RNA-binding networks determined by CLIP-seq

Institute for Genomic Medicine,UCSD Stem Cell Program,Dept of Cellular and Molecular Medicine, UCSD Molecular Engineering Lab, Singapore

Gene Yeo, Ph.D.

RNA binding proteins mediate many aspects of RNA processing

mRNA processing miRNA processing

RNA binding

proteins

RNA binding proteins are key players in Post-transcriptional Gene Regulation

RNAiAlternativeSplicingLocalizationRNA-editing

Development& Reproduction

Stem CellBiology

Cancer

RBPs

TranslationPolyadenylation

StabilitymiRNA Processing& targeting Transcription

Genetic Disease

Information transmission

Signaling

Behavior

~500 RBPs in Elegans, ~700-1000 in Human

RBPs are often evolutionarily conserved

Each RBP has one or more RNA binding domains

RNA binding proteins have multiple functional domains

What are the RNA targets of RBPs?

Finding bona fide RNA targets has been a substantial barrier to our progress in understanding the functions of RBPs.

Cross-linking, IP and sequencing to identify direct binding sites

Yeo et al, Nature Structural Molecular Biology, 2009

Sanford et al, Genome Research, 2009Licatalosi et al, Nature 2008 HITS-CLIP,

CLIP-seq

Unbiased, global search for Fox2 targets in stem cells

Conserved

GCAUG

motifs

Yeo, .., Fu, Gage, 2009

Splicing Position-specific rules can be generated from RBP-RNA maps

Darnell lab

Corrionero A & Valcarcel J, Mol Cell 2009

Yeo lab, Fu lab

Yeo, Fu, Gage

lin-14 mRNA

Comprehensive discovery of Argonaute binding sites in C. elegans

miRNA

targetmRNA

lin-4 miRNA

Zisoulis, Lovci…Pasquinelli and Yeo, 2010

From Song et al., 2004

RNA binding proteins in disease and development

hnRNP A1

hnRNP KSF2/ASF

eIF4E

Sam68

Sarcomas

Translocation

QKI

TLS/FUSRBFOX-1

Argonautes

NOVA

AutoimmunityLIN28

RBFOX-2

RBPs

TET

EWS

MSI1, MSI2

IGFBP1,2,3

hnRNP H

CancerStem Cells,

Development

2010

2009

Ataxias

ATXN2

PEM/SN

FXS

FMRP hnRNP A2

FXTAS

DM

MBNLSMN

SMA

OPMDPABN1

DMPK

CUGBP1

POMA

Neurological

Disorders

Muscular

Atropies

TDP-43

ALSEpilepsy

2011

Yeo Lab, 2008-2011

Bioinformatics

ALS, hnRNP

Neurobiology

RBFOX

Technology

Small RNAs

Evolution

CLIP, SequencinghnRNP, CLIP

Bioinformatics

ALS, hnRNP

RNA editing

BMS

RBFOX, Autism

Materials

Nanoparticles

Biology

Virus-host,

Small RNA

BMS

LIN28, RBPs in Stem

Cells

Don Cleveland

Frank Bennett

Ed Wancewicz

Curt Mazur

Yalda Sedaghat

Gene Hunt

TDP-43 in collaboration with Cleveland Lab

Clotilde Lagier-Tourenne Magdalini Polymenidou

Bing Ren’s lab

Gene Hunt

Sue Freier

Zhen Ye

Samantha Kuan

Lee Edsall

Jacqueline Moran

Devesh Vashishtha

Shuo-Chien Ling

Eveline Arnold

Holly Kordasiewicz

Melissa Mcalonis

Sandrine Da Cruz

aka Lou Gehrig’s disease in the US

adult-onset disease

upper and lower motor neuron degeneration

muscle weakness leading to paralysis

invariably progressing to death within 1-5 years

from onset

ubiquitin-positive cytoplasmic protein

inclusions

Amyotrophic Lateral Sclerosis

from onset

no available treatment

more than 1 per 100,000 get ALS each year

can be associated with Frontotemporal Lobar

Dementia (FTLD) Arai et al., 2009

90% sporadic and 10% familial

20% of the familial cases caused by

mutations in SOD1

Mutations within TDP-43 cause ALS

Lagier-Tourenne C., Polymenidou M, and Cleveland D.W.

Gitcho et al, 2008; Sreedharan et al, 2008; Kabashi et al, 2008; Kwiatkowski, 2009; Vance et al, 2009

TDP-43 is involved in multiple steps of RNA processingTDP-43 plays multiple roles in RNA processing

Adapted from Lagier-Tourenne, Polymenidou, Cleveland. HMG (2010)

It is now crucial to identify RNA targets of TDP-43

Igaz et al. Am. J. Pathol (2008)

Loss of TDP-43 nuclear function

RNA targets of TDP-43 will (hopefully) reveal underpinnings of

ALS

Elder et al. Nature (2010)

RNA binding is necessary for TDP-43 toxicity

No RNA binding

CLIP-seq for TDP-43 in normal mouse brain

Protocol adapted from Ule et al. Science (2003) andYeo et al. Nat Struct Mol Biol (2009)

CLIP-seq of TDP-43 in mouse brains

CLIP-seq for TDP-43 in normal mouse brainReproducibility of CLIP-seq

Is there an enriched motif in TDP-43 binding sites ?Clusters are highly enriched for GU-rich sequences

(UG)11-13-(T)5-9 9 108

TDP43

CFTR mRNA Buratti et al, EMBO J, 2001

Buratti & Baralle, JBC, 2001

Wang et al, Genomics 2004

Ayala et al, JMB, 2005

Ayala et al, FEBS Letters, 2006

Buratti & Baralle, 2008

CLIP-seq for TDP-43 in normal mouse brainGU-rich motif is neither sufficient nor necessary for TDP43 binding

Depletion of TDP-43 in vivo

Construction of strand-specific RNA-seq libraries

Protocol from Parkhomchuk et al., 2009

Quantitative measurements of gene expression

RPKM (reads per kilobase exon per million mapped reads)

TDP-43 targets genes containing long introns

TDP-43

clusters

Long genes are regulated targets of TDP-43

Targets are conserved in human neurons depleted of TDP-43

Inferring alternative splicing from RNA-seq data

TDP-43 plays a widespread role in alternative splicing regulation

Validation of alternative isoforms regulated by TDP-43

FUS/TLS also a target of TDP-43

Auto-regulation of TDP-43

Auto-regulation of TDP-43

Igaz et al. (2008)

Cytoplasmic aggregationsLoss of nuclear TDP-43

Working model

Yeo & Cleveland Labs

Abnormal metabolism of long

transcripts involved in synaptic transmission

Neuronal sensitivity

Alternative splicing of targets,

including TDP-43

Reduces levels of TDP-43

Neuronal sensitivity

Summary of TDP-43

CLIP-seq analysis confirmed that TDP-43 binds UG rich sequences

and determined that binding occurs mainly in distal introns far from exon-intron junctions

We have generated an RNA-protein interaction map for TDP-43 by CLIP-seq

Binding of TDP-43 on long transcripts related to synaptic activity is required to

sustain their normal mRNA levels

Alternative splicing events are targets of TDP-43

TDP-43 autoregulates by binding its own transcript

TDP-43 binds ~50% of transcribed genes in the brain

TDP-43 targets genes containing long introns, important for

synapse function

TDP-43

Cytoplasmic aggregationsLoss of nuclear TDP-43

NMD

Auto-regulation of TDP-43 is likely important for disease

���� 3’UTR splicing���� TDP-43

qRT-PCR validation of candidate genes

TDP-43 are distributed in the distal introns