2_microRNA mRNA Control of Gene Expression

7/30/2019 2_microRNA mRNA Control of Gene Expression

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microRNA/mRNA control of gene expression

in tissue re-modelling and regeneration

Dr N Botchkareva


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What is RNA?

Ribonucleic Acid

Types

Coding: messenger RNA (mRNA) -

information formaking proteins

Non-coding regulation of protein formation:

Ribosomal RNA (rRNA)

Transfer RNA (tRNA)

Small nuclear RNA (snRNA)

Small nucleolar RNA (snoRNA)

Short interfering RNA (siRNA)

Long nc RNA

MicroRNA


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Gene Expression

microRNA


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MicroRNAs are a new class of non-protein-coding,

endogenous, very small molecules ~22 nt

Negative regulators of eukaryotic gene expression by interactingwith target messenger RNAs (mRNA) in a sequence-specific

manner

Protein expression is repressed or the coding message is

degraded when miRNAs are bound to the 3-untranslated regions(UTRs) of the target (mRNAs)

What is microRNA (miRNA)?


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Each miRNA may directly regulate expression of up to

200 different mRNA targets;

Conversely, a gene could be a common target of many

miRNAs.

Some miRNAs may also target another miRNAs andantagonize their effects on gene expression

MicroRNAs:

a comprehensive post-transcriptional regulatory network

mediated by miRNAs


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In mammals, over 800 miRNAs have been identified (over 1000miRNAs are predicted)

Over 30% of protein-coding genes may directly be regulated by

miRNAs

Therefore, miRNAs are likely to be master switches in many

biological pathways

miRNAs control diverse pathways: development, celldifferentiation, cell proliferation, apoptosis, hormone secretion,

stem cell maintenance, and tumorigenesis


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First discovered in 1993 in the Lab of Prof V Ambros at

Harvard University: lin-4 regulates mRNA translation duringworm development

The C. elegans heterochronic gene lin-4 encodes small

RNAs with antisense complementarity to lin-14 Cell. 1993

7 years break: Reinhart, Slack et al.,The 21-nucleotide let-7

RNA regulates developmental timing in Caenorhabditis

elegans. Nature. 2000

Now: thousands of microRNA molecules

Discovery


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1998-Fire and Mello, experiments in C. elegans, first to show that dsRNA is

much more potent at inhibiting gene expression than antisense RNA

(published in Nature, 1998)

Set the stage for understanding the role of miRNAs in development and gene regulation


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RNA interference:

Two Phase Process

Initiation (nucleus)

Generation of mature miRNA

Execution (cytoplasm)

Silencing of target gene

by

mRNA degradation orInhibition of translation

Nucleus Cytoplasm


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miRNAs are transcribed by the RNApolymerase II enzyme to produce a

primary-microRNA (pri-miRNA) forming specific hairpin secondary structures

Pri-miRNAs is cropped by a microprocessor complex, composed of the RNaseIII enzyme Drosha and the molecular anchor Di George syndrome critical

region 8 (DGCR8) producing precursor-miRNA (pre-miRNA)

Pre-miRNA is recognized by the Exportin-5 and transported to the cytoplasm

Cleaved by Dicer into mature miRNA

Biogenesis - Initiation


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2 mechanisms of action: mRNA cleavage OR inhibition of translation

miRNAs are incorporated into

RNA-induced silencing complex

(RISC)

Guided by base complementarity

of the miRNA, the RISC targets

mRNA for degradation

RNA interference/Execution

RISC

multiprotein complex,

containing Dicer,

Ago proteins,

miRNA, and

complementary mRNA


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The duplex for miR-579 and its target LRIG3 is partitioned into

two parts, the seed part and the out-seed part

seed region of miRNA: Six to eight nucleotides at the 5 end of

the mature miRNA sequence are very important in the selection of

target site

Target recognition

Example:

- 3UTR

miRNA target: a messenger RNA encoding a protein, containing

target sites for and regulated by an miRNA


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Lim (2003) Genes & Dev.17: 991-1008

Evolutionary conservation of miRNAs

Lim et al. compared microRNA sequences from

C. elegans to the human genome, and found that over

1/3 of these genes have homologs in humans.

