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, GCB 2011

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, GCB 2011

4. Problems of gene expression analysis on total RNA level
Caroline C. Friedel, GCB 2011

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, GCB 2011

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, GCB 2011

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, GCB 2011

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, GCB 2011

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, GCB 2011

10. Gene expression analysis on newly transcribed RNA
Solution:
Gene expression analysis on newly transcribed RNA
Caroline C. Friedel, GCB 2011

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, GCB 2011

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, GCB 2011

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, GCB 2011

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, GCB 2011

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, GCB 2011

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, GCB 2011

17. 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, GCB 2011

18. 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, GCB 2011

19. 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, GCB 2011

20. Combining RNA-Seq and RNA tagging
Caroline C. Friedel, GCB 2011

21. From microarrays to RNA-seq
Previous 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
Newly transcribed RNA not yet completely processed
Analysis of RNA processing by combining RNA-seq with RNA tagging
Caroline C. Friedel, GCB 2011

22. RNA-seq of newly transcribed RNA
Pilot study w/o rRNA depletion (1 cell line)
60 min newly transcribed 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, GCB 2011

23. Sequencing output 10-15% rRNA reads for newly transcribed RNA
~42% in total RNA w/o rRNA depletion
~10% in total and newly transcribed 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, GCB 2011

24. Unspliced transcripts in newly transcribed RNA
Caroline C. Friedel, GCB 2011

25. 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 transcription changes from changes in decay
Transcript half-lives determined from newly transcribed RNA
More precise than previous approaches
Support analysis of gene regulation
RNA tagging compatible with RNA-seq
Newly transcribed RNA contains large fractions of unspliced RNA
Caroline C. Friedel, GCB 2011

26. 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 Molecular Cell.
Caroline C. Friedel, GCB 2011

27. Acknowledgements Lars Dölken Ulrich Koszinowski Ralf Zimmer
Lukas Windhager
Florian Erhard
Gergely Csaba
Friedel Group
Thomas Bonfert
Caroline C. Friedel, GCB 2011

28. Thank you for your Attention !
Questions ?
Caroline C. Friedel, GCB 2011