Download Transcriptome profiling

OWS 2013
Transcriptome profiling
--Past, Present and Future
Wei Chen
Berlin Institute for Medical Systems Biology
Max-Delbrueck-Center for Molecular Medicine
Why RNA?
DNA
Transcription
5’ capping
Splicing
RNAi (yeast)
LincRNA
Promoter associated RNA
enhancer associated RNA
…
snRNA
3’ poly A
RNA editing
Localization
…
(m)RNA
degradation
Translation
Protein
tRNA
rRNA
miRNA …
Transcriptome profiling
•  Past
–  Pre-genome era
–  Genome era
•  Present
•  Ongoing and further development
Pre-genome era (1960s)
?
DNA
Protein
rRNA tRNA mRNA
Fractionation technique (Count Concurrent Distribution for tRNA
isolation )
During his (R.Holley) 3 years of work on the structure of the alanine tRNA, Holley used a
total of only 1 g of highly purified material, which he isolated from approximately 200 g
of bulk yeast tRNA, which in turn was obtained by phenol extraction of approximately 140
kg of commercial bakers' yeast.
RNA sequencing
Specifically, Holley, George A. Everett, James T. Madison, and Ada Zamir first used
pancreatic ribonuclease to cleave the RNA chain next to pyrimidine nucleotides and then
used takadiastase ribonuclease T1 to cleave the RNA chain at guanylic acid residues. They
isolated the resulting fragments by ion-exchange chromatography. The components of
dinucleotide fragments were then identified by chromatographic and electrophoretic
properties and spectra…
Pre-genome era (1970s and 1980s)
•  Reverse transcriptase (Temin and Baltimore,
1970, Nobel prize 1975)
•  PCR (Mullis, 1983, Nobel Prize 1993)
•  Sanger Sequencing (Sanger, 1977, Nobel Prize
1980)
•  Northern Blot (Alwine, Kemp, and Stark, 1977 )
Genome era (1990s, 2000s)
•  Expressed sequence tags (EST) sequencing
–  one-shot sequencing of a clone cDNA/mRNA
–  Several hundred bps, 3’, 5’ or random
• Discovery of expressed (m)RNAs from different tissues
• Physical mapping of genes into chromosome
• Design of expression microarray
Genome era (1990s, 2000s)
•  Series Analysis of Gene expression (SAGE)
Genome era (1990s, 2000s)
•  Microarray
Limitations
a.  Available annotation
b.  Cross hybridization
c.  Limited dynamic
range/sensitivity
Massive parallel RNA sequencing (2005-present)
• Small RNA sequencing
• miRNA, piRNA, siRNA…
• RNA-seq
•  >200nt
Small RNA library prep (miRNA, PiRNA...)
• Ligation: 5’ phosphate and 3’ OH, ligation bias
• RT-PCR: strong bias due to 2nd structure
Small RNA sequencing result
10-40nt
40-90nt
Li et.al, NAR 41(6) 3619-3634
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RNA-seq library prep (>200nt)
rRNA –
poly A+
UNG treatment
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RNA-seq vs Array
Wang et.al, Nature Review Genetics (10) 57-63
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Findings
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Novel miRNAs
Novel PiRNAs
Endo-siRNAs
Novel isoforms (5’/3’ end, alternative splicing)
Promoter associated RNAs
Enhancer RNAs
LincRNAs
Circular RNAs
LincRNAs
•  Negative definition
•  Not protein coding
•  Not overlapping with other defined transcripts
•  PolII transcripts
–  Cap, polyA, often splicing
•  A heterogeneous group with diverse properties and
functions
LincRNA detection
•  FANTOM project (cDNA
clone and Sanger seq)
–  >34000 in different
mouse tissues
•  Tiling array
–  define transcribed region
w/o transcript model
•  RNA-seq & de novo
assembly
•  Chromatin map
•  Other supporting data
–  CAGE, 3-P
Igor Ulitsky and David P. Bartel Cell (154) 26-46
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Non-coding vs coding
Igor Ulitsky and David P. Bartel Cell (154) 26-46
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LincRNA association with Ribosome
Guttman et.al. Cell (154) 240-251
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LincRNA genomics
•  Preferentially surrounding developmental TFs
–  Regulate gene is cis (e.g. HOTTIP)
–  Act in concert and benefit from co-regulation (e.g. Six3 and
Six3os)
–  Accommodating environment for the emergence of new
lincRNAs
•  Low expression and tissue specific (brain and testis)
–  median 1/10 protein-coding
•  Subcellular localization
–  both nuclear and cytoplasme
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Diverse functions of lincRNAs
Cis-regulation
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Association with PRC2, CTCF…
Direct chromatin modifying complex to
DNA via nascent transcript or triplex
interaction
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Malat1 binds multiple proteins in
paraspeckles
• 
Gadd7 & TDP-43
Paring ofAlu-repeat induces STAU1
action.