Hundreds of conserved microRNAs
http://www.ncbi.nlm.nih.gov/pubmed/12672692?dopt=Citationhttp://www.ncbi.nlm.nih.gov/pubmed/12672692?dopt=Citationhttp://www.ncbi.nlm.nih.gov/pubmed/12672692?dopt=Citationhttp://www.ncbi.nlm.nih.gov/pubmed/12672692?dopt=Citationhttp://www.ncbi.nlm.nih.gov/pubmed/12672692?dopt=Citationhttp://www.ncbi.nlm.nih.gov/pubmed/12672692?dopt=Citation


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Expression pattern of microRNAs

He et al., 2004


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miR-124a is restricted to the brain and spinal cord in fish and

mouse or to the ventral nerve cord in fly

miR-1 is restricted to the muscles and the hart in mouse

Most MicroRNA genes are tissue-specific


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miRNAs Affect Everything

Frank Slack [email protected]
http://webinar.sciencecareers.org/miRNA/lobby.htmlhttp://webinar.sciencecareers.org/miRNA/lobby.html


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Cell / tissue Phenotype

mES cells Reduced proliferation rate, impaired differentiation: lack of

differentiation markers along with high levels of pluripotent

markers

Mouse embryonic

fibroblasts

inhibited proliferation, and induced a premature senescence

Neurogenesis massive hypotrophy of the postnatal cortex; neuroepithelial cells

(primary neural progenitors) are largely unaffected, defect of

neuronal differentiation, neuronal apoptosis

Skeletal development: reduction in skeletal size: reduced proliferation, acceleration of

hypertrophic differentiation of proliferating chondrocytes

Limb (deletion from

mesoderm)

Reduced in size limb due to massive apoptosis; no defects in basic

patterning or in tissue-specific differentiation

Skeletal muscle a decrease in muscle mass resulted from skeletal muscle hypoplasia;

increased apoptosis of myogenic cells

Cardiomyogenesis 1) stem cell depletion

2) heart malformation, due to greatly decreased mesenchymal

apoptosis in the outflow tract

Global role of microRNAs: Dicer deficiency

D l ti f Di


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Deletion of Dicer causes

dramatic alterations in skin development

By P5.5, Dicer knockout miceShow weight loss compared due

to defect in barrier formation

Abnormal hair follicles and cysts

(arrows) in the Dicer1

conditional knockout skins

D l ti f Di


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Apoptosis/Caspase-3

Dicer1 knockout skin is enriched in

apoptosis detected in abnormalcysts in the epidermis (marked by

active caspase-3 staining).

Deletion of Dicer causes

dramatic alterations in skin development

iRNA E i R lt i T l d


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miRNA Expression Results in Temporal and

Spatial Reciprocity with Target Expression

Annu. Rev. Cell Dev. Biol. 2007.23:175-205

Mutually exclusive expression of miRNAs and their targets.

S ti t l i f iR 203 d it t t 63
http://www.ncbi.nlm.nih.gov/pubmed/17506695?dopt=Citationhttp://www.ncbi.nlm.nih.gov/pubmed/17506695?dopt=Citationhttp://www.ncbi.nlm.nih.gov/pubmed/17506695?dopt=Citationhttp://www.ncbi.nlm.nih.gov/pubmed/17506695?dopt=Citation


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miR-203

restriction of miR-203 todifferentiating suprabasal

layers of skin

p63

restriction of p63 toproliferating basal

layer of skin

Spatiotemporal expression of miR-203 and its target p63

during skin development

miR-203 overexpression (TG) (low p63):

thinner epidermis, basal cell depletionmiR-203 antagonist

increases p63 expression

Yi et al., Natur e, 2008

p63 transcr ipt io n factor is

a cr i t ical regulator of epid ermal development and dif ferent iat ion

Ti d ti ifi i f iRNA


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Tissue and time specific expression of miRNAs

miRNA expression profiling in skin by microarray analysis

Aged skin

Young skin

Anagen

Catagen

Telogen

The heat map shows miRNAs that are down-regulated (Green)

and miRNAs that are up-regulated (Red)

