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microRNAs Small Non-coding RNAs with Big Impact in Biology. Hua-Chien Chen Ph.D. Molecular Medicine Research Center Chang Gung University. Numbers of protein coding genes do not scale strongly with complexity. yeastWormFlyHuman Genes 6,00018,50013,50030,000

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microRNAs Small Non-coding RNAs with Big Impact in Biology

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Micrornas small non coding rnas with big impact in biology l.jpg

microRNAsSmall Non-coding RNAs with Big Impact in Biology

Hua-Chien Chen Ph.D

Molecular Medicine Research Center

Chang Gung University


Numbers of protein coding genes do not scale strongly with complexity l.jpg

Numbers of protein coding genes do not scale strongly with complexity

yeastWormFlyHuman

Genes6,00018,50013,50030,000

Genome15 Mb100 Mb120 Mb3,300 Mb

Cell types11,1002,0001 X 1012

Biological complexity


Slide3 l.jpg

Biological complex may come from non-coding region


Slide4 l.jpg

Type of RNA molecules

RNA

mRNA

Protein-coding RNA

ncRNA: non-coding RNAs

Transcribed RNA with a structural,

functional or catalytic role

tRNA

Transfer RNA

Interface between

mRNA &

amino acids

snRNA

Small nuclear

RNA

RNA thatform

part of the

spliceosome

snoRNA

Small nucleolar

RNA

Found in nucleolus,

involved in

modification

of rRNA

rRNA

Ribosomal RNA

Participate in

protein synthesis

RNAi

RNA interference

Small non-coding

RNA involved

in regulation

of gene expression

Other

Including large RNA

with roles in

chromotin structure

and imprinting

siRNA

Small interfering RNA

Active molecules in

RNA interference

miRNA

MicroRNA

Small RNA involved

in regulation

of protein-coding gene

Modified from Dr Morten Lindow slide


C elegans lin 4 first identified microrna l.jpg

lin-4 precursor

lin-4 RNA

target mRNA

“Translational repression”

C. elegans lin-4 : first identified microRNA

lin-4 RNA

V. Ambros lab

  • lin-4 encodes two small RNA molecules, a more abundant 22 nt that are processed from a rare 61 nt pre-lin-4 . These hairpin precursor is a characteristic feature of the miRNA class of regulatory RNAs.

  • One of lin-4’s target genes, lin-14, encodes a novel nuclear protein and is a putative transcription factor. The lin-4 microRNA regulates lin-14 through specific sequences in the 3’ UTR of the lin-14 mRNA

  • Upon lin-4 expression, lin-14 protein levels are reduced. Although transcription from the lin-14 gene still occurs, it is of no consequence. (Posttranscriptional control).


Lin 4 and let 7 are funding members of microrna l.jpg

lin-4 and let-7 are funding members of microRNA

  • Seven years later, let-7 (another non-coding gene) was shown to regulate development in worms

  • A homolog of let-7 was identified in humans and Drosophila

  • Lin-4 and let-7 became founding members of a group of endogenous small RNA molecules with regulatory functions

Lin-4: regulates heterochronic development at L1 to L2 stage

Let-7: regulates heterochronic development at L4 to adult stage

Nature (2000) 403: 901-906


Let 7 sequence and gene regulation l.jpg

Let-7 sequence and gene regulation

Nature (2000) 403: 901-906


Major differences between sirna and microrna l.jpg

Major differences between siRNA and microRNA

  • miRNA: microRNA, 21-25 nt

    • Encoded by endogenous genes

    • ssRNA with stem-loop structure

    • Partial complement to the 3’UTR of target genes

    • Recognize multiple targets

    • Regulate translation and RNA stability

  • siRNA: short-interfering RNA, 21-25 nt

    • Mostly exogenous origin

    • dsRNA precursors

    • May be target specific

    • Regulate mRNA stability


Micrornas at a glance l.jpg

microRNAs at a glance

microRNA precursor

  • Small, single-stranded forms of RNA (~22 nucleotides in length)

  • generated from endogenous hairpin-shaped transcripts encoded in the genomes

  • Negatively regulate protein-coding genes through translational repression or targeting mRNA for degradation

  • More than 500 microRNAs encoded in human genenome constitute a largest gene family

  • It has been estimate that more than 30% of protein-coding genes can be regulated by microRNAs


More than 4 000 mirnas in public databases l.jpg

More than 4,000 miRNAs in public databases

  • Homo sapiens (541)

  • Mus musculus (443)

  • Rattus norvegicus (287)

  • Drosophila melanogaster (152)

  • Caenorhabditis elegans (137)

  • Arabidopsis thaliana (184)

