Régulation génique par les microARNs Gene regulation by microRNAs - M. Simard... · Régulation...

Régulation génique par les microARNs

Gene regulation by microRNAs

Martin Simard Ph.D.Oncologie-CRCHU de Québec (Hôtel-Dieu de Québec)Centre de Recherche sur le cancer de l’Université Laval

[email protected]

• Discovery of microRNA (small temporal RNA)

• Example of function

• microRNA and Cancer

• microRNA and therapy

• Where they come from?

• Biogenesis of microRNA

• Regulation of the microRNA pathway

• Regulation of gene expression

Outlines

microRNAs: a very unique class of

gene uncovered in 1993

• Control developmental timing in specific cells and thus, insure the p r o p e r a n i m a l development. lin-4

lin-14

lin-28

Lee et al, Cell

Wightman et al, Cell

egg

L1

L2L3

L4

Adult

Heterochronic genes

• Gene that does not code for any protein

• 21 nucleotides long RNA

• Abrogates protein synthesis

• Partially complementary to its target RNA

Well! A weird phenomenon only seen in worms!

lin-4: A very special gene

Pasquinelli et al, Nature, 2000

A second example: let-7

Science, 2001

2001: More than 100 are identified

Major breakthrough

• C. elegans: Development, neuronal asymmetry

• Fruit fly: Apoptosis, cell proliferation

• Plants: leaves and flowers morphogenesis

• Vertebrates: Brain morphogenesis, hematopoiesis, neuronal and heart development

Over 10 000 microRNAs to date

Precise expression profile

Pena et al, Nature Methods 2009

Precise expression profile: hematopoiesis

Gangaraju and Lin, Nature Reviews MCB 2009

The loss of microRNAs lead to fatality

Zhao et al, Cell 2007

Ventura et al, Cell 2008

Loss of miR-1-2 leads to

overproduction of muscle cells

Problème majeur dans la

formation de cellules B

Loss of miR-17-92 cluster is embryonic

lethal

microRNAs and Cancer

microRNAs are frequently located in altered genomic regions associated to various

cancers

Calin et al, PNAS 2004

Interaction between microRNAs with tumor suppressors and oncogenes

microRNAs are part of the chain of reaction leading to tumorigenesis

• microRNAs can regulates their expressions

• microRNAs can be regulated by tumor suppressors genes and oncogenes

p53, p21, PTEN, p27, etc

p53, MYC

MYC: a key transcriptional regulation of microRNAs

Nature Genetics, 2008

Interactions of microRNAs with MYC

Lujambio et al, Nature 2012

miR-34

BCL2 CDK4 CDK6CyclinE2

Cell proliferationSurvival

p53

He et al. Nature 2007 (+4)

Interaction of microRNAs with the p53 pathway

miR-34

BCL2 CDK4 CDK6CyclinE2

Cell proliferationSurvival

miR-16-1

miR-143

miR-145

p53

Suzuki et al. Nature 2009He et al. Nature 2007 (+4)

Interaction of microRNAs with the p53 pathway

Hermeking, Nat. Rev. Cancer 2012

Implication of the p53 pathway: Multiple levels

Interaction of microRNA with BRCA1

J. Cell Biology 2012

• Oncogenes

TumourSuppressor

Tumour


Oncogene 2006

One example: miR-10b

Nature, 2007

microRNA as Oncogene : miR-10b

• Overexpression increases angiogenesis

• Induces metastasis formation

• Correlation between mir-10b overexpression and metastatic tumours caused by breast cancer

Ma et al, Nature 2007

Cancers tumors can become addicted to the overexpression of microRNAs: the miR-21 case

Medina et al, Nature, 2010

The overexpression of miR-21 in the

hematopoietic system lead to lymphoma

formation

Medina et al, Nature, 2010

Blocking miR-21 overexpression:

the tumors shrink!

Cancers tumors can become addicted to the overexpression of microRNAs: the miR-21 case

• Oncogenes

• Tumour suppressors

TumourSuppressor

Tumour

Oncogene

Tumour


Oncogene 2006

microRNA as Tumour suppressor : let-7

• let-7 level is altered in lung cancer

• let-7 controls RAS expression

• HMGA2 is another target of let-7

Johnson et al, Cell 2005

Mayr et al, Science 2007

Nature, 2008

One example: miR-335 et miR-126

Tavazoie et al, Nature, 2008

Re-establishing their expressions diminish the metastasis formation

microRNA as Tumour suppressor : miR-335 and miR-126

miR-335 controls cell migration

miR-126 controls cell proliferation

Ventura and Jacks, Cell 2009

microRNAs and Cancer: more and more examples

A new term in medical biology: oncomiRs

microRNAs with altered expression in tumours leading to affect cellular transformation, carcinogenesis and metastases formation: oncogene and tumour suppressors.

