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

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

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

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

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

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

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

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

Mapping of sequencing reads >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 … Problem: Sequencing errors lead to a completely wrong sequence when decoding this way Decoding only by alignment to genome or transcriptome T32131320331122103222022330223022233 T A G T … Caroline C. Friedel, Recess internal workshop

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 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

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

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

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

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

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

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

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

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

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

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

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

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

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

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, et al. Cell Host Microbe. 2010, 7(4):324-34. 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

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