Best practices for genome-wide RNA structure analysis: combination of mutational profiles and drop-off information

Genome-wide RNA structure maps have recently become available through the coupling of in vivo chemical probing reagents with next-generation sequencing. Initial analyses relied on the identification of truncated reverse transcription reads to identify the chemically modified nucleotides, but recent studies have shown that mutational signatures can also be used. While these two methods have been employed interchangeably, here we show that they actually provide complementary information. Consequently, analyses using exclusively one of the two methodologies may disregard a significant portion of the structural information. We find that the identity and sequence environment of the modified nucleotide greatly affects the odds of introducing a mismatch or causing reverse transcriptase drop-off. Finally, we identify specific mismatch signatures generated by dimethyl sulfate probing that can be used to remove false positives typically produced in RNA structurome analyses, and how these signatures vary depending on the reverse transcription enzyme used.

[1]  John S. Mattick,et al.  The RNA modification landscape in human disease , 2017, RNA.

[2]  M. Rutenberg-Schoenberg,et al.  Interpreting Reverse Transcriptase Termination and Mutation Events for Greater Insight into the Chemical Probing of RNA. , 2017, Biochemistry.

[3]  Christopher E. Mason,et al.  Charting the unknown epitranscriptome , 2017, Nature Reviews Molecular Cell Biology.

[4]  R. Spitale,et al.  Measuring RNA structure transcriptome-wide with icSHAPE. , 2017, Methods.

[5]  Michael H. Schwartz,et al.  Selective Enzymatic Demethylation of N2 ,N2 -Dimethylguanosine in RNA and Its Application in High-Throughput tRNA Sequencing. , 2017, Angewandte Chemie.

[6]  Yuri Motorin,et al.  Detecting RNA modifications in the epitranscriptome: predict and validate , 2017, Nature Reviews Genetics.

[7]  S. Kalantry,et al.  Functional Dissection of the m6A RNA Modification. , 2017, Trends in biochemical sciences.

[8]  T. Lowe,et al.  Small RNA Modifications: Integral to Function and Disease. , 2016, Trends in molecular medicine.

[9]  Zhao Su,et al.  Genome-Wide Analysis of RNA Secondary Structure. , 2016, Annual review of genetics.

[10]  J. Weissman,et al.  DMS-MaPseq for genome-wide or targeted RNA structure probing in vivo , 2016, Nature Methods.

[11]  T. Bredy,et al.  Evolving insights into RNA modifications and their functional diversity in the brain , 2016, Nature Neuroscience.

[12]  Kaoru Inoue,et al.  SHAPE reveals transcript-wide interactions, complex structural domains, and protein interactions across the Xist lncRNA in living cells , 2016, Proceedings of the National Academy of Sciences.

[13]  Kyle E. Watters,et al.  Characterizing RNA structures in vitro and in vivo with selective 2'-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq). , 2016, Methods.

[14]  W. Gilbert,et al.  Messenger RNA modifications: Form, distribution, and function , 2016, Science.

[15]  Chengqi Yi,et al.  Transcriptome-wide mapping reveals reversible and dynamic N(1)-methyladenosine methylome. , 2016, Nature chemical biology.

[16]  Gideon Rechavi,et al.  The dynamic N1-methyladenosine methylome in eukaryotic messenger RNA , 2016, Nature.

[17]  S. Oliviero,et al.  RNA structure framework: automated transcriptome-wide reconstruction of RNA secondary structures from high-throughput structure probing data , 2016, Bioinform..

[18]  Howard Y. Chang,et al.  Transcriptome-wide interrogation of RNA secondary structure in living cells with icSHAPE , 2016, Nature Protocols.

[19]  Walter N. Moss,et al.  Probing Xist RNA Structure in Cells Using Targeted Structure-Seq , 2015, PLoS genetics.

[20]  Pavel P. Kuksa,et al.  Chemical Modifications Mark Alternatively Spliced and Uncapped Messenger RNAs in Arabidopsis[OPEN] , 2015, Plant Cell.

[21]  Kevin M Weeks,et al.  Selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) for direct, versatile and accurate RNA structure analysis , 2015, Nature Protocols.

[22]  Andreas Hildebrandt,et al.  The reverse transcription signature of N-1-methyladenosine in RNA-Seq is sequence dependent , 2015, Nucleic acids research.

[23]  Chengqi Yi,et al.  Efficient and quantitative high-throughput transfer RNA sequencing , 2015, Nature Methods.