miRNA sponge
Igor Ulitsky and David P. Bartel Cell (154) 26-46
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Circular RNAs
Jeremy E. Wilusz and Phillip A. Sharp Science (340) 440
1. 
Cocquerelle, C., et al, Mis-splicing yields circular RNA molecules. FASEB J. 7, 155–160 (1993). 2. 
Capel, B. et al. Circular transcripts of the testis-determining gene Sry in adult mouse testis. Cell 73, 1019–1030 (1993). 3. 
Chao, C. W., et al., The mouse formin (Fmn) gene: abundant circular RNA transcripts and gene-targeted deletion analysis. Mol. Med. (1998). 4. 
Suzuki, H. et al. Characterization of RNase R-digested cellular RNA source that consists of lariat and circular RNAs from pre-mRNA splicing. Nucleic Acids Res. (2006). 5. 
Burd, C. E. et al. Expression of linear and novel circular forms of an INK4/ARF- associated non-coding RNA correlates with atherosclerosis risk. PLoS Genet. (2010). 6. 
Hansen, T. B. et al. miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA. EMBO J. 30, 4414–4422 (2011). 7. 
Salzman,J.et al. , Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types. PLoS ONE 7, e30733 (2012). 8. 
Jeck, W. R. et al. Circular RNAs are abundant, conserved, and associated with ALU repeats. RNA 19, 1–17 (2013).
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Detection of circular RNA
Memczak et.al Nature. (7441):333-8
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CDR1as—a circular miR-7 sponge
Memczak et.al Nature. (7441):333-8
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CDR1as—a circular miR-7 sponge
Memczak et.al Nature. (7441):333-8
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CDR1as—a circular miR-7 sponge
Memczak et.al Nature. (7441):333-8
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Possible functions of circular RNAs
Matthias W Hentze and Thomas Preiss Embo J (32) 923–925
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Ongoing and further development
•  Full length RNA sequencing
•  Single cell transcriptome profiling
•  Direct RNA sequencing
•  Discovery and Profiling of RNA modification
•  In situ RNA sequencing
Transcriptome assembly- state-of-art
State-of-Art transcriptome assembly using short reads
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Full length cDNA sequencing
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Full length cDNA sequencing—a hybrid
approach
You et.al. Unpublished
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Single cell RNA-seq
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Single cell RNA-seq (Fluidigm)
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Direct RNA-seq (Helicos)
Fatih Ozsolak and Patrice M. Milos, Nature Review Genetics (12) 87-98
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Direct RNA-seq (Helicos)—mapping 3’ end
Ozsolak et.al. Nature. (461) 814-8
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Direct RNA-seq (PacBio)
Vilfan et.al. Journal of Nanobiotechnology 11:8
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PacBio RNA-seq—RNA modification
Vilfan et.al. Journal of Nanobiotechnology 11:8
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In situ RNA-seq
Ke et.al. Nature Methods (2013) doi:10.1038/nmeth.2563
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In situ RNA-seq (2)
Ke et.al. Nature Methods (2013) doi:10.1038/nmeth.2563
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