1. 3rd Internal RECESS workshop Caroline C. Friedel
Into the deep: Characterization of RNA splicing by high-throughput sequencing of newly transcribed RNA
3rd Internal RECESS workshop
Caroline C. Friedel

2. Metabolic tagging of newly transcribed RNA
Pre-existing RNA
Newly transcribed RNA
Simultaneous measurements of RNA decay and de novo transcription
RNA quantification
microarrays / RNA-seq
Caroline C. Friedel, Recess internal workshop

3. Metabolic tagging of newly transcribed RNA
4-thiouridine (4sU)
Kenzelmann et al., PNAS, 2007
Tagging of newly transcribed RNA U U U U U U
Dölken et al., RNA, 2008
Thiol-mediated purification
Newly transcribed RNA
Pre-existing RNA U U U U U U
Caroline C. Friedel, Recess internal workshop

4. Advantages Gene expression profiling on newly transcribed RNA
Increases sensitivity to differential expression
No bias towards differential expression of short-lived transcripts
Can distinguish primary from secondary responses
Can discriminate transcription changes from changes in decay
Allows determination of mRNA Half-life
Caroline C. Friedel, Recess internal workshop

5. From microarrays to RNA-seq
Most existing studies used microarrays
Only one value per gene
Even with exon arrays only some parts of a gene are probed
Sequencing of RNA (RNA-seq) allows the analysis of all (expressed) parts of a gene
Nascent RNA not yet completely processed
Analysis of RNA processing by combining RNA-seq with RNA tagging
Caroline C. Friedel, Recess internal workshop

6. Exon-intron junction reads
RNA-Seq vs. 4sU-seq
Exon-intron junction reads
Intronic reads
Caroline C. Friedel, Recess internal workshop

7. RNA-seq of nascent RNA (4sU-seq)
Poly-A selection not applicable for nascent RNA
Random primer
RNA samples contain large fractions of rRNA
Previous studies combining RNA tagging and RNA-seq:
45 min (Rabani et al., Nat. Biotech., 2011)
2 h (Schwanhäusser et al., Nature, 2011)
Pilot study w/o rRNA depletion (1 cell line)
5, 10, 15, 20 and 60 min nascent RNA
Total RNA, pre-existing RNA
Follow-up study w/ rRNA depletion (2 cell lines)
Ultra-short and progressive 4sU-seq
Caroline C. Friedel, Recess internal workshop

8. Sequencing output 10-15% rRNA reads for nascent RNA
~42% in total RNA w/o rRNA depletion
~10% in total and nascent RNA w/ rRNA depletion
rRNA depletion necessary for analysis of total and pre-existing RNA only
# rRNA reads
Total # reads
Pilot study
DG75
Follow-up
DG75 DG75-10/12
Caroline C. Friedel, Recess internal workshop

9. Mapping of sequencing reads
>1_56_481_F3 AY60U length=35 filter=1 lane=1 T
>1_56_1075_F3 AY60U length=35 filter=1 lane=1 T
>1_58_1978_F3 AY60U length=35 filter=1 lane=1 T
>1_60_892_F3 AY60U length=35 filter=1 lane=1 T …
Problem:
Sequencing errors lead to a completely
wrong sequence when decoding this way
Decoding only by alignment to genome or transcriptome T T A G T …
Caroline C. Friedel, Recess internal workshop

10. Exon-intron junctions
Mapping pipeline
>1_56_481_F3 AY60U length=35 filter=1 lane=1 T
>1_56_1075_F3 AY60U length=35 filter=1 lane=1 T
>1_58_1978_F3 AY60U length=35 filter=1 lane=1 T
>1_60_892_F3 AY60U length=35 filter=1 lane=1 T …
Reads in color code
Alignment to rRNA
Unaligned reads
Mapped rRNA reads
Alignment to transcriptome
Exons
Exon-exon junctions
Unaligned reads
Alignment to genome
Introns
Exon-intron junctions
Unaligned reads
Novel splicing isoforms, contaminations, noise
Caroline C. Friedel, Recess internal workshop