Hair Cycle

Skin Ageing

iR 31 i i k dl i d d i


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0

5

10

15

20

25

30

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d 0 d3 d5 d12 d16 d17 d19

** **

0

5

10

15

20

25

30

35

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d 0 d3 d5 d12 d16 d17 d19

** **

Telogen Late-anagen CatagenMid-anagen

*

Telogen Late-anagen CatagenMid-anagen

*

miR-31 expression is markedly increased during anagen

and decreased in catagen and telogen

Tel An CatqRT-PCR

in situ hibridization

Inhibition of anti-miR-31 leads to


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Inhibition of anti-miR-31 leads tohyperplasia of the outer root sheath

and defects in the hair shaft

Day 8

Control anti-mir-31

Day 8

Changes in gene expression

program In keratinocytes

due to miR-31 inhibition

(microarray analysis)

Inhibition of miR 31 results in elevated expression of


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qRT-PCR

Western blot

FGF10 BAMBI Sclerostin

Inhibition of miR-31 results in elevated expression of

FGF10, BAMBI, Sclerostin, and

cytokeratins


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Mir-31 Krt14Krt16

Krt17

Hair matrix

Mir-31

Wnts,

BMPs

Sclerostin

FGF10

BAMBI

miR-31 is required for proper hair follicle growth and hair

fiber formation by controlling hair cycleassociated gene

expression (Mardary ev et al, FASEB J, 2010)

Alterations in miRNAs are found in


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George Calin

Alterations in miRNAs are found in

every type of human disease

miRNA-155 impaired

T and B cell differentiation

miR-208

pathological cardiac

growth


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MicroRNAs Regulate Cell Growth and Death

Frank Slack [email protected]

Roles in cancer


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De-regulation in microRNA expression results in the

development of pathological conditions, including cancer.

miRNAs related to cancer pathogenesis considered as

onco-miRs: The overexpression of such miRNAs is frequently

observed in cancer cell lines (for example miR-21)

Some miRNAs can act as tumor suppressors miRNAs by

repressing the expression of oncogenes: their under-

expressions were also found in cancer cells (for example let-7)

Roles in cancer

K t d t


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Known tumor supressors and protooncogenes

are regulated by microRNAs

miRNA Oncogenes or Tumour Suppressor Genes


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miRNA Oncogenes or Tumour Suppressor Genes

Croce Nat Rev Genet. 2009 Oct;10(10):704-14

Oncogenes

Tu

moursuppressors

miR 21
http://www.ncbi.nlm.nih.gov/pubmed/19763153?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=1http://www.ncbi.nlm.nih.gov/pubmed/19763153?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=1http://www.ncbi.nlm.nih.gov/pubmed/19763153?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=1http://www.ncbi.nlm.nih.gov/pubmed/19763153?itool=EntrezSystem2.PEntrez.Pubmed.Pubmed_ResultsPanel.Pubmed_RVDocSum&ordinalpos=1


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miR-21

miR-21 is a well recognized oncogenic miRNA that is

overexpressed in various tumours, and has been classified

as an oncomirProfiling of miRNA s in 540 tumor samples including lung, breast, stomach,prostate, colon, and pancreatic tumors showed that miR-21 was the only

miRNA up-regulated in all these tumors

miR-21 inhibits apoptosis by targeting tumour-suppressorgenes (PTEN, PDCD4)Knockdown of miR-21 in cultured glioblastoma cells activates caspases

leading to apoptotic cell death

miR-21 affects cell cycle progression and DNA damage-inducedcheckpoint function through the Cdc25a target gene (in colon

cancer cells)

miR-21 promotes cell migration by targeting TPM1, TIMP3


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Trangenic mouse model revealed- over-expression ofmiR-21 enhances tumorigenesis and

- deletion ofmiR-21 partially protects against tumor formation

(lung cancer)

miR-21-null mice: a significant reduction in skin tumorformations in response to tumor promoter treatment.

papilloma formation compared with wild-type mice; increase

in apoptosis and reduced cell proliferation; up-regulation of

miR-21 target genes expression, such as Spry1, Pten, andPdcd4

miRNAs and Cancer A Summary


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miRNAs and Cancer A Summary

miRNAs control cell cycle, cell differentiation and

apoptosis by regulating oncogenes and tumorsuppressor genes

miRNAs are misexpressed in cancer and are therefore

excellent diagnostic/prognostic markers in cancer

MicroRNAs could augment current cancer therapies.

