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RNA Framework for Assaying the Structure of RNAs by High-Throughput Sequencing

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2284))

Abstract

RNA structure is a key player in regulating a plethora of biological processes. A large part of the functions carried out by RNA is mediated by its structure. To this end, in the last decade big effort has been put in the development of new RNA probing methods based on Next-Generation Sequencing (NGS), aimed at the rapid transcriptome-scale interrogation of RNA structures. In this chapter we describe RNA Framework, the to date most comprehensive toolkit for the analysis of NGS-based RNA structure probing experiments. By using two published datasets, we here illustrate how to use the different components of the RNA Framework and how to choose the analysis parameters according to the experimental setup.

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References

  1. Incarnato D, Oliviero S (2017) The RNA epistructurome: uncovering RNA function by studying structure and post-transcriptional modifications. Trends Biotechnol 35:318–333

    Article  CAS  Google Scholar 

  2. Siegfried NA, Busan S, Rice GM et al (2014) RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP). Nature Methods. 11:959–965

    Article  CAS  Google Scholar 

  3. Homan PJ, Favorov OV, Lavender CA et al (2014) Single-molecule correlated chemical probing of RNA. Proc Natl Acad Sci USA 111:13858–13863

    Article  CAS  Google Scholar 

  4. Zubradt M, Gupta P, Persad S et al (2017) DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo. Nat Methods 14:75–82

    Article  CAS  Google Scholar 

  5. Simon LM, Morandi E, Luganini A et al (2019) In vivo analysis of influenza A mRNA secondary structures identifies critical regulatory motifs. Nucleic Acids Res 36:3960

    Google Scholar 

  6. Incarnato D, Morandi E, Simon LM et al (2018) RNA framework: an all-in-one toolkit for the analysis of RNA structures and post-transcriptional modifications. Nucleic Acids Res 46:e97–e97

    Article  Google Scholar 

  7. Rouskin S, Zubradt M, Washietl S et al (2014) Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo. Nature 505:701–705

    Article  CAS  Google Scholar 

  8. Langmead B, Trapnell C, Pop M et al (2009) Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol 10:R25

    Article  Google Scholar 

  9. Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–359

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  11. Lucks JB, Mortimer SA, Trapnell C et al (2011) Multiplexed RNA structure characterization with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). Proc Natl Acad Sci USA 108:11063–11068

    Article  CAS  Google Scholar 

  12. Incarnato D, Neri F, Anselmi F et al (2014) Genome-wide profiling of mouse RNA secondary structures reveals key features of the mammalian transcriptome. Genome Biol 15:491

    Article  Google Scholar 

  13. Deigan KE, Li TW, Mathews DH et al (2009) Accurate SHAPE-directed RNA structure determination. Proc Natl Acad Sci 106:97–102

    Article  CAS  Google Scholar 

  14. Lorenz R, Bernhart SH, Höner Zu Siederdissen C et al (2011) ViennaRNA Package 2.0. Algorith Mol Biol 6:26

    Article  Google Scholar 

  15. Reuter JS, Mathews DH (2010) RNAstructure: software for RNA secondary structure prediction and analysis. BMC Bioinformatics 11:129

    Article  Google Scholar 

Download references

Acknowledgments

This work was supported by the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO; OCENW.XS3.044) and by funding of the University of Groningen (Groningen, the Netherlands) and the Groningen Biomolecular Sciences and Biotechnology Institute (GBB) to D.I.

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Correspondence to Danny Incarnato .

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Marinus, T., Incarnato, D. (2021). RNA Framework for Assaying the Structure of RNAs by High-Throughput Sequencing. In: Picardi, E. (eds) RNA Bioinformatics. Methods in Molecular Biology, vol 2284. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1307-8_5

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  • DOI: https://doi.org/10.1007/978-1-0716-1307-8_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1306-1

  • Online ISBN: 978-1-0716-1307-8

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