11. Nascent RNA contains unspliced transcripts
Novel splicing isoforms ?
Retained introns ?
Splicing ?
Sequencing problems ?
Caroline C. Friedel, Recess internal workshop

12. Progressive RNA-seq visualizes splicing
Gap between intron and exon-intron junction reads
Gap between exon and exon-exon junction reads
Caroline C. Friedel, Recess internal workshop

13. Long introns are faster degraded
Normalization by intron length
Caroline C. Friedel, Recess internal workshop

14. Clustering of intron decay patterns
Intron/gene expression ratios over time
K-means clustering (k=10) …
Network calculation
Final stable clusters
Caroline C. Friedel, Recess internal workshop

15. Classes of intron splicing
Caroline C. Friedel, Recess internal workshop

16. Classes of intron splicing
„Normal“ splicing
„Faster“ splicing
„Slower“ splicing
Intron retention
Caroline C. Friedel, Recess internal workshop

17. Examples Class 1: „Normal“ intron decay Class 4: Intron retention
Caroline C. Friedel, Recess internal workshop

18. Retained introns are eventually degraded
Core exons
Known retain introns
Secondary splicing or nonsense-mediated decay ?
Caroline C. Friedel, Recess internal workshop

19. snoRNAs
small RNA molecules guiding chemical modifications of other RNAs
Mostly encoded within introns of other genes
Splicing independent
Splicing dependent
Caroline C. Friedel, Recess internal workshop

20. Microarray probes measure short half-lives of snoRNA precursors
Fast decay of snoRNAs ?
Microarray probes measure short half-lives of snoRNA precursors
Caroline C. Friedel, Recess internal workshop

21. Slower decay of snoRNA containing introns
Decay of snoRNA precursor introns slowed down
To allow for snoRNA processing?
Short half-lives indicates inefficiency of snoRNA processing
Splicing and decay faster than processing after splicing ?
Caroline C. Friedel, Recess internal workshop

22. Distinguishing precursors from mature snoRNAs
Long precursor
Mature snoRNA
non-intronic snoRNA
Caroline C. Friedel, Recess internal workshop

23. Summary
Short-term and progressive 4sU-seq for analysis of RNA splicing and processing
Distinct classes of splicing can be identified
Retained introns are eventually degraded:
Secondary splicing or nonsense-mediated decay ?
Microarrays measure short half-lives of snoRNA precursors
Inefficient processing of snoRNAs?
Caroline C. Friedel, Recess internal workshop

24. References
High-resolution gene expression profiling for simultaneous kinetic parameter analysis of RNA synthesis and decay. Dölken L, Ruzsics Z, Rädle B, Friedel CC, Zimmer R, Mages J, Hoffmann R, Dickinson P, Forster T, Ghazal P, Koszinowski UH. RNA, 2008, 14(9),
Conserved principles of mammalian transcriptional regulation revealed by RNA half-life. Friedel CC, Dölken L, Ruzsics Z, Koszinowski U, Zimmer R. Nucleic Acids Research, Nucleic Acids Res., 2009, 37(17):e115
Metabolic tagging and purification of nascent RNA: Implications for transcriptomics. Friedel CC and Dölken L. Molecular BioSystems, Mol Biosyst., 2009, 5(11):
Systematic analysis of viral and cellular microRNA targets in cells latently infected with human gamma-herpesviruses by RISC immunoprecipitation assay. Dölken L, Malterer G, Erhard F, Kothe S, Friedel CC, et al. Cell Host Microbe. 2010, 7(4):
Ultra short and progressive 4sU-tagging reveals key characteristics of RNA processing at nucleotide resolution. Windhager L. et al. Submitted to Genome Research.
Caroline C. Friedel, Recess internal workshop

25. Acknowledgements Lars Dölken Ulrich Koszinowski Dirck Eick
Kaspar Burger
Philip Rosenstiel
Markus Schilhabel
Ralf Zimmer
Lukas Windhager
Florian Erhard
Gergely Csaba
Thomas Bonfert
Caroline C. Friedel, Recess internal workshop