Download Biologie des ARN/RNA Biology

Biologie des ARN/RNA Biology
Eric Lécuyer, PhD
Director, RNA Biology Lab
Systems Biology Axis, IRCM
Professeur-Chercheur Adjoint, Département de Biochimie
Université de Montréal
Associate Membre, Division of Experimental Medicine,
McGill University
Cours BIM-6026
16 Octobre, 2014
Presentation Outline
• Overview of eukaryotic RNA populations
• Biogenesis of messenger RNAs (mRNAs): capping,
~1h cleavage and polyadenylation, splicing.
• Biogenesis of non-coding RNAs: ribosomal RNAs, transfer
RNAs, micro RNAs, circular RNAs, long non-coding RNAs.
• Functions and mechanisms of RNA Localization
~1h • Methods to Study RNA Localization
• RNA Localization in Disease
2
Central Dogma of Molecular Biology
Francis Crick
Crick, Nature 227: 561 (1970)
(1916 - 2004)
Nobel Prize 1962
3
Various Categories of Eukaryotic RNAs
Wahl et al. (2009) Cell,
136: 701-718.
• Eukaryotic cells have diverse varieties of functional RNA molecules.
• Often require specific modifications to be active: nucleolytic cleavages, sequence
additions in 5’ ou 3’ of the RNA, nucleotide modifications.
4
Maturation Steps of Messenger RNAs
Hieronymus et Silver (2004)
Genes & Dev, 18: 2845-60.
5
Biogenesis of Messenger RNAs
6
Cap Structure at the 5’ Extremity of mRNAs
Can be O2’-methylated
• 7-Methyl Guanosine added
via 5’-5’ triphosphate bridge.
• Added before the mRNA
reaches 30 nt in length by
the
enzyme
Guanylyl
Transferase.
7
Functions of the Cap Structure
• Protects mARNm from degradation
by 5’ 3’ exonucleases.
• Contributes to the nuclear export of
mRNAs.
• Increases translational efficiency.
8
Cleavage and Polyadenylation the 3’ End of mRNAs
• Involves 2 reactions:
- Clivage 10-35nt after AAUAAA or
<50nt before G/U rich sequence.
- Synthesis of a poly-A chain by
the Poly-A polymerase (PAP),
~200nt in length.
aval
9
Functions of PolyA Tails
• Protect mRNA dégradation
3’ 5’ exonucleases.
by
• Stimulation of tranlsation via
interaction with Cap bound factors
(circularization of the mARN)
• Nuclear export of mRNAs.
• PolyA tails are also sometimes
added differnet types of noncoding RNAs (i.e. tRNA, snRNA,
rRNA, snoRNA, bacterial ARN).
• In prokaryotes, polyA tails can
promote degradation of both
coding and non-coding RNAs.
10
Eukaryotic mRNAs Undergo Extensive Splicing
Prix Nobel 1993
Phillip Sharp
Richard Roberts
11
Splicing is Controlled by Consensus Sequences
Patel et Steitz (2003) Nat Rev Mol Cell Biol
12
Molecular Mechanism of Splicing
13
Splicing is Performed by Ribonucleoprotein
Complexes that From the Spliceasome
U2AF
Joan Steitz
Yale University
14
Transcripts Can Undergo Alternative Splicing
Nilsen and Graveley (2010) Nature
a)
b)
c)
d)
Use of an alternative 5’ splice site.
Use of an alternative 3’ splice site.
Inclusion or exclusion of ‘cassette’ exons.
Exclusion ou retention of introns.
15
Cool Examples of Alternative Splicing
a)
b)
c)
Nilsen and Graveley (2010)
Nature
Splicing of the human KCNMA1 mRNA.
The Dscam mRNA of Drosophila.
The Drosophila mod(mdg4) mRNA can undergo alternative
splicing in trans from exons transcribed on different DNA strands.
