GLOBAL ANALYSES of RNPs

(1)

GLOBAL ANALYSES of RNPs

(2)

OLD-FASHIONED BIOCHEMICAL PURIFICATION

(3)

The RNA insert (red) is expressed in the context of RNA vector sequences (black) tethered upstream of lacZ (brown) and HIS3 reporter genes via a MS2 coat–LexA fusion protein (blue and black). Gene activation depends on binding of the Gal4 activation domain (yellow) –prey fusion protein (green).

GENETIC SCREEN- YEAST THREE HYBRID

very artefactual

(4)

U snRNPs with anti-TMG cap antibody

Bochnig et al, Eur. J Biochem. 1987 (Luhrmann’s lab)

WITH SPECIFIC ANTIBODIES or USING TAGGED PROTEINS

Markham et al, Anal Bioanal Chem. 2007

RNA analysed by:

- pCp labeling - northern blot - primer extension - RT-PCR

- RNASeq

RNP IMMUNOPRECIPITATION

(5)

IMMUNOPRECIPITATION of U1 snRNP with anti-70K Ab (U1 specific protein)

Immunoaffinity +ion exchange

Electron Microscopy

IMMUNOPRECIPITATION of snRNPs

with anti-TMG cap antibody

(6)

TANDEM AFFINITY PURIFICATION (TAP)

usual yield:

1mg of protein

complex from 300mg of total protein

(100 000 X purification

at 30% efficiency)

(7)

MODIFIED TAP tags

mammalian cells

Drakas et al., Proteomics, 2005

plant cells

Van Leene et al., TiPlSci, 2008; Gloeckner et al, Proteomics, 2007

(8)

Diameter 2.8 um volume 40 000 smaller than agarose/sepharose

MAGNETIC BEADS vs SEPHAROSE

(9)

METHODS TO STUDY TRANSCRIPTOMES

• SAGE - serial analysis of gene expression

sequencing of small cDNA tags generated by type II restriction enzymes

• CAGE - cap analysis of gene expression

sequencing of small cDNA tags derived from capped transcripts

• 3’ long SAGE

identification of SAGE tags that originate from 3’ ends of transcripts

• RNA Seq - high throughput sequencing of cDNAs

• GRO-seq - genomic run-on sequencing

sequencing of cDNA tags extended from nascent transcripts

• tiling arrays

microarrays with overlapping probes that cover the complete genome

(10)

METHODS TO STUDY TRANSCRIPTOMES

• ChIP (ChIP-chip, ChIP-Seq) - chromatin immunoprecipitation

indirectly reveal unknown ncRNAs

• RIP-Seq - RNA immunoprecipitation-sequencing

• ChIRP – Chromatin isolation by RNA Purification (+RNA-Seq)

• ChART - Capture Hybridization Analysis of RNA targets (+RNA- Seq)

biotinylated oligonucleotides used to enrich for DNA sequences associated with a particular RNA

• CRAC - CRosslinking and Analysis of cDNA

• PAR-CLIP - PhotoActivatable ribonucleoside–enhanced CrossLinking and ImmunoPrecipitation

• HITS-CLIP - High-Throughput Seq CLIP

(11)

Chu et al., Mol. Cell, 2011; Simon et al., PNAS’11

Captu re Hybridi za tion Analy sis of RNA T arget s

Chroma tin Iso lati on by RNA Purifica tion

ChIRP

CHART

(12)

CRAC technique:

CRosslinking and Analysis of cDNA

Granneman et al., PNAS, 2009

(13)

CLASH (intra- and intermolecular RNA-RNA interactions) Crosslinking

Ligation and Sequencing of Hybrids

Kudla et al., PNAS, 2011

U3- 18S rRNA interactions

(14)

http://www.jove.com/index/details.stp?ID=2034

Hafner et al., Cell, 2010

PAR-CLIP

PhotoActivatable

ribonucleoside–enhanced CrossLinking and

ImmunoPrecipitation

HITS-CLIP:

High-Throughput Seq CLIP

Creamer et al., PLOS Genet, 2011

(15)

Ascano et al., WIREs RNA, 2012 iCLAP

RBP is Strep- and polyHis tagged

PAR-CLIP 4-thioU UV 365 nm

HiTS-CLIP iCLIP ssDNA is circularized UV 254 nm

(16)

Ascano et al., WIREs RNA, 2012

(17)

Darnell, WIREsRNA, 2010

(18)

4-thioU bromoU

Core et al., Science, 2010

human plant insect worm

ANALYSIS OF NASCENT TRANSCRIPTS

(19)

ANALYSIS OF NASCENT TRANSCRIPTS

Miller et al., Mol Syst Biol, 2010

Expression of hENT1 nucleoside transporter enables uptake of UTP derivatives

yeast

Non-perturbing RNA labeling in yeast

Allows dynamic transcriptome analysis: sythesis and decay rates

and the study of nascent transcripts

(20)

possible detection in living cells

at transcription sites (nucleus)

Larson et al., TiCB, 2009

RNA LOCALIZATION: FISH

• Constitutively expressed genes are transcribed by single events separated in time; regulated genes (e.g. by SAGA) are expressed by transcriptional bursts

• Transcription of functionally related constitutive genes is not coordinated (regulated post-transcriptionally or post-translationally) /Singer lab/

Complex quantification allows analysis of single-molecule gene expression,

e.g. transcription/splicing in real time, RNA level in single cells.