Slack, Nature Rev. Cancer, 2006

Different ways microRNAs can lead to cancer

Lujambio et al, Nature 2012

microRNAs and Cancer: Metastase formation

Kasinski and Slack, Nat. Rev. Cancer 2011

Different expression profile between healthy and cancer tissue

samples

Lu et al, Nature 2005

microRNAs and Cancer: new biomarkers


Nature Biotechnology 2008


• Stable and easily detectable in body fluids

• Specific miRNA signature

Weber et al, Clinical Chemistry 2010


Cortes et al, Nat.Rev. Clinical Oncology 2011

Different ways to export microRNAs

Cortes et al, Nat.Rev. Clinical Oncology 2011

• Exosomes

• HDL

• Coupled with proteins

“field in progress”

Export of microRNAs: very promising research avenue

Nature 2015

microRNAs as biomarkers: Advantages

• Very efficient to distinguish different tissues and tumours

• Simpler to identify good biomarkers from a set of hundreds microRNAs than 40 000 mRNAs

• microRNAs are extremely stable

• This stability allows retrospective analyses with conserved tissues

• microRNA can be visualized in tissues and cells with conventional techniques

• Viral infections (Herpes virus, SV40, HIV)

- Viruses encodes microRNAs that target viral mRNAs to regulate various stages of the viral life cycle

- Viral microRNAs suppress expression of specific host genes

- Viral infections induce expression of host microRNAs that inhibits expression of cellular genes

- Upon viral infections, host cells express specific microRNA that suppress viral mRNA expression

• May be important for gene imprinting

• Cardiac, immune, neurological and metabolic disorders

microRNA pathway associated to various diseases

Viruses highjack host microRNAs Cazalla et al, Science 2010

• Herpesvirus saimiri infects T cells of primates and causes aggressive leukemias and ymphomas

• non-coding RNA expresses by HVS

• Binding of host miRNA to HSURs lead to their degradation

New therapies: Controlling microRNA expression

Controlling microRNA expression: different methods

Garzon et al, Nature Reviews Drug Discovery, 2010

A nice proof of principle

Cell, 2009

miR-26a expression is decreased in liver tumours

Kota et al. Cell, 2009

microRNA as a Cancer therapy: miR-26a

Expression of miR-26a in vivo

Kota et al. Cell, 2009


Kota et al. Cell, 2009Decrease in tumour mass


microRNA as a Cancer therapy: many examples

Kasinski and Slack, Nat. Rev. Cancer 2011

Slack’s group Nature, 2014

Cancers specific targeting: promising approach

low pH-induced transmembrane structure (pHLIP)

Mix therapy: Modulate microRNAs levels to sensitize tumoral cells to other conventional therapy

Molecular Cell, 2011

Role of microRNAs in drug resistance

Migliore et al, Trends in Molecular medicine, 2013

• Small non-coding RNAs (21 to 23 nucleotides long)

• First discovered in Caenorhabditis elegans

• Found in nearly all eukaryotes

• microRNA as new biomarkers

• Modulation of microRNA expression: new therapeutic strategy

• mRNAs from cancer cells as shorter 3’UTR

To summarize microRNAs: A new class of regulatory molecules

More than 10,000 microRNAs found in 103 species!

How microRNA are produced?

Where microRNAs come from?

Kim et al, Nature Reviews MCB, 2009

Two types of microRNAs


1st processing step: the RNAseIII Drosha

Kim’s lab; Nguyen et al, Cell, 2015

• The microprecessor

• DGCR8 determine the precise cleavage site

2nd processing step: the RNAseIII Dicer

• TRBP and PACT are interacting with Dicer but are not essential for the activity

• Also important for the RNAi pathway


How accurate processing of miRNAs work?

Park et al, Nature, 2011

Dicer recognizes the 5’ of pre-miRNA for efficient and accurate miRNA

production

Mutations in Dicer leads to cancer

Foulkes et al, Nature Rev. Cancer, 2014

Pleuropulmonary blastoma is the first cancer in which the responsible gene is

associated to microRNA pathway

And now 2nd processing step without Dicer!

Cell Research, 2010

• One example: miR-451

• Observed in mouse and zebrafish

Cheloufi et al, Nature, 2010

Cifuentes et al, Science, 2010

Science, 2005

• Highly conserved gene family

• Implicated in RNA silencing pathways

Argonautes: key players in the microRNA pathway

Nature Reviews MCB, 2008

Argonautes: Four conserved domains

Schirle and MacRae, Science 2012Elkayam et al, Cell 2012

Link between Argonaute and cancer

Yang et al, Molecular Cell, 2014

Argonaute is part of chain of reaction leading to tumorigenesis

Argonautes important for miRISC formation

Nature Reviews MCB, 2008

SlicingAGO

NAR, 2012

Asymmetry rule: guide which strand will be incorporated into the active complex

Schwarz et al and Khvorova et al, Cell 2003

Stability of the 5’ end dictateswhich strand will be associatedto Argonaute complex

• let-7 microRNA only appears after differentiation of embryonic stem cells

• the levels of pri- and pre-let-7 are identical in un- and differentiated ES cells