(For each example, constitutive exons are colored in bleu et alternative exons
are colored in yellow, red or violet)
16
DSCAM: an Extreme Case of Alternative Splicing
DSCAM = Down Syndrome Cell Adhesion Molecule
Protein involved in axon guidance.
62 Kb
7.8 Kb
Domaines Ig
Domaine Transmembranaire
270 KDa
Mutually exclusive alternative splicing of cassette exons 4, 6, 9 et 17.
Each neuron will express a different mRNA isoform.
12 x 48 x 33 x 2 = 38016 potential isoforms of DSCAM
Schmucker et al (2000) Cell, 101: 671-84.
17
DSCAM Protein Prevents Interactions Between
Dendrites of the Same Cell
Gilbert (2010) Developmental Biology
18
Phosphorylation of the C-Terminal Domain of RBP1:
An Interface to Coordinate RNA Maturation Steps
Meinhart A et al. Genes Dev. 2005;19:1401-1415
The C-terminal Domain (CTD) of RPB1 of eukaryotic RNA Polymerase II:
-Contains heptapeptide repeats of the YSPTSPS sequence (26x in
yeast, 52x in humans) that are phosphorylated on Sérine, Thréonine et
Tyrosine residues.
-CTD phosphorylation is dynamic during transcription and different
phosphorylation signatures allow the CTD to interact with proteins involved
in RNA capping, splicing and cleavage/polyadenlyation.
19
Role of the CTD in Recruiting RNA Maturation
Factors Co-Transcriptionally
Phosphorylated CTD
Weaver (2012) Molecular Biology
20
RNA Maturation Involves the Interplay of
Trans-Acting Factors and Cis elements
• Trans-regulators (green) can either be RNA Binding Proteins (RBPs)
or regulatory RNAs (e.g. microRNAs).
The human genome encodes >1000 RBPs
(Curration from Gene Yeo’s Lab)
Biogenesis of Various Non-Coding RNAs
23
Biogenesis of Ribosomal RNA (rRNAs)
Non-Transcribed Sequences
• Ribosomal RNA genes are clustered
as repeats.
• Mature rRNAs derive from an
extensive-processed precursor.
24
Maturation Steps of rRNAs
• The 5.8S, 18S and 28S rRNAs are produced
and matured in the nucleolus, transcribed by
RNA Polymerase I.
• Maturation of the primary rRNA transcript
requires modifications by small nucleolar RNAs
(snoRNAs). SnoARNs are 60-100nt RNAs that
derive from introns of mARNs. They hybridize to
specific regions of pre-rRNAs and guide 2 main
types of modifications:
- Methylation of 2’OH of ribose.
- Pseudouridylation of sprecific Uridines.
• These modifications will dictate the maturation
steps of the rRNA precursors.
• The 5S rRNA is synthesized in the nucleoplasm
by RNA polymérase III.
25
Biogenesis of Transfer RNAs (tRNAs)
• Mature tRNAs are ~80nt in length and have a
clover leaf secondary structure.
• They are transcribed by RNA Pol III as longer
precursors, sequences trimmed from 5’/3’
ends.
• In eukaryotes, tRNAs are produced form
individual tRNA gene loci, some will also
undergo splicing.
26
Maturation of Transfer RNAs
• Étape 1: coupure du côté 5’ par
l’ARNase P (enzyme formé de
sous-unité catalytique ARN:
ribozyme).
• Étape 2: clivage du côté 3’ par
ARNases. (3’tARNses chez
eucaryotes).
• Étape 3: ajout de la séquence
CCA à l’extrémité 3’ par tRNA
nucléotdidyl transférase. Site ou
les acides aminés seront
attachés.
• Modifications
de
plusieurs
bases par méthylation.
27
Discovery of Micro RNAs (miRNAs)
Victor Ambros et Rosalind Lee
28
Mode of Action of miRNAs
• Partial Base Pairing: inhibits
translation of the target mRNA.
• Pefect Base Pairing: induces
degradation of target mRNAs.
