microRNA/mRNA control of gene expression in tissue re-modelling and regeneration

Dr N Botchkareva

What is RNA?
Ribonucleic Acid Types Coding: messenger RNA (mRNA) information for making 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

Gene Expression

microRNA

What is microRNA (miRNA)?

MicroRNAs are a new class of non-protein-coding, endogenous, very small molecules ~22 nt
Negative regulators of eukaryotic gene expression by interacting with 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)

MicroRNAs: a comprehensive post-transcriptional regulatory network mediated by miRNAs

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 and antagonize their effects on gene expression

In mammals, over 800 miRNAs have been identified (over 1000 miRNAs 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, cell differentiation, cell proliferation, apoptosis, hormone secretion, stem cell maintenance, and tumorigenesis

Discovery
First discovered in 1993 in the Lab of Prof V Ambros at Harvard University: lin-4 regulates mRNA translation during worm 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

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

RNA interference: Two Phase Process

Nucleus Cytoplasm Initiation (nucleus) Generation of mature miRNA Execution (cytoplasm) Silencing of target gene by mRNA degradation or Inhibition of translation

Biogenesis - Initiation

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 RNase III 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

RNA interference/Execution miRNAs are incorporated into RNA-induced silencing complex (RISC) Guided by base complementarity of the miRNA, the RISC targets mRNA for degradation

RISC multiprotein complex, containing Dicer, Ago proteins, miRNA, and complementary mRNA

2 mechanisms of action: mRNA cleavage OR inhibition of translation

Target recognition
miRNA target: a messenger RNA encoding a protein, containing target sites for and regulated by an miRNA 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
Example:

- 3UTR

The duplex for miR-579 and its target LRIG3 is partitioned into two parts, the seed part and the out-seed part

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

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

Expression pattern of microRNAs

He et al., 2004

Most MicroRNA genes are tissue-specific

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

miRNAs Affect Everything

Frank Slack frank.slack@yale.edu

Global role of microRNAs: Dicer deficiency

Cell / tissue mES cells Phenotype Reduced proliferation rate, impaired differentiation: lack of differentiation markers along with high levels of pluripotent markers inhibited proliferation, and induced a premature senescence massive hypotrophy of the postnatal cortex; neuroepithelial cells (primary neural progenitors) are largely unaffected, defect of neuronal differentiation, neuronal apoptosis reduction in skeletal size: reduced proliferation, acceleration of hypertrophic differentiation of proliferating chondrocytes Reduced in size limb due to massive apoptosis; no defects in basic patterning or in tissue-specific differentiation

Mouse embryonic fibroblasts Neurogenesis

Skeletal development: Limb (deletion from mesoderm)

Skeletal muscle
Cardiomyogenesis

a decrease in muscle mass resulted from skeletal muscle hypoplasia; increased apoptosis of myogenic cells
1) stem cell depletion 2) heart malformation, due to greatly decreased mesenchymal apoptosis in the outflow tract

Deletion of Dicer causes dramatic alterations in skin development

By P5.5, Dicer knockout mice Show weight loss compared due to defect in barrier formation

Abnormal hair follicles and cysts (arrows) in the Dicer1 conditional knockout skins

Deletion of Dicer causes dramatic alterations in skin development

Dicer1 knockout skin is enriched in apoptosis detected in abnormal cysts in the epidermis (marked by active caspase-3 staining).

Apoptosis/Caspase-3

miRNA Expression Results in Temporal and Spatial Reciprocity with Target Expression
Mutually exclusive expression of miRNAs and their targets.

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

Spatiotemporal expression of miR-203 and its target p63 during skin development
restriction of miR-203 to differentiating suprabasal layers of skin restriction of p63 to proliferating basal layer of skin

miR-203

p63

p63 transcription factor is a critical regulator of epidermal development and differentiation miR-203 antagonist increases p63 expression miR-203 overexpression (TG) (low p63): thinner epidermis, basal cell depletion

Yi et al., Nature, 2008

Tissue and time specific expression of miRNAs miRNA expression profiling in skin by microarray analysis
The heat map shows miRNAs that are down-regulated (Green) and miRNAs that are up-regulated (Red)
Hair Cycle Anagen Catagen Telogen Skin Ageing

Aged skin
Young skin

miR-31 expression is markedly increased during anagen and decreased in catagen and telogen
qRT-PCR
** **
45 40 35 30 25 20 15 10 5 0

Tel

An
**

Cat
**

12

P 17

P 19

P 20

P 21

P 22

P 23

d0

d3

d5

d12

d16

d17

d19

in situ hibridization Telogen

Mid-anagen

Late-anagen

Catagen

Inhibition of anti-miR-31 leads to hyperplasia of the outer root sheath and defects in the hair shaft
Control

anti-mir-31

Day 8

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 FGF10, BAMBI, Sclerostin, and cytokeratins
qRT-PCR
FGF10 BAMBI Sclerostin

Western blot

miR-31 is required for proper hair follicle growth and hair fiber formation by controlling hair cycleassociated gene expression (Mardaryev et al, FASEB J, 2010)

Hair matrix
FGF10 Sclerostin Mir-31

Krt14 Krt16 Krt17

BAMBI

Wnts, BMPs

Alterations in miRNAs are found in every type of human disease

miR-208 pathological cardiac growth miRNA-155 impaired T and B cell differentiation

George Calin

MicroRNAs Regulate Cell Growth and Death

Frank Slack frank.slack@yale.edu

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

Known tumor supressors and protooncogenes are regulated by microRNAs

miRNA Oncogenes or Tumour Suppressor Genes

Oncogenes

Tumour suppressors

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

miR-21
miR-21 is a well recognized oncogenic miRNA that is overexpressed in various tumours, and has been classified as an oncomir
Profiling 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-suppressor genes (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-induced checkpoint function through the Cdc25a target gene (in colon cancer cells) miR-21 promotes cell migration by targeting TPM1, TIMP3

Trangenic mouse model revealed - over-expression of miR-21 enhances tumorigenesis and - deletion of miR-21 partially protects against tumor formation (lung cancer) miR-21-null mice: a significant reduction in skin tumor formations 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, and Pdcd4

miRNAs and Cancer A Summary

miRNAs control cell cycle, cell differentiation and apoptosis by regulating oncogenes and tumor suppressor 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?

Several computational approaches have been developed to facilitate experimental design and predicting miRNA targets. Computational target prediction identifies potential binding sites according to base-pairing rules and across species conservation conditions.

miRBase
http://www.mirbase.org/

miRBase Human let-7a-1

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

miRBase::MicroCosm miRNA Targets

http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/#

miRBase::MicroCosm miRNA Targets

http://www.ebi.ac.uk/enright-srv/microcosm/htdocs/targets/v5/#

predicted targets of let-7a

Predicted targets require experimental validation!

Reporter assays
RNA levels of predicted target (qRT-PCR) Protein levels of predicted targets (Western blot, ect.)

Summary
Revolution in the understanding of cell biology MicroRNA genes represent probably 1-5% of the predicted genes in humans Because of their ability to target multiple mRNAs, about 10% - 30% protein-coding genes are predicted targets regulated by miRNAs.

Identification of new miRNAs and their biological roles will provide important insights into current knowledge of the molecular biology, and lead to development of new therapeutical approaches by targeting distinct microRNAs

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/

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.