[24]  Sarah M Assmann,et al.  Genome-wide profiling of in vivo RNA structure at single-nucleotide resolution using structure-seq , 2015, Nature Protocols.

[25]  Ryan T Fuchs,et al.  Bias in Ligation-Based Small RNA Sequencing Library Construction Is Determined by Adaptor and RNA Structure , 2015, PloS one.

[26]  Yin Tang,et al.  The RNA structurome: transcriptome-wide structure probing with next-generation sequencing. , 2015, Trends in biochemical sciences.

[27]  P. Agris.  The importance of being modified: an unrealized code to RNA structure and function , 2015, RNA.

[28]  Howard Y. Chang,et al.  Structural imprints in vivo decode RNA regulatory mechanisms , 2015, Nature.

[29]  J. Satterlee,et al.  Novel RNA Modifications in the Nervous System: Form and Function , 2014, The Journal of Neuroscience.

[30]  A. Ferré-D’Amaré,et al.  Structural basis for activity of highly efficient RNA mimics of green fluorescent protein , 2014, Nature Structural &Molecular Biology.

[31]  Steven Busan,et al.  RNA motif discovery by SHAPE and mutational profiling (SHAPE-MaP) , 2014, Nature Methods.

[32]  J. Doudna,et al.  Insights into RNA structure and function from genome-wide studies , 2014, Nature Reviews Genetics.

[33]  K. Morris,et al.  The rise of regulatory RNA , 2014, Nature Reviews Genetics.

[34]  Kevin Struhl,et al.  Global Analysis of mRNA Isoform Half-Lives Reveals Stabilizing and Destabilizing Elements in Yeast , 2014, Cell.

[35]  Qiangfeng Cliff Zhang,et al.  Landscape and variation of RNA secondary structure across the human transcriptome , 2014, Nature.

[36]  Manolis Kellis,et al.  Genome-wide probing of RNA structure reveals active unfolding of mRNA structures in vivo , 2013, Nature.

[37]  Yuk Yee Leung,et al.  HAMR: high-throughput annotation of modified ribonucleotides , 2013, RNA.

[38]  V. Iyer,et al.  Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing , 2013, RNA.

[39]  J. Mattick,et al.  Structure and function of long noncoding RNAs in epigenetic regulation , 2013, Nature Structural &Molecular Biology.

[40]  Lior Pachter,et al.  SHAPE–Seq: High‐Throughput RNA Structure Analysis , 2012, Current protocols in chemical biology.

[41]  Daniel Lai,et al.  R-chie: a web server and R package for visualizing RNA secondary structures , 2012, Nucleic acids research.

[42]  Steven L Salzberg,et al.  Fast gapped-read alignment with Bowtie 2 , 2012, Nature Methods.

[43]  Hashim M. Al-Hashimi,et al.  Functional complexity and regulation through RNA dynamics , 2012, Nature.

[44]  Sergey Melnikov,et al.  The Structure of the Eukaryotic Ribosome at 3.0 Å Resolution , 2011, Science.

[45]  Steve Hoffmann,et al.  Traces of post-transcriptional RNA modifications in deep sequencing data , 2011, Biological chemistry.

[46]  T. Fennell,et al.  Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries , 2011, Genome Biology.

[47]  Howard Y. Chang,et al.  Genome-wide measurement of RNA secondary structure in yeast , 2010, Nature.

[48]  K. Weeks.  Advances in RNA structure analysis by chemical probing. , 2010, Current opinion in structural biology.

[49]  David H. Mathews,et al.  RNAstructure: software for RNA secondary structure prediction and analysis , 2010, BMC Bioinformatics.

[50]  Phillip A Sharp,et al.  The Centrality of RNA , 2009, Cell.

[51]  D. Mathews,et al.  Accurate SHAPE-directed RNA structure determination , 2009, Proceedings of the National Academy of Sciences.

[52]  D. Mathews.  Using an RNA secondary structure partition function to determine confidence in base pairs predicted by free energy minimization. , 2004, RNA.

[53]  D. Chen,et al.  Reverse transcriptase adds nontemplated nucleotides to cDNAs during 5'-RACE and primer extension. , 2001, BioTechniques.

[54]  T. Cech,et al.  A preorganized active site in the crystal structure of the Tetrahymena ribozyme. , 1998, Science.

[55]  J. Ebel,et al.  Probing the structure of RNAs in solution. , 1987, Nucleic acids research.

[56]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[57]  R. Russell,et al.  DMS footprinting of structured RNAs and RNA–protein complexes , 2007, Nature Protocols.