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

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, 25.5.2011

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, 25.5.2011

Problems of gene expression analysis on total RNA level Caroline C. Friedel, 25.5.2011

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, 25.5.2011

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, 25.5.2011

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, 25.5.2011

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, 25.5.2011

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, 25.5.2011

Gene expression analysis on newly transcribed RNA Solution: Gene expression analysis on newly transcribed RNA Caroline C. Friedel, 25.5.2011

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, 25.5.2011

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, 25.5.2011

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, 25.5.2011

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, 25.5.2011

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, 25.5.2011

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, 25.5.2011

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, 25.5.2011

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, 25.5.2011

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, 25.5.2011

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, 25.5.2011

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, 25.5.2011

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, 25.5.2011

Combining RNA-Seq and RNA tagging Caroline C. Friedel, 25.5.2011

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, 25.5.2011

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, 25.5.2011

Exon-intron junctions Mapping pipeline >1_56_481_F3 AY60U length=35 filter=1 lane=1 T32131320331122103222022330223022233 >1_56_1075_F3 AY60U length=35 filter=1 lane=1 T23031011033033033002033030000002002 >1_58_1978_F3 AY60U length=35 filter=1 lane=1 T30331122103122002320023022113232222 >1_60_892_F3 AY60U length=35 filter=1 lane=1 T32102032101330013231331210311132212 … 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, 25.5.2011

Nascent RNA contains unspliced transcripts Caroline C. Friedel, 25.5.2011

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, 25.5.2011

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, 25.5.2011

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, 25.5.2011

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, 25.5.2011

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), 1959-72 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):1271-8. 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):324-34. Caroline C. Friedel, 25.5.2011

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, 25.5.2011

Thank you for your Attention ! Questions ? Caroline C. Friedel, 25.5.2011