census of human RNA-binding proteins. Nat. Rev. Genet. 15, 829–845 (2014).

  2. 2.

    Glisovic, T., Bachorik, J. L., Yong, J. & Dreyfuss, G. RNA-binding proteins and post-transcriptional gene regulation. FEBS Lett. 582, 1977–1986 (2008).

  3. 3.

    Witten, J. T. & Ule, J. Understanding splicing regulation through RNA splicing maps. Trends Genet. 27, 89–97 (2011).

  4. 4.

    Zhao, J., Hyman, L. E. & Moore, C. Formation of mRNA 3′ ends in eukaryotes: mechanism, regulation, and interrelationships with other steps in mRNA synthesis. Microbiol. Mol. Biol. Rev. 63, 405–445 (1999).

  5. 5.

    Jensen, T. H., Patricio, K., McCarthy, T. & Rosbash, M. A block to mRNA nuclear export in S. cerevisiae leads to hyperadenylation of transcripts that accumulate at the site of transcription. Mol. Cell 7, 887–898 (2001).

  6. 6.

    Ito, D., Hatano, M. & Suzuki, N. RNA binding proteins and the pathological cascade in ALS/FTD neurodegeneration. Sci. Transl. Med. 9, eaah5436 (2017).

  7. 7.

    Maziuk, B., Ballance, H. I. & Wolozin, B. Dysregulation of RNA binding protein aggregation in neurodegenerative disorders. Front. Mol. Neurosci. 10, 89 (2017).

  8. 8.

    Darnell, J. C. et al. FMRP stalls ribosomal translocation on mRNAs linked to synaptic function and autism. Cell 146, 247–261 (2011).

  9. 9.

    Garzia, A., Morozov, P., Sajek, M., Meyer, C. & Tuschl, T. PAR-CLIP for discovering target sites of RNA-binding proteins. Methods Mol. Biol. 1720, 55–75 (2018).

  10. 10.

    Darnell, R. B. HITS-CLIP: panoramic views of protein-RNA regulation in living cells. Wiley Interdiscip. Rev. RNA 1, 266–286 (2010).

  11. 11.

    Hrvatin, S. et al. Single-cell analysis of experience-dependent transcriptomic states in the mouse visual cortex. Nat. Neurosci. 21, 120–129 (2018).

  12. 12.

    Artegiani, B. et al. A single-cell RNA sequencing study reveals cellular and molecular dynamics of the hippocampal neurogenic niche. Cell Rep. 21, 3271–3284 (2017).

  13. 13.

    Ule, J., Jensen, K., Mele, A. & Darnell, R. B. CLIP: a method for identifying protein-RNA interaction sites in living cells. Methods 37, 376–386 (2005).

  14. 14.

    Hafner, M. et al. PAR-CliP--a method to identify transcriptome-wide the binding sites of RNA binding proteins. J. Vis. Exp. 2, 2034 (2010).

  15. 15.

    Gilbert, C. & Svejstrup, J.Q. RNA immunoprecipitation for determining RNA-protein associations in vivo. Curr. Protoc. Mol. Biol. chap. 27: unit 27.4 (2006).

  16. 16.

    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).

  17. 17.

    van Steensel, B. & Henikoff, S. Identification of in vivo DNA targets of chromatin proteins using tethered dam methyltransferase. Nat. Biotechnol. 18, 424–428 (2000).

  18. 18.

    Southall, T. D. et al. Cell-type-specific profiling of gene expression and chromatin binding without cell isolation: assaying RNA Pol II occupancy in neural stem cells. Dev. Cell 26, 101–112 (2013).

  19. 19.

    Lehmann, K. A. & Bass, B. L. Double-stranded RNA adenosine deaminases ADAR1 and ADAR2 have overlapping specificities. Biochemistry 39, 12875–12884 (2000).

  20. 20.

    Macbeth, M. R. et al. Inositol hexakisphosphate is bound in the ADAR2 core and required for RNA editing. Science 309, 1534–1539 (2005).

  21. 21.

    O’Connell, M. A. et al. Cloning of cDNAs encoding mammalian double-stranded RNA-specific adenosine deaminase. Mol. Cell. Biol. 15, 1389–1397 (1995).

  22. 22.

    Kim, U., Wang, Y., Sanford, T., Zeng, Y. & Nishikura, K. Molecular cloning of cDNA for double-stranded RNA adenosine deaminase, a candidate enzyme for nuclear RNA editing. Proc. Natl. Acad. Sci. USA 91, 11457–11461 (1994).