(21)

RNA LOCALIZATION: FISH

www-bioc.rice.edu/bios576/immuno/immuno.html

Pagano et al., RNA, 2011

(22)

RNA LOCALIZATION: FRAP and FLIP

FRAP - fluorescence recovery after photobleaching FLIP - fluorescence loss after photobleaching

to analyse molecule kinetics in living cells

MS2x24

GFP- MS2

cleavage and polyadenylation

mRNA release

Edouard Bertrand, Montpellier, RIBOSYS

(23)

RNA LOCALIZATION: FRAP

MS2x24

GFP- MS

2

bleach

cleavage and polyadenylation

mRNA release

(24)

cleavage and polyadenylation

mRNA release

MS2x24

GFP- MS2 recovery

FRAP curve

fl uoresc enc e

time

Analysis of: - transcription rates - 3’-end formation - transcript release

transcription

polyadenylation and release

0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 1,1

0 50 100 150 200 250 300

Série1 Série1

complex mathematical modeling

trx longation rate: 2 kb/min

RNA LOCALIZATION: FRAP

(25)

Hegarat et al., NAR, 2010; Michlewski and Caceres, RNA, 2010

RNA/D NA

linker biotin beads (e.g. magnetic

streptavidin)

separation

specific proteins

contaminants

RNase-assisted RNA chromatography

SDS/PAGE MS

+ RNases

RNA CHROMATOGRAPHY ^{in vitro}

(26)

RNA CHROMATOGRAPHY in vivo

Higg and Collins, RNA, 2007; Srisawat and Engelke, Methods, 2002; Bachler et al., RNA, 1999; Weil et al., TiCB, 2010;

Piekna-Przybylska et al., Meth Enzymol, 2007

Streptavidin

Streptomycin

RNP purification from cells expressing RNA with affinity tags

(27)

RNA-protein binding using fluorescent probes

RNA/DNA labeling: 5’- 3’-

Pagano et al., RNA, 2011

periodate cleavage

fluorescein 5-thio- semicarbazide

Fluorescent Polarization Assay

- varying [protein] + fixed low [RNA]

- reaction equilibration

- sample excited with polarized light - emitted light measured with a plate reader + polarizers

- extent of polarization is proportional to the fraction of bound probe

Fluorescent

EMSA

(28)

PARS – Parallel Analysis of RNA Structure

measuring RNA structural properties by deep sequencing

Kertesz et al., Nature, 2010

- PARS confirmed for known RNA structures

- used to establish structures of > 3000 yeast transcripts

- unexpected conclusion: coding mRNA regions are more structured than UTRs!

(29)

PARS – Parallel Analysis of RNA Structure

(30)

NEW

- CHART ncRNA binding to chromatin - NET-seq

- CRISPR Cas9 gene modification

- 3′READS 3’ region extraction and deep sequencing for identification of pA sites - 3’T-fill, identification of pA sites

- DRS Direct RNA Sequencing, identification of pA sites - HITS-KIN high-throughput sequencing kinetics

- ChIA-PET Chromatin Interaction Analysis using Paired-End Tag sequencing - Hi–C

- m

⁶

A-Seq and MeRIP-Seq to identify m

⁶

A (methylated) positions in mRNAs - PAR-CLIP SILAC (qMASS-SPEC) to identify RNA-protein interactions

- iPAR-CLIP (in vivo)

(31)

GENE LOOPING (long range) 3C chromosome conformation capture

Ansari and Hampsay, GDev, 2005; El Kaderi et al., JBC, 2009

Loop formation requires interaction between factors at the promoter (THIIB) and terminator (Rna15 from CF1) /in mammals: transcription factors, nuclear receptors, insulators, chromatin remodellers, Polycomb, architectural proteins/

Loop function: facilitation of transcription reinitiation of PolII, but also repression of gene expression (PcG, DNA methylation)

activated transcription

Scaffold = transcription factors

(TFIID, A, E, H)

(32)

3C 4C 5C …..

3C Chromosome Conformation Capture

4C Circularized CCC (enhanced 3C)

5C Carbon-Copy CCC with multiple ligation-mediated amplification (LMA)

GCC Genome CC (Hi-C – deep Seq), ChIP version of GCC as 6C

ChIP Loop (indirect ChIP)

6C ChIP GCC

(33)

ChIA-PET

Chromatin Interaction

Analysis using Paired-End Tag sequencing

Zang et al., Methods, 2012

ChIP for Pol II  enrichment

Nuclear Proximity Ligation

Paired-End Tag sequencing

(34)

Hi-C

Belton et al., Methods, 2012

- chromatin crosslinking, digestion, re-ligation, PCR amplification/deep sequencing

- biotin-labeled nucleotide incorporated at the ligation junction for

selective purification of chimeric DNA ligation junctions

(35)

MTRIPs and proximity ligation

So¨derberg et al., Nat. Methods 2006; Jung et al., NAR, 2012

proximity probes:

oligo probes attached to antibodies

MTRIP enter cell via streptolysin O Cy3B-conjugated

oligo

anti-FLAG

Ab anti-RBP

Ab proximity

probes

proximity probes

oligo