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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=Citation7/30/2019 2_microRNA mRNA Control of Gene Expression
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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.html7/30/2019 2_microRNA mRNA Control of Gene Expression
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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=Citation7/30/2019 2_microRNA mRNA Control of Gene Expression
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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
35
40
45
d 0 d3 d5 d12 d16 d17 d19
** **
0
5
10
15
20
25
30
35
40
45
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
http://webinar.sciencecareers.org/miRNA/lobby.htmlhttp://webinar.sciencecareers.org/miRNA/lobby.html7/30/2019 2_microRNA mRNA Control of Gene Expression
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MicroRNAs Regulate Cell Growth and Death
Frank Slack [email protected]
Roles in cancer
http://webinar.sciencecareers.org/miRNA/lobby.htmlhttp://webinar.sciencecareers.org/miRNA/lobby.html7/30/2019 2_microRNA mRNA Control of Gene Expression
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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=17/30/2019 2_microRNA mRNA Control of Gene Expression
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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
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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=MI00000607/30/2019 2_microRNA mRNA Control of Gene Expression
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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/v5/7/30/2019 2_microRNA mRNA Control of Gene Expression
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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/v5/7/30/2019 2_microRNA mRNA Control of Gene Expression
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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/7/30/2019 2_microRNA mRNA Control of Gene Expression
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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.