1. High resolution profiling of RNA synthesis and decay
Caroline C. Friedel
Institut für Pharmazie und Molekulare Biotechnologie
Ruprecht-Karls-Universität Heidelberg

2. Introduction Standard differential gene expression analysis:
based on changes in total cellular RNA
New method:
Metabolic tagging of newly transcribed RNA
Direct analysis of differential de novo transcription
Caroline C. Friedel,

3. 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,

4. Problems of gene expression analysis on total RNA level
Caroline C. Friedel,

5. Low sensitivity and bias
Delay between changes in transcription and detectable effect on total RNA
10x Up-regulation
of transcription
Short
transcript half-life =
High basal transcription rates
Fast
up-regulation
of total RNA
Long
transcript half-life =
Low basal transcription rates
Slow
up-regulation
of total RNA
Caroline C. Friedel,

6. Low sensitivity and bias
Delay between changes in transcription and detectable effect on total RNA
Down-regulation by
transcriptional arrest
Short
transcript half-life =
Fast
transcript decay
down-regulation
of total RNA
Long
transcript half-life =
Slow
transcript decay
down-regulation of total RNA
Caroline C. Friedel,

7. Low sensitivity and bias
Same transcription changes for transcripts with different half-lives
 different changes in total RNA
Differential expression of short-lived transcripts detected sooner than for long-lived transcripts
Bias for differential expression of short-lived transcripts
To increase sensitivity, total RNA extracted several hours after the stimulus
Caroline C. Friedel,

8. Low temporal resolution
Cannot distinguish between
Primary (immediate) induction of long-lived transcripts
Secondary (down-stream) induction of short-lived transcripts
total RNA
newly transcribed RNA
T1/2=10 h
T1/2=2 h
Temporal development observed in total RNA
True temporal development
Caroline C. Friedel,

9. Alteration in RNA transcription or stability ?
Changes in total RNA:
due to alterations in RNA transcription and/or alterations in RNA stability
2-fold down-regulation of total RNA
t=t1/2=1 h
Normal RNA stability
Transcription shut-off
Normal RNA transcription
RNA stability highly decreased
Caroline C. Friedel,

10. Gene expression analysis on newly transcribed RNA
Solution:
Gene expression analysis on newly transcribed RNA
Caroline C. Friedel,

11. 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,

12. Metabolic tagging of newly transcribed RNA
Pre-existing RNA U U U U U U
RNA quantification
Microarrays / RNA-seq
Standard Workflow
Normalization
Detection of differentially expressed genes
Caroline C. Friedel,

13. Metabolic tagging of newly transcribed RNA
Directly observe changes in de novo transcription
Increased sensitivity
Observed changes relative to basal transcription rate
Independent of transcript half-life
No bias towards short-lived transcripts
Newly transcribed RNA U U U U U U
Detection of differentially expressed genes
Caroline C. Friedel,

14. Case study: IFN stimulation
1 h IFN treatment
Tagging of newly transcribed RNA:
0-30 min
0-60 min
30-60 min
Total RNA: after 1 h
Short half-life
Long
half-life
Caroline C. Friedel,

15. Temporal resolution Transcription changes can be detected promptly
total RNA
newly transcribed RNA
T1/2=10 h
T1/2=2 h
Transcription changes can be detected promptly
Both for short- and long-lived transcripts
Temporal development of the cell response can be determined
Caroline C. Friedel,

16. Alteration in RNA transcription or stability ?
Analysis of total and newly transcribed RNA:
Changes in transcription may occur together with changes in decay
Changes only in newly transcribed RNA
Changes only in total RNA
Alterations (mostly) in RNA
transcription
Alterations in RNA
stability / decay
Changes in newly transcribed and total RNA
Caroline C. Friedel,

17. Alteration in RNA transcription or stability ?
Alteration both in transcription and stability ?
Additionally analyze pre-existing RNA
Use RNA half-lives to correlate changes in transcription to changes in total RNA
Observed change in total RNA
Expected change in total RNA
Determined from newly transcribed RNA
Caroline C. Friedel,

18. Determination of transcript half-life
Transcript half-life ~ speed of RNA turnover
Previously identified by inhibition of transcription and monitoring of decay
Cell-invasive
Stabilization of transcripts
Non-invasive determination from ratios of newly transcribed/total RNA
Caroline C. Friedel,

