Gene Expression Regulation in Subcellular Space

Eric Lécuyer, PhD

Associate Professor and Axis Director, RNA Biology Lab Systems Biology Axis, IRCM Associate Research Professor, Département de Biochimie Université de Montréal Associate Member, Division of Experimental Medicine, McGill University

VizBi 2018 Meeting The Central Dogma in Subcellular Space

Crick, Nature 227: 561 (1970) Biological Functions of Localized mRNAs

mRNA Protein

Extracellular Vesicles

Cody et al. (2013). WIREs Dev Biol Raposo and Stoorvogel (2013) J Cell Biol Cis-Regulatory RNA Localization Elements

Van De Bor and Davis, 2004. Curr.Opin.Cell.Biol. Global Screen for Localized mRNAs in Drosophila

http://fly-fish.ccbr.utoronto.ca (Lécuyer et al. Cell, 2007) Diverse RNA Subcellular Localization Patterns

RNA DNA Correlations in mRNA-Protein Localization

Localization Patterns

Terms

Ontology Gene

RNA Protein DNA

(Lécuyer et al. Cell, 2007) Models and Approaches to Decipher the mRNA Localization Pathways

Drosophila & RNA/Protein High-Content Screening Human Cell Models Imaging & RNA Sequencing

+ + Cell Fractionation and RNA Sequencing (CeFra-seq) to Study Global RNA Distribution Cell Fractionation-Seq to Study RNA Localization

RNA and Protein Extraction, RiboDepletion or PolyA+

RNA-seq and MS profiling (K562, HepG2 and D17) Wang et al (2012) Cell https://www.encodeproject.org/ Lefebvre et al (2017) Methods Benoît Bouvrette et al (2018) RNA Interesting Examples of RNA Localization

ANKRD52 (mRNA and ciRNA)

Total Nuclear Cytosolic ciRNA Membrane Insoluble mRNA ANKRD52 DANCR (lncRNA)

Total Nuclear Cytosolic Membrane Insoluble DANCR (Neal Cody) Frac-seq Reveals Extensive Asymmetric Distribution of Cellular RNAs

FPKM Distribution by Biotype Examples of Localized Transcripts HepG2 Ribo-Depleted mRNAs lncRNAs

lincRNAlincRNA miRNAmiRNA TMEM107 ● ● ● RPPH1 ● ● ● n= 1913n= 1913 ● ● ● ● n= 451n= 451 100100 ● ● ● ● ● ● ● ● ● ● ● ● ● ● TAS2R19 RP4−802A10.1 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● SNHG20 RP11−473I1.9 ● 75 75 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● SMIM1 ● ● ● ● ● ● RP11−310J24.3 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● PRKACA ● ● ● 50 50 ● ● ● ● RP11−290D2.6 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● PLS3 ● ● RP11−19C24.1 ● ● ● ● ● ● ● ● ● log(FPKM) log(FPKM) ● ● ● ● ● ● 15 25 25 ● ● 15 ● ● ● ●

● PGK1 ● ● RP11−156E6.1 ● ● ● ● ) ) 10 ● 10

MT−ND6 ● ● RP1−40E16.9 ● ● ● ●

%

% ( ( 0 0 5 5

●

LINC00641 ● ● PLCB1−IT1 ● ● M

M protein_codingprotein_coding snoRNAsnoRNA 0 0 K K IRAK1 ● ● ● n= 10801n= 10801 ● ● n= 331n= 331 LINC00526 ● ● ● ● ● ● ● 100100 ● ● ● ● ● ● ● ●