(Imperfect Base Pairing)
(Perfect Base Pairing)
29
IV.D. Le phénomène de l’interférence par ARN
RNA Interference (RNAi)
RNAi is the process by which small RNAs, either originating from exogenous or
endogenous sources, can modulate gene expression, typically by causing the
degradation of complementary cellular RNAs.
microRNA /
‘Source Endogène’
‘Source Exogène’
Dégradation ou répression
traductionnel de l’ARN cible (vert)
30
RNAi: A Revolutionary Research Tool for
Functional Genomic Screening
Boutros and Ahringer
(2008) Nature Review
Genet, 9: 554-566
31
Long Non-Coding RNAs (lncRNAs)
• LncARNs are defined as non-coding RNAs with a length >200nt. Deep sequencing studies
have revealed thousands of dynamically expressed lncRNAs transcribed from the human
genome. LncRNAs are often organized in close association with protein coding genes and
share similar features to mRNAs (e.g. trx by RNA Pol II, capped, polyA, spliced).
Mercer and Mattick (2013) NSMB, 20: 300-307.
32
Suspected Functions of LncRNAs
Paralkar & Weiss
(2013) Blood
33
Example of lncRNA: Xist and X Inactivation
FISH
sonde
pour X
Lee (2012) Science
DAPI
L’ADN du Xi est
plus condensé.
• In female human cells, one of the X chromosomes is inactivated (Xi) transcriptionally. This
repression is modulated by the Xist RNA transcribed from the Xi chromosome This lncRNA
of ~20kb coats the Xi and recruits proteins involved in heterochromatin formation.
34
Biogenesis of Circular RNAs (ciRNAs)
• ciARNs and an emerging class of RNA species taht result from ‘head-to-tail’ splicing by the
5’splice junction of one exon and a donor site at the 3’end of a downstream exon. These
were discovered by looking for junction reads in deep sequencing datasets.
Hentze and Priess (2013) EMBO Journal, 32: 923-925
35
Possible Functions of ciRNAs
Hentze and Priess (2013) EMBO Journal, 32: 923-925
36
General Principles and Functions of RNA
Intracellular Localization
37
Traditional View of Protein Targeting
Alternative Mechanism: Localizing the mRNA prior to
translation
38
Why Localize mRNAs?
• Saving energy: A single mRNA can undergo several rounds
of translation.
• Prevents proteins from accumulating in the wrong
subcellular compartment. (e.g. MBP mRNA, oskar mRNA)
• Rapid tuning of protein expression in specific subcellular
compartments in response to (e.g. β-actin mRNA)
• Promoting the co-translational assembly of protein
complexes. (e.g. co-localization of functionally related
mRNAs)
39
Biological Functions of Localized mRNAs
Cody et al., 2013. WIREs Dev Biol
40
Loss of Anterior Structures in Bicoid Mutants
Exosquelettes de Larves
Phénotype
‘Bicaudal’
Frohnhöfer & Nüsslein-Volhard (1986) Nature
• La localization antérieure de l’ARNm de bicoid est essentielle pour
sa fonction et contrôlée par les séquences de son 3’UTR.
41
Anterior Nanos Targeting Causes Bicaudal Phenotype
Gavis & Lehmann (1992) Cell
• RNAs detected by in situ hybridization (ISH).
42
RNA Secretion Within Extracellular Vesicles
Raposo et al (2013) J. Cell Biol
43
Cis-Regulatory RNA Localization Elements
Van De Bor and Davis, 2004. Curr.Opin.Cell.Biol.
44
Mechanisms of RNA Localization
• RNAs can be localized via distinct mechanisms, including: 1) Active
transport on cytoskeletal elements; 2) Random diffusion and capture;
3) Vectorial export from the nucleus and local cytoplasmic diffusion;
and 4) General degradation coupled to localized protection.
(1)
(3)
(2)
(4)
Reviewed by: Lipshitz and Smibert, 2000. Curr.Opin.Cell.Biol.