  23. 23.

    McMahon, A. C. et al. TRIBE: hijacking an RNA-editing enzyme to identify cell-specific targets of RNA-binding proteins. Cell 165, 742–753 (2016).

  24. 24.

    Bass, B. L. & Weintraub, H. An unwinding activity that covalently modifies its double-stranded RNA substrate. Cell 55, 1089–1098 (1988).

  25. 25.

    Matthews, M. M. et al. Structures of human ADAR2 bound to dsRNA reveal base-flipping mechanism and basis for site selectivity. Nat. Struct. Mol. Biol. 23, 426–433 (2016).

  26. 26.

    Eggington, J. M., Greene, T. & Bass, B. L. Predicting sites of ADAR editing in double-stranded RNA. Nat. Commun. 2, 319 (2011).

  27. 27.

    Kuttan, A. & Bass, B. L. Mechanistic insights into editing-site specificity of ADARs. Proc. Natl. Acad. Sci. USA 109, E3295–E3304 (2012).

  28. 28.

    Xu, W., Rahman, R. & Rosbash, M. Mechanistic implications of enhanced editing by a HyperTRIBE RNA-binding protein. RNA 24, 173–182 (2018).

  29. 29.

    Lapointe, C. P., Wilinski, D., Saunders, H. A. & Wickens, M. Protein-RNA networks revealed through covalent RNA marks. Nat. Methods 12, 1163–1170 (2015).

  30. 30.

    Kwak, J. E. & Wickens, M. A family of poly(U) polymerases. RNA 13, 860–867 (2007).

  31. 31.

    Lim, J. et al. Uridylation by TUT4 and TUT7 marks mRNA for degradation. Cell 159, 1365–1376 (2014).

  32. 32.

    Abruzzi, K., Chen, X., Nagoshi, E., Zadina, A. & Rosbash, M. RNA-seq profiling of small numbers of Drosophila neurons. Methods Enzymol. 551, 369–386 (2015).

  33. 33.

    Khodor, Y. L. et al. Nascent-seq indicates widespread cotranscriptional pre-mRNA splicing in Drosophila. Genes Dev. 25, 2502–2512 (2011).

  34. 34.

    Huang, A.M., Rehm, E.J. & Rubin, G.M. Quick preparation of genomic DNA from Drosophila. Cold Spring Harb. Protoc. 2009, 10.1101/pdb.prot5198 (2009).

  35. 35.

    Rodriguez, J., Menet, J. S. & Rosbash, M. Nascent-seq indicates widespread cotranscriptional RNA editing in Drosophila. Mol. Cell 47, 27–37 (2012).

  36. 36.

    van Gurp, T. P., McIntyre, L. M. & Verhoeven, K. J. F. Consistent errors in first strand cDNA due to random hexamer mispriming. PLoS ONE 8, e85583 (2014).

  37. 37.

    Mahmood, T. & Yang, P.-C. Western blot: technique, theory, and trouble shooting. North Am. J. Med. Sci. 4, 429–434 (2012).

Download references

Acknowledgements

We thank K. Abruzzi and J. Sherk for their helpful comments on improving the manuscript. We thank J. Rodriguez for his contribution in developing some of the scripts that are now part of the HyperTRIBE software. This work was supported by the Howard Hughes Medical Institute, NIH EUREKA grant DA037721 and NIH grant 5R01AG052465.

Author information

Affiliations

  1. Department of Biology, Howard Hughes Medical Institute and National Center for Behavioral Genomics, Brandeis University, Waltham, MA, USA

    • Reazur Rahman
    • , Weijin Xu
    • , Hua Jin
    •  & Michael Rosbash

Authors

  1. Search for Reazur Rahman in:

  2. Search for Weijin Xu in:

  3. Search for Hua Jin in:

  4. Search for Michael Rosbash in:

Contributions

R.R., W.X., H.J. and M.R. wrote the manuscript. W.X. and H.J. developed the experimental protocol and wrote the relevant sections of the protocol. R.R. developed the software and wrote the bioinformatics sections of the protocol.

Competing interests

The authors declare that a PCT patent application (PCT patent application no. PCT/US2016/05425) has been filed based on the HyperTRIBE method described in this paper.

Corresponding author

Correspondence to Michael Rosbash.

Supplementary information

About this article

Publication history

Published

DOI

https://doi.org/10.1038/s41596-018-0020-y

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.