19. Normalization
Standard normalization methods assume overall equal intensities
Second normalization:
Estimate normalization factors with linear regression using that
Newly transcribed + pre-existing RNA=total RNA
Normalization based on thousands of genes from one time point
Allows quality control on measurements
Depending on deviation from regression line
Caroline C. Friedel,

20. Calculation of RNA half-life
Calculate RNA half-life from exponential decay model
RNA half-lives determined from de novo transcription are more precise than from RNA decay
De novo transcription
RNA decay
Caroline C. Friedel,

21. Why are RNA half-lives interesting ?
Determine the effect on total RNA for a specific change in de novo transcription
How fast can genes be regulated on the transcriptional level ?
Fast decay = fast regulation
Slow decay = slow regulation
Correlated to gene function
Caroline C. Friedel,

22. Regulation of protein complexes
Conserved regulatory principles for protein complexes
by transcriptional regulation of key regulatory subunits
“Just-in-time assembly” (de Lichtenberg et al. Science 2005)
PBRM1
9.0 / NA
ACTL6A
8.2 / 6.3
SMARCA4
5.1/ 5.8
SMARCC2
9.7 / 5.6
SMARCD1
5.4 / 3.3
SMARCC1
11.9 / 6.1
ARID2
2.3 / 1.7
Common with BAF complex
SMARCB1
13.3 / 13.7
9.2 / 5.7
SMARCE1
Specific
for PBAF
complex
Caroline C. Friedel,

23. Combining RNA-Seq and RNA tagging
Caroline C. Friedel,

24. RNA-seq of nascent RNA Pilot study w/o rRNA depletion (1 cell line)
60 min nascent RNA
Total RNA, pre-existing RNA
Follow-up study w/ rRNA depletion (2 cell lines)
Previous studies combining RNA tagging and RNA-seq:
45 min (Rabani et al., Nat. Biotech., 2011)
2 h (Schwanhäusser et al., Nature, 2011)
Caroline C. Friedel,

25. 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
# rRNA reads
Total # reads
Pilot study
DG75
Follow-up
DG75 DG75-10/12
Caroline C. Friedel,

26. 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
Mapped rRNA reads
Unaligned reads
Alignment to transcriptome
Exons
Exon-exon junctions
Unaligned reads
Alignment to genome
Introns
Exon-intron junctions
Unaligned reads
Caroline C. Friedel,

27. Nascent RNA contains unspliced transcripts
Caroline C. Friedel,

28. Identifying experimental artefacts
nascent RNA cell line 2
Total RNA cell line 2
Nascent RNA cell line 1
Total RNA cell line 1
Short non-coding RNAs
Caroline C. Friedel,

29. Identifying experimental artefacts
Nascent and pre-existing RNA ≠ total for snoRNAs
 Artefact, probably during size selection in library preparation
Total RNA cell line 2
Total RNA cell line 1
Normalized sum of nascent and pre-existing RNA
Total RNA cell line 1
Total RNA cell line 2
Caroline C. Friedel,

30. Conclusions 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 changes in transcription from changes in decay
Transcript half-lives determined from newly transcribed RNA
More precise than previous approaches
Support analysis of gene regulation
Caroline C. Friedel,

31. Conclusions RNA tagging compatible with RNA-seq
Nascent RNA contains large fractions of unspliced RNA
Experimental artefacts can be identified using inherent quality control
Caroline C. Friedel,

32. 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, Suffert G, Marcinowski L, Motsch N, Barth S, Beitzinger M, Lieber D, Bailer SM, Hoffmann R, Ruzsics Z, Kremmer E, Pfeffer S, Zimmer R, Koszinowski UH, Grässer F, Meister G, Haas J. Cell Host Microbe. 2010, 7(4):
Caroline C. Friedel,

33. Collaborators Jürgen Haas Lars Dölken Susanne Bailer Zsolt Ruzsics
Albrecht von Brunn
Ekaterina Dall'Armi
Even Fossum
Lars Dölken
Zsolt Ruzsics
Ulrich Koszinowski
Herbert Schiller
Ania Muntau
Søren Gersting
Mathias Woidy
Peter Ghazal
Paul Dickinson
Thorsten Forster
Reinhard Hoffmann
Kevin Robertson
Steven Watterson
Manfred Koegl
Caroline C. Friedel,

34. Thank you for your Attention !
Questions ?
Caroline C. Friedel,