Yeast Functional Genomics pp 141-160 | Cite as
Genome-Wide Probing of RNA Structures In Vitro Using Nucleases and Deep Sequencing
- 4 Citations
- 1 Mentions
- 3.2k Downloads
Abstract
RNA structure probing is an important technique that studies the secondary and tertiary conformations of an RNA. While it was traditionally performed on one RNA at a time, recent advances in deep sequencing has enabled the secondary structure mapping of thousands of RNAs simultaneously. Here, we describe the method Parallel Analysis for RNA Structures (PARS), which couples double and single strand specific nuclease probing to high throughput sequencing. Upon cloning of the cleavage sites into a cDNA library, deep sequencing and mapping of reads to the transcriptome, the position of paired and unpaired bases along cellular RNAs can be identified. PARS can be performed under diverse solution conditions and on different organismal RNAs to provide genome-wide RNA structural information. This information can also be further used to constrain computational predictions to provide better RNA structure models under different conditions.
Key words
RNA Structure Biochemistry Genomics High-throughput sequencingNotes
Acknowledgements
This work is supported by NIH R01-HG004361 (H.Y.C.) and Agency for Science, Technology and Research of Singapore (Y.W.).
References
- 1.Wan Y, Kertesz M, Spitale RC, Segal E, Chang HY (2011) Understanding the transcriptome through RNA structure. Nat Rev Genet 12:641–655CrossRefPubMedGoogle Scholar
- 2.Weeks KM (2010) Advances in RNA structure analysis by chemical probing. Curr Opin Struct Biol 20:295–304PubMedCentralCrossRefPubMedGoogle Scholar
- 3.Ehresmann C et al (1987) Probing the structure of RNAs in solution. Nucleic Acids Res 15:9109–9128PubMedCentralCrossRefPubMedGoogle Scholar
- 4.Mitra S, Shcherbakova IV, Altman RB, Brenowitz M, Laederach A (2008) High-throughput single-nucleotide structural mapping by capillary automated footprinting analysis. Nucleic Acids Res 36, e63PubMedCentralCrossRefPubMedGoogle Scholar
- 5.Lucks JB et al (2011) Multiplexed RNA structure characterization with selective 2′-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). Proc Natl Acad Sci U S A 108:11063–11068PubMedCentralCrossRefPubMedGoogle Scholar
- 6.Underwood JG et al (2010) FragSeq: transcriptome-wide RNA structure probing using high-throughput sequencing. Nat Methods 7:995–1001PubMedCentralCrossRefPubMedGoogle Scholar
- 7.Kertesz M et al (2010) Genome-wide measurement of RNA secondary structure in yeast. Nature 467:103–107CrossRefPubMedGoogle Scholar
- 8.Wan Y et al (2012) Genome-wide measurement of RNA folding energies. Mol Cell 48:169–181PubMedCentralCrossRefPubMedGoogle Scholar
- 9.Wan Y et al (2014) Landscape and variation of RNA secondary structure across the human transcriptome. Nature 505:706–709PubMedCentralCrossRefPubMedGoogle Scholar
- 10.Ouyang Z, Snyder MP, Chang H (2013) SeqFold: genome-scale reconstruction of RNA secondary structure integrating high-throughput sequencing data. Genome Res 23(2):377–387PubMedCentralCrossRefPubMedGoogle Scholar
- 11.Guo F, Gooding AR, Cech TR (2004) Structure of the Tetrahymena ribozyme: base triple sandwich and metal ion at the active site. Mol Cell 16:351–362PubMedGoogle Scholar
- 12.Quail MA et al (2008) A large genome center’s improvements to the Illumina sequencing system. Nat Methods 5:1005–1010PubMedCentralCrossRefPubMedGoogle Scholar