Kim (2004) and Hammond (2006) groups

pri-let-7

pre-let-7

let-7

Regulation of microRNA biogenesis: The let-7 case

• Determination of the protein associated to pre-let-7 lead to the discovery of Lin28 (RNA binding protein highly expressed in ES cells)

• Lin28 leads to the uridylation of pre-let-7 by Tut4


Gregory, Wulczin, Hammond and Kim groups

Heo et al. Cell, 2009


Molecular Cell, 2011

Regulation of microRNA biogenesis: MCPIP1 acts as a more general inhibitors

Winter et al. Nature Cell Biology, 2009

Regulation of microRNA biogenesis: SMAD proteins and hnRNPA1 facilitates cleavage

Regulation of the microRNA biogenesis: the beginning

Krol et al. Nature Reviews Genetics, 2010

• Subsets of microRNAs

• Sequence specificity

hnRNP A1 and KSRP

• Compete with the machinery

Controls of the turnover of microRNA pathway components emerge as a key player in the regulation

Bossé et al. Mol. Cell, 2013

• microRNAs stability

• Recycling of miRISC components

AGO

Dicer

AGO AAAAAAA

AGO

AGO

AGO

XRN DCS-1

Meziane et al. Sci. Rep, 2015

Modification of microRNAs: RNA editing

• Deamination from adenosine to inosine

• Alters base-pairing and stability

• Enzymes responsible for edition: ADAR1 and ADAR2

• pri-miR editing inhibits Drosha and Dicer cleavage and favors degradation by Tudor-SN

• Some pri-miRs editing favors processing by Drosha

• Can impact microRNA target specificity

Modification of microRNAs: Uridylation

• Terminal uridylyl transferases (TUTs)

• oligo-uridylation leads to pre-miRNA degradation (let-7 case)

• mono-uridylation favors pre-miRNA processing

• uridylation appears to favor turnover of degradation intermediates

Kim et al, EMBO J 2015

Post-translational modifications of microRNA pathway components: example Argonaute

Meister, Nature Rev. Genetics 2013

The microRNA pathway is regulated at many levels

Ha, Nature Rev. MCB 2014

How microRNA regulates gene expression?

MicroRNA binds by base complementarity to 3’UTRs

• Partial complementarity

• “seed” sequence defines microRNA family

• More microRNAs, more effect: cooperativity

• Abrogate protein synthesisOne microRNA may regulate up to 100 different genes!

Filipowicz Nature Reviews Genetics, 2008

Extended complementarity with the mRNA leads to endonucleolytic cleavage

Shin et al. Mol. Cell, 2010

MicroRNA serves as guide for Argonaute...

Tethering Argonaute to the 3’UTR is sufficient to induce

translational repression

Pillai et al. RNA, 2004

...but how it finds its mRNA target with such a low basepair complementarity?

Jannot et al. EMBO Rep., 2011rack-1 RNAi

ctl let-7 lin-41 daf-12

Rel

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e as

soci

atio

nto

pol

ysom

es

0.2

0.6

1.0

P<0.005

RACK1 is a docking platform to recruit miRISC to the translation site

Discrepancy might be explained by the Argonautes

• 4 in humans and 2 in drosophila and C. elegans

• Binding partners

• fully complementary or bulged Dicer products

Iwasaki et al.Mol. Cell, 2009

Translational repression: Different models


Maybe more mRNA degradation then we thought...

...Well not settle yet!

Science, 2012

It most likely reflect the difference between different cellular states.

• Cytoplasmic granules

• Enriched in proteins involved in RNA catabolism and translation repression

• Many components interacts the miRISC complex

• Argonautes, GW182 and microRNA are found in these granules

The P bodies or GW-bodies


Fabian et al, NSMB, 2011

• GW182 recruits deadenylases

• Facilitates removal of the poly(A) tail by CCR4-NOT1

Chekulaeva et al, NSMB, 2011Braun et al, Mol. Cell, 2011

How GW182 contributes to microRNA gene regulation?

Release of the microRNA repression: rapid turnover

Cell, 2010

Extremely rapid turnover in neurons to respond quickly to stimuli

Modulators of microRNA function

AAAAAAAAgo

• Conditions specific

• Tissues specific

• Development specific

Interplay between microRNA complex and mRNA associated proteins: more and more examples

van Kouwenhove et al , Nat. Rev. Cancer 2011

Another function of microRNA: RNA binding proteins decoy

Beitzinger and Meister, Cell, 2010

A new class of RNA molecule called circular RNA competition for microRNA binding

Nature, 2013

Extremely abundant molecules in cells that contain complementary sites for microRNAs

To summarize

• Mainly produced by RNA polymerase II

• 1st maturation in the nucleus: microprocessor

• 2nd maturation in the cytoplasm: Dicer complex

• Active complex: miRISC Argonaute and microRNA

• In animals, binds non-coding regions (3’UTR)

• Abrogate protein synthesis

• What are the targets of microRNAs?

• How the microRNA pathway is controlled?

• How microRNAs regulate gene expression?

• Are microRNAs can travel from cell to cell and act in trans?

Questions to be answered

Questions/ comments [email protected]

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