Landscape and variation of RNA secondary structure across the human transcriptome

Presentation of: Wan Y. et al. (2014) Landscape and variation of RNA secondary structure across the human transcriptome. Nature, 505(7485), 706-9.

DOI: http://dx.doi.org/10.1038/nature12946

Background: M.Sc. Bioinformatics RNA course

Presented by : Fritz Lekschas

Transcript

1. LANDSCAPE AND VARIATION OF RNA SECONDARY STRUCTURE ACROSS THE HUMAN TRANSCRIPTOME Y. Wan et al. (2014) 12. November 2014 Fritz Lekschas 1
2. INTRODUCTION 12. November 2014 Fritz Lekschas 2
3. THE STUDY Transciptome-wide study of RNA secondary structure (RSS) in a family trio (mother, father and son) •  RSS landscape of mRNA •  Influence of RSS on gene regulation •  Effects and importance of SNVs on RSS 12. November 2014 Fritz Lekschas 3
4. OVERVIEW 1.  Background to RSS 2.  How to technically assess RSS 3.  How to measure RSS 4.  How to study effects of RSS 5.  Results & Lookout 12. November 2014 Fritz Lekschas 4
5. BACKGROUND 12. November 2014 Fritz Lekschas 5
6. RNA SECONDARY STRUCTURE Sequence à Secondary Structure à Tertiary Structure 12. November 2014 Fritz Lekschas 6
7. INFORMATION FLOW 12. November 2014 Fritz Lekschas 7
8. INFLUENCE OF RSS •  Gene regulation – Accessibility of miRNA •  Splicing – Accessibility of spliceosome •  Folding – Guidance after translation 12. November 2014 Fritz Lekschas 8
9. METHODS 12. November 2014 Fritz Lekschas 9
10. WORKFLOW Wet laboratory 1.  RNA extraction 2.  RNA structure probing 3.  Library preparation 4.  Deep Sequencing Dry laboratory 1.  Read mapping to human transcriptome 2.  PARS score 3.  Structural differences score 4.  Impact of SNVs on RSS 12. November 2014 Fritz Lekschas 10
11. PAIRWISE ANALYSIS OF RNA STRUCTURE (PARS) 12. November 2014 Kertesz et al. (2010) 11 * Number of normalised mapped reads for RNase V1 and S1 nuclease
12. PARS SCORE Less Structure = more unpaired = score < 0 More structure = more paired = score > 0 12. November 2014 Fritz Lekschas 12
13. STRUCTURAL DIFFERENCE SCORE Normalised, absolute difference between PARS score of 2 conditions in a 5 bp window. 12. November 2014 Fritz Lekschas 13
14. STRUCTURAL DIFFERENCE SCORE 12. November 2014 Fritz Lekschas 14
15. RIBOSNITCHES SNV that might significantly alter RSS are called riboSNitchs. 1.  Structural Difference Score > 1 2.  P-value < 0.05 3.  FDR less than 0.1 4.  Local read coverage > 10 and at least 3 single / double-stranded reads in 11 bp window 12. November 2014 Fritz Lekschas 15
16. RIBOSNITCHES 12. November 2014 Fritz Lekschas 16
17. RESULTS 12. November 2014 Fritz Lekschas 17
18. RSS LANDSCAPE (1) 12. November 2014 Wan et al. (2014) 18
19. RSS LANDSCAPE (2) •  High similarity re-folded vs native deproteinised à RSS mainly determined by sequence •  Highly structured RNA have less variations à RSS important for RNA function 12. November 2014 Fritz Lekschas 19
20. POST-TRANSCRIPTIONAL REGULATION (1) Splicing miRNA-related AGO binding 12. November 2014 Fritz Lekschas 20
21. POST-TRANSCRIPTIONAL REGULATION (2) •  Unique RSS signature around exon-exon junctions •  Structural accessibility drives miRNA binding possibility – miRNA binding drives AGO / RISC binding à Gene silencing 12. November 2014 Fritz Lekschas 21
22. RIBOSNITCHES •  The more RSS is functionally important the less riboSNitches were found! •  RiboSNitches influes gene regulation, splicing and diseases 12. November 2014 Fritz Lekschas 22
23. DISCUSSION 12. November 2014 Fritz Lekschas 23
24. DISCUSSION •  Native deproteinised != in vivo –  Issues raised earlier still not addressed –  18S RNA PARS score only agrees to 50% –  9 of 14 tRNA structures predicted incorrectly •  Secondary != tertiary structure •  PARS scores useful for folding algorithms (SeqFold) •  Importance of SNVs in non-coding regions 12. November 2014 Fritz Lekschas 24
25. SUMMARY 12. November 2014 Fritz Lekschas 25
26. SUMMARY •  RSS determines accessibility of RNA – Translation and gene regulation – Splicing •  PARS: method for genome-wide study of RNA secondary structure •  SNVs can alter RSS & function of RNA (riboSNitches) 12. November 2014 Fritz Lekschas 26
27. THANK YOU! 12. November 2014 Fritz Lekschas 27 Questions?
28. APPENDIX 12. November 2014 Fritz Lekschas 28
29. RECOMMENDED READING 1.  Mauger & Weeks (2010) Toward global RNA structure analysis. PMID:21057487 2.  Lokody (2014) RNA: riboSNitches reveal heredity in RNA secondary structure. PMID:24535248 3.  What do your riboSNitches tell about you? (2014) 4.  Wan et. al. (2014) Landscape and variation of RNA secondary structure across the human transcriptome. PMID:24476892 5.  Kertesz & Wan et. al. (2010) Genome-wide Measurement of RNA Secondary Structure in Yeast. PMID:20811459 6.  Ouyang et al. (2013) SeqFold: genome-scale reconstruction of RNA secondary structure integrating high-throughput sequencing data. PMID:23064747 12. November 2014 Fritz Lekschas 29 * Ordering: easy to hard to additional
30. STRUCTURAL DIFFERENCE SCORE Z-score: (StrucDiff – mean) / s.d. of pos. control1 P-value: Estimated likelihood that a 5-base average permuted2 PARS structural difference is greater than the 5-base average of a SNVs PARS structural difference. FDR: Multi-hypothesis in R using p.adjust 12. November 2014 Fritz Lekschas 30 1 Tetrahymena ribozyme acts as positive control 2 1000 permutations of abs. PARS score differences of transcript containing SNVs
31. STUDY: RSS & GENE REGULATION •  Argonaute (AGO) is an important catalyst in RISC •  RISC is responsible for gene silencing •  Predict miRNA target sites and assess PARS score •  Compare true and false AGO-binding sites •  Compare real AGO-binding with iCLIP •  Study repression of mRNA with high and low miRNA accessibility in HeLa cells 12. November 2014 Fritz Lekschas 31
32. STUDY: RIBOSNITCH •  Predict riboSNitches in silico •  Experimentally confirm using RNA structure probing (e.g. SHAPE) •  Compare PARS score derived RSS with experimental data 12. November 2014 Fritz Lekschas 32
33. RIBOSWITCH VS SNITCH Riboswitch: mRNA binding sites which cause a significant structural and functional change of the mRNA. RiboSNitch: a SNV / SNP that putatively alters the RSS significantly and might point us to a possible Riboswitch. 12. November 2014 Fritz Lekschas 33