● ● P P ● ● ● ● ● ● ● ● −5 ● ● ● ● −5

F ● F ● ● ● ● ● ● ● ● ● HSP90AA1 ● ● FGD5−AS1 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● pFPKM ● ● ● ● pFPKM ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● HIST1H2AK ● ● 75 75 ● ● ● ● CTD−2651B20.7 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 0.25 ● ● ● ● ● ● ● 0.25 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● GLUD2 ● ● ● ● ● ● ● ● ● ● CTD−2651B20.6 ● ● ● ● ● ● 0.50 0.50 ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● 50 50 ● ● FOXM1 ● ● ● ● CTD−2267D19.3 ● ● ● ● 0.75 ● 0.75 ● ● ● ● ● EPB41L1 ● ● CTD−2196E14.9 ● ● ● ● ● ● ● ● ● ● ● 25 25 ● ● ● ELK1 ● CTD−2196E14.4 ● ● ● ● ● ● ● ● ● ● ● ● ● ●

● ● CDYL ● CTD−2035E11.3 ● ● ● 0 0 ALDH1A1 ● ● ● CTC−444N24.11 ● ● c c e e e e r r c c e e e e r r ● ● ● li li n n l l a a li li n n l l a a b b e e b b e e ● ● ● o o l l o o l l ● ● s s ra ra lu lu c c s s ra ra lu lu c c AL121963.1 BCYRN1 ● to to b b o o u u to to b b o o u u y y m m s s N N y y m m s s N N n n n n ● ● C C e e I I C C e e I I AC005493.1 AC017002.2 ● ● ● M M M M

c e e r li n l a ic e le r o a b le l n b a s r lu c o a u le to b o u s r l c y m s to b o u e n N y m s N C I C e In M M (Philip Bouvrette) Most RNAs are Asymmetrically Distributed

HepG2 Transcriptome Asymmetric (+Nucleus) Asymmetric (Cyto Fractions) 100 Highly enriched in 1 fraction

80

60

40

Percent Percent 20

0

• RNA asymmetric when exhibiting ≥2-fold enrichment in FPKM values in one fraction compared to one or all other fractions. (Louis Philip B. Bouvrette) Correlations in mRNA and Protein Distribution Systematic Characterization of Human RBP Subcellular Distribution Patterns Subcellular Regulation of Gene Expression

Subnuclear Compartments Transcription Exon 1 Intron 1 Exon 2 Intron 2 Exon 3 pA Machinery

n 3 Exo Ex 1 on 2 Exon Cleavage/ Polyadenylation Splicing Capping RNA Editing NucleusNucleus 7meGpppG Exon 1 Exon 2 Exon 3 AAAAAAAAA RNA Cytoplasmic Destinations

Export

CytoplasmCytoplasm TranRNAslati on Localization, Transl ationRNA & S Stabilitytability

Where are RNAs and RBPs localized?

RNA DNA Shiels et al (2007) PLoS Comp Biol; Lecuyer et al (2007) Cell “RNA binding proteins”

(Gene Yeo’s Lab) The ENCyclopedia Of DNA Elements (ENCODE) Consortium

ENCORE Team

Brent Graveley, Lead Gene Yeo (UConn Heath Center) (UCSD)

Chris Burge Xiang-Dong Fu (MIT) (UCSD) Graveley Team Project: Identifying Functional RNA Elements Encoded in the Human Genome

(K562 & HepG2 cells)

Gene Yeo Brent Graveley Xiang-Dong Fu Chris Burge

Lecuyer Lab Objectives: -Establish RBP imaging database - Fractionation-seq to look at RNA localization globally Paper Detailing Optimized ‘RBP’ Resources

Sundararaman et al. (2016) Molecular Cell, 61: 903-13. https://www.encodeproject.org/ Progress - RBP Imaging The RBP Image Database http://rnabiology.ircm.qc.ca/RBPImage

(Xiaofeng Wang, Philip Bouvrette) Browse by ‘Google Image-Like’ Format

http://rnabiology.ircm.qc.ca/RBPImage Human RBPs Display Broad Distribution Patterns http://rnabiology.ircm.qc.ca/RBPImage

RBPs DNA Controlled Vocabulary Annotations Enable RBP Clustering Into Localization Groups

Hela_P.bodies Hela_Compartment.Organelle HepG2_Mitochondria HepG2_Compartment.Organelle HepG2_Nice.image HepG2_Nuclear.release.mitosis HepG2_Mitosis.related Hela_Nucleolus HepG2_Nucleolus Hela_Speckles HepG2_Very.cool.pattern Hela_Nice.image Hela_Mitochondria Hela_Focal.Adhesions Hela_Cytoskeleton HepG2_Actin HepG2_Microtubule HepG2_Cytoskeleton HepG2_ER HepG2_Spindle HepG2_Mitotic.Apparatus HepG2_Variable.Nuclear.Intensity HepG2_Unknown.Cyto.Foci HepG2_Golgi Hela_ER HepG2_Unknown.Nuclear.Foci Hela_Nuclei..UB.

Patterns Hela_Ubiquitous Hela_Cytoplasm..UB. Hela_Mitosis.related Hela_Mitotic.Apparatus Hela_Spindle HepG2_P.bodies HepG2_Mutually.Exclusive.from.ER Hela_Very.cool.pattern HepG2_Unidentified HepG2_Cell.Cortex Hela_Unknown.Nuclear.Foci HepG2_Cell.Junctions HepG2_PML.bodies Hela_Unidentified HepG2_Nuclei..UB. HepG2_Ubiquitous Hela_Microtubule Hela_Golgi Hela_Unknown.Cyto.Foci Hela_Cajal.bodies Hela_Nuclear.release.mitosis Hela_Mutually.Exclusive.from.ER Hela_PML.bodies HepG2_Unknown.Cyto.Territory HepG2_Centrosomes HepG2_Focal.Adhesions HepG2_Endosomal HepG2_Unknown.Nuclear.Territory HepG2_Cajal.bodies HepG2_Nuclear.Envelope HepG2_Cytoplasm..UB. Hela_Cell.Cortex Hela_Centrosomes Hela_Cell.Junctions Hela_Unknown.Cyto.Territory Hela_Endosomal Hela_Unknown.Nuclear.Territory Hela_Nuclear.Envelope Hela_Variable.Nuclear.Intensity Hela_Actin Hela_Cytoplasm HepG2_Cytoplasm Hela_Marker.Co.Localized HepG2_Marker.Co.Localized Hela_Cytosolic HepG2_Cytosolic HepG2_Nucleoplasm HepG2_Nuclei HepG2_Speckles HepG2_Mutually.Exclusive.from.Nucleolus Hela_Nuclei Hela_Nucleoplasm Hela_Mutually.Exclusive.from.Nucleolus GOLGB1 DNAJC17 SND1 CDC40 ZNF622 UBAP2L UBE2L3 XRN1 EIF2B ZFC3H1 ADD1 CKAP4 CALR EIF3G NUFIP2 EEF2 NOLC1 P PKM KIF1C RBM6 EIF4B DDX6 AIMP1 AKAP1 EIF2S1 RA EIF4G2 SERBP1 RBM27 EIF3A G3BP2 IGF2BP3 DDX3Y G3BP1 FXR1 DDX3X TNRC6A EIF4A3 AD RBM5 PUM2 A T SUPV3L1 YTHDC2 PTBP1 PNPT1 GRSF1 KRR1 FXR2 MTP F GNL3 DDX59 TBRG4 CASC3 HSPD1 LARP4 FKBP4 CNO DHX33 DDX5 A PRPF6 EIF3H POLK PSPC1 LARP7 LIN28B NKRF DDX1 F NFX1 DDX55 MARK2 A TRIP6 CCNL1 UCHL5 PRRC2C NAA15 BCCIP A SRP68 RRP9 EXOSC9 SRFBP1 TRA2A RPL23A RPS11 RPS5 RPS3 RPS24 MET ZONAB ASCC1 DNAJC2 SMN1 RECQ1 XPO5 NSUN2 XRCC5 SUB1 XRCC6 EIBAP5 DDX24 DDX20 RCC2 NIP7 GR DKC1 P ABCF1 BOP1 WDR3 UTP18 WDR43 ZFP106 UTP3 CCDC86 NOL12 DDX52 CEBPZ AARS AA ABT1 MAK16 ILF3 NPM1 KHDRBS2 RBM34 IGF2BP2 PSIP1 SL KHDRBS1 A PPIG CSTF2T DR SRPK2 COR NUSAP1 PUM1 TRIM56 DNAJC21 CCDC124 DHX30 TFIP11 RBM25 PRPF8 A SNRNP200 UPF2 SF3A3 GEMIN5 SCAF4 TR NCBP2 SRSF4 EIF4G1 PCBP2 PUS1 RBFO GNB2L1 RBM17 PNN EIF3D B T SUPT6H RPLP0 RECQL5 NUP35 RBMX2 SUGP2 ZC3H8 A PES1 PHF6 DDX19B ZC3H11A DEAF1 CSTF50 AKAP8 CPSF7 CPEB4 R CCAR1 CUGBP1 T CAPER PUF60 CPSF6 BCLAF1 AL QKI SF3B4 PCBP1 DDX42 LARP1 XPO1 HNRNPC HNRNPK EWSR1 EFTUD2 U2AF2 DGCR8 U2AF1 SAFB2 XRN2 LSM11 KHSRP FUS KIAA0082 ZRANB2 DDX27 HDGF NONO D P FIP1L1 CSTF2 SFPQ ARDBP AF1168 ASTKD2 AM120A A2G4 TXN2 TXN1 ABPC4 TP5C ABPN1 OE1 UH GGF1 QR CO1 UD13 AZAP1 TF1 Y TM TF VER1 O AR OSHA

WD1 Localization AP AP2 VE2 T7 O1A X2 Proteins

(Xiaofeng Wang & Srishti Jain) RBP Periodic Table

(Design: Brent Graveley, CSHL Meeting on Eukaryotic mRNA Processing) Other Screens Using the RBP Antibody Collection RBP Targeting to the Mitotic Apparatus

RBM39 XPO1 PSIP1 NUSAP1 RBM27 CPSF6 PABPC4

KHDRBS1 WDR43 CCDC86 AQR SLTM RPS3 GRWD1

entrin

C RBP

eIF4G2 IGF2BP1 CUGBP1 KHDRBS2 CDC40 ZNF622 AKAP1 DAPI

PUF60 HNRNPUL1 FXR2 LARP1 PUM1 SND1 DDX5

Centrin RBP DNA (Sulin Oré Rodriguez and Dhara Patel) RBP Targeting to Disease-Associated Repeat RNA Nuclear Foci: Myotonic Dystrophy Type 1

CUG repeats for RNA Hairpin CUG repeats RBP DNA

• DMPK mRNA with long CUG repeats forms extended stem loop structure. • Aberrant RNA forms nuclear aggregates and exerts a toxic gain-of-function effect by sequestering RNA binding proteins in the nucleus. RBP Localization to Stress-Induced Structures

Control Stress (Arsenite)

Cytoplasmic Stress Granules

TIA1 G3BP1 DNA Control Stress (Etoposide)

Nuclear DNA- Damage Foci

eIF3D 53BP DNA (Sahar Soltanieh, Markmiller et al Cell 2018; Samantha Bovaird) Cells With Personnality… Acknowledgements

-Lécuyer Lab- -Collaborators- Current ENCODE Project Dr. Julie Bergalet Brenton Graveley (UConn) Philip Bouvrette Gene Yeo (UCSD) Samantha Bovaird Xiang-Dong Fu (UCSD) Ashley Chin Christopher Burge (MIT) Dr. Cédric Diot MgGill University Fabio Alexis Lefebvre Mathieu Blanchette Juan Carlos Padilla Nada Jabado Dhara Patel Paul Lasko Lilyanne Perras Janusz Rak Dr. Sahar Soltanieh Jerome Waldispühl Xiaofeng Andy Wang IRCM/UdeM Former Members Pascal Chartrand Dr. Neal Cody Marlene Oeffinger Adda Graham-Paquin Alexis Blanchet-Cohen Srishti Jain Odile Neyret Xuan-Tam Nguyen Dominic Filion Sulin Oré Rodriguez -Funding- Yang Olivia Zhang (Samantha Bovaird) 32