How do we find miRNA targets?


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How do we find miRNA targets?

Several computational approaches have been developed to

facilitate experimental design and predicting miRNA targets.

Computational target prediction identifies potential bindingsites according to base-pairing rules and across species

conservation conditions.


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miRBasehttp://www.mirbase.org/

miRBase Human let 7a 1
http://www.mirbase.org/http://www.mirbase.org/http://www.mirbase.org/


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miRBase Human let-7a-1

http://www.mirbase.org/cgi-bin/mirna_entry.pl?acc=MI0000060
http://www.mirbase.org/cgi-bin/mirna_entry.pl?acc=MI0000060http://www.mirbase.org/cgi-bin/mirna_entry.pl?acc=MI0000060http://www.mirbase.org/cgi-bin/mirna_entry.pl?acc=MI0000060http://www.mirbase.org/cgi-bin/mirna_entry.pl?acc=MI0000060


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miRBase::MicroCosm miRNA Targetshttp://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/#

iRB Mi C iRNA T t
http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/


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miRBase::MicroCosm miRNA Targetshttp://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/#

predicted targets of let-7a
http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/


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Predicted targets require experimental validation!

Reporter assays

RNA levels of predicted target (qRT-PCR)

Protein levels of predicted targets (Western blot, ect.)

Summary


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MicroRNA genes represent probably 1-5% of thepredicted genes in humans

Because of their ability to target multiple mRNAs, about

10% - 30% protein-coding genes are predicted targetsregulated by miRNAs.

Revolution in the understanding of cell biology

Summary

Id en ti f ic at io n o f new m iRNAs and their b io lo gic al ro les

w il l p ro vide importan t in sights in to curren t k now ledge o f

th e mo lecu lar b io lo gy, and lead to develo pmen t o f n ew

therapeu ti cal app roaches by target ing d is tinc t m ic roRNAs


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Important MicroRNA Web Sites

Diana Lab: http://diana.cslab.ece.ntua.gr/

miRBase: http://microrna.sanger.ac.uk/

miRBase: http://www.mirbase.org/

MicroCosm: http://www.ebi.ac.uk/enright-srv/microcosm/

miRNAminer: http://groups.csail.mit.edu/pag/mirnaminer

miRviewer: http://people.csail.mit.edu/akiezun/miRviewer Patrocles: http://www.patrocles.org/

PicTar: http://pictar.mdc-berlin.de/

TargetRank: http://hollywood.mit.edu/targetrank

TargetScanS: http://www.targetscan.org/
http://diana.cslab.ece.ntua.gr/http://microrna.sanger.ac.uk/http://www.mirbase.org/http://www.ebi.ac.uk/enright-srv/microcosm/http://groups.csail.mit.edu/pag/mirnaminerhttp://people.csail.mit.edu/akiezun/miRviewerhttp://www.patrocles.org/http://pictar.mdc-berlin.de/http://hollywood.mit.edu/targetrankhttp://www.targetscan.org/http://www.targetscan.org/http://hollywood.mit.edu/targetrankhttp://pictar.mdc-berlin.de/http://pictar.mdc-berlin.de/http://pictar.mdc-berlin.de/http://www.patrocles.org/http://people.csail.mit.edu/akiezun/miRviewerhttp://groups.csail.mit.edu/pag/mirnaminerhttp://www.ebi.ac.uk/enright-srv/microcosm/http://www.ebi.ac.uk/enright-srv/microcosm/http://www.ebi.ac.uk/enright-srv/microcosm/http://www.mirbase.org/http://microrna.sanger.ac.uk/http://diana.cslab.ece.ntua.gr/


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Further Reading

Bushati N, Cohen SM. microRNA functions. Annu Rev Cell Dev Biol.

2007;23:175-205. Review

.

Sayed D, Abdellatif M. MicroRNAs in development and disease.

Physiol Rev. 2011 Jul;91(3):827-87. Review.

Krichevsky AM, Gabriely G. miR-21: a small multi-faceted RNA. J

Cell Mol Med. 2009 Jan;13(1):39-53. Review.

Documents

2_microRNA mRNA Control of Gene Expression