45
Coding-Dependent and Coding-Independent
Functions of Localized RNAs
1. Two mRNAs encoded by functionally
related genes (G1 and G2).
2. Bound by RNA binding protein that
recognizes common element in
mRNAs.
3. mRNAs are exported from the
nucleus into the cytoplasm.
4. Then they are transported to the cell
cortex, where they are translated
locally. Encoded proteins assemble
into complex.
5. Localized mRNAs could also play
coding-independent role (e.g.
scaffolding function).
6. Sites of mRNA accumulation may
also serve as storage depots ready
Lécuyer et al., Curr Opin Cell Biol (2009)
to deploy mRNA pools for local
translation.
46
Mathematical Model of Localized Protein
Complex Assembly
Protein Half-life (min):
Batada et al., 2004. PNAS
• Is co-localization important for efficient assembly of protein
complexes or to prevent cross-talk between pathways?
• Conclusions:
- Random protein interactions are rare, cross-talk is insignificant.
- Co-localized sources are likely to be essential to increase the probability
or protein interactions, diffusion is not enough.
47
Methods for Studying RNA Localization
48
Methods to Study mRNA Expression Localization:
In Situ Hybridization (ISH)
1
Make labeled DNA or RNA probe
complementary to target mRNA
• Different nucleotide labels available
(e.g. Dig, DNP, FITC, Biotin).
• Different methods (e.g. DNA nick
labeling, PCR, IVT transcription).
2
Hybridize probe to fixed and
permeabilized cells, tissues or
whole mount specimens.
3
Add enzyme-conjugated antibody
that recognized nucleotide label.
(e.g. anti-Dig antibody coupled to
HRP or AP enzymes)
4
Add chromogenic or fluorescentlyconjugated enzyme substrate.
(e.g. NBT-BCIP = AP substrate).
49
Fluorescent In Situ Hybridization (FISH)
Posterior mRNA in Drosophila
Tyramide Signal Amplification (TSA)
TSA
RNA
CG14217
DNA
NTB-BCIP
DIG
Lécuyer et al., Methods Mol. Biol. (2008)
Lécuyer et al., CSHL Protocols (2008)
50
Diverse mRNA Localization Patterns in Fly Embryos
• ~70% of mRNA are Localized during early embryogenesis, as
assessed by a global FISH study of ~4000 mRNA species.
51
Lécuyer et al. (2007) Cell
Correlations in mRNA/Protein Distribution
DNA
RNA
Protein
Lécuyer et al. (2007) Cell
52
Multiplex FISH for Simultaneous
Detection of Multiple mRNAs
Fly Patterning Genes
Fly Hox Genes
sog ind msh wg en
lab Dfd Scr Antp Ubx abd-A abd-B
Kosman et al., Science (2004)
53
Multiplex DNA-FISH for Chromosome Painting
Schrock et al., 1996. Science
Speicher et al., 1996. Nature Genet
Method used for karyotyping human chromosomes.
54
Fluorescent In Situ Sequencing (FISSEQ)
Outline:
55
Lee et al. (2014) Science, 343: 1360-1363
Assays for RNA Expression/Localization Profiling
Ginart and Raj (2014)
Nature Methods
56
Identifying Organelle-Enriched RNAs
Separated Pseudopodia (Ps) from Cell
Bodies (CB), then micro-arrays
Confirmed by RT-PCR
Used MS2 tagging
system to show
3’UTR dependent
Blower et al., J Cell Biol (2007)
Mili et al., Nature (2009)
Micro-array analysis reveals mRNAs
associated with frog microtubules,
confirmed by FISH
Identified ~50 mRNAs localized to
pseudopodia in fibroblasts
57
Other Global Methods to Profile Localized mRNAs
Purification of RNA-binding proteins (RBPs) followed by
microarrays or deep sequencing (e.g. RIP-Chip, RIP-Seq, CLIP).
Keene, Nat Rev Genet (2007)
Gerber et al., PLoS Biol (2004)
Yeast Puf proteins associate with
functionally related mRNAs
58
Methods to Image mRNAs in Live Cells
Van de Bor and Davis, 2004. Curr.Opin.Cell.Biol.
a)
b)
c)
Direct injection of fluorescently labeled mRNA molecules
MS2-GFP labelling system (Bertrand et al. 1998, Mol Cell)
Molecular beacon technology (Bratu et al. 2003, PNAS)
59
Live Imaging of nanos mRNA in Drosophila Oocytes
Via MS2 Tagging Approach
MCP-GFP
nanos-(MS2)6
Forrest and Gavis, 2003. Curr.Biol.
Posterior targeting of nanos mRNA is established during oogenesis by
60
diffusion and entrapment mechanism
New Method for RNA Live Imaging: FluorophoreBinding RNA Aptamers
HBI = fluorophore 4-hydroxybenzlidene imidazoline.
Paige et al (2011) Science
• Used multiple rounds of in vitro selection (SELEX) to screen 5x10E13 RNA
molecules (70 nt in length) to identify RNA aptamers with the capacity to
bind and activate various fluorophores.
61
The Spinach Aptamer System
5S-Spinach : 5S Ribodomal
RNA fused to the Spinach RNA
aptamer
• Addition of sucrose to the cells induces stress granule formation.
Paige et al (2011) Science
62
Genetic Disorders Impacting RBPs and RNA
Localization Pathways
63
Degenerative Diseases With RNP Perturbations
Ramaswami M. et
al. (2013) Cell
64
Synaptic Translation Often Disrupted in
Neurodegenerative Disorders
65
Liu-Yesucevitz et al. (2011) J. Neurosci.
Trinucleotide Repeat Expansion Diseases (TREDs)
Krzyzosiak W J et al. (2011) Nucl. Acids Res.
66
Fragile X Syndrome
• Most frequent inherited form of mental retardation.
• Impairment ranges from learning disorders to severe
cognitive disabilities.
• Unusual X-linked disorder, inheritance from nonpenetrant male carriers to their non-penetrant
daughters with affected sons.
• Results from trinucleotide (CGG) repeat expansion
within the 5’UTR of FMR1 gene. Repeats expanded
~800 times in affected individuals.
• FMR1 encodes FMRP, an mRNA binding protein
that regulates transport and localized translation of
mRNAs.
67
FMRP Required for Stimulus-Induced mRNA
Transport and Translation
Dictenberg et al., Dev Cell (2008)
FMRP function important for dendritic spine maturation
68
Dominant Effects of Toxic RNA Repeats in
Different TREDs
Krzyzosiak W J et al. (2011) Nucl. Acids Res.
• Mutant CUG and CAG repeat RNAs form nuclear foci (red) in Type 1
Muscular Dystrophy (DM1) and Huntington’s disease (HD)
69
Amyotrophic Lateral Sclerosis (ALS) and
Frontotemporal Dementia (FTD)
• ALS and FTD share similarities in disease
mechanisms, show co-morbidity.
• ALS/Lou Gherig’s Disease: muscle weakness and
atrophy due to motor neuron degeneration.
• ALS affects 1-2/100,000 people, usually manifests
from 40-60 years of age.
• Both familial (10%) and sporadic (90%) cases with
recurring mutations.
• FTD affects 3-4/100,000 people, defined by
abnormal behaviour and language.
• In both ALS and FTD, neurons exhibit inclusions of
aggregated proteins known as ‘Stress Granules’
that contain signature RBPs.
Ling S.-C. et al. (2013) Neuron
70
Genetic Causes of ALS/FTD
Robberecht and Philips (2013) Nat. Rev. Neurosci.
71
Functional Disruption of TDP43 and FUS/TLS
Ling S.-C. et al. (2013) Neuron
72
What Explains Selective Sensitivity of Neurons
Ramaswami M. et al. (2013) Cell
73
Myotonic Dystrophy Type I (Steinert’s Disease)
•Most common form of muscle disease
(1/8000 affected worldwide;
1/500 in Saguenay)
•Clinical features: Multisystemic, muscle
wasting and myotonia, cataracts, heart
defects, catacracts, apathy, cognitive
impairments.
•Autosomal dominant inheritance
•Expansion of CTG repeats in 3’UTR of
DMPK gene on Chr 19:
Normal: 5-37 repeats
Pre-mutation: 38-49 repeats
Symptomatic: >50 repeats
74
Expanded CUG-Repeat mRNA Sequesters
Muscleblind Family Proteins in the Nucleus
CUG repeats for RNA Hairpin
Krzyzosiak W J et al. (2011) Nucl. Acids Res.
• 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.
75
Eric Wang
(MIT)
Chris Burge
(MIT)
Neal Cody
Defining Transcriptome Binding Properties of
Mbnl Proteins
(Eric Wang)
• Cross-Linking and IP and RNA sequencing (CLIP-Seq) experiments performed
to define transcriptome binding profile of Mbnl proteins.
Wang et al (2012) Cell, 150: 710-24.
3’UTRs Binding Properties of Mbnl1
Mbnl1 binding along 3’UTRs
from CLIP datasets
Enriched ‘Cellular Component’
GO categories for genes with
Mbnl binding in 3’UTR
Wang et al (2012) Cell, 150: 710-24.
• Potential roles of Mbnl proteins in RNA localization?
Wang et al (2012) Cell, 150: 710-24.
(Eric Wang)
Cell Fractionation and RNA Sequencing
Biochemical fractionation of mouse myoblasts and fly cells, followed by RNA sequencing.
(Eric Wang and Neal Cody)
Wang et al (2012) Cell, 150: 710-24.
Muscleblind Depletion Impacts the
Targeting of Membrane mRNAs
(Eric Wang and Neal Cody)
Wang et al (2012) Cell, 150: 710-24.
Mbnl Functions in Translation
and Protein Secretion
Ribosome Footprint
Profiling
Luciferase Assays
Wang et al (2012) Cell, 150: 710-24.
(Eric Wang)
Mbl Functions in mRNA Splicing and Localization
Wang et al., Cell (2012)
Approaches to Decipher the
‘mRNA Localization Code’
Drosophila
Genetics
RNA Imaging in Fly
and Human Cells
+
High-Content Screening
And RNA Sequencing
+
Participation in ENCODE Project: Identifying Functional
RNA Elements Encoded in the Human Genome
K562 & Mcf7 cells
Gene Yeo
Xiang-Dong Fu
Brent Graveley
Chris Burge
Eric Lecuyer
Lecuyer Lab Objectives:
-Defining RBP Subcellular
Localization Properties
- Assess impact of RBP depletion on mRNA localization
84
Fractionation-Seq to Study Transcriptome Localization
Nuclear
K562 Cells
RNA Extraction, RiboDepletion (Ribozero)
Illumina TruSeq Stranded Kit Library Generation and RNA-seq
Replicates of Total, Nuclear, Cytosolic, Membrane and Insoluble fractions
Wang et al (2012) Cell
Western Blotting
15
Tubulin
50
KDEL
100
Ninein
190
Expression Relative
to Total (%)
His H3
RT-qPCR
100
75
100
100
XIST
75
ANLN
50
50
50
25
25
25
0
N C M I
0
H3
75
0
N C M I
N C M I
(Neal Cody & Xiaofeng Wang)
85
Broad Asymmetric Distribution of Functionally Coherent
RNA Populations in K562 Cells
Cell Component GO Terms
M
Total
Transcriptome
I
C
M
LncRNA
C
I
N
P-Value
(Neal Cody)
86
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
87
Progress - RBP Imaging
RBP Imaging Database
http://csb.cs.mcgill.ca/RBPImage
88
HNRNPK / DCP1a / DAPI
89
Diverse RBP Subcellular Localization Patterns
DNA Marker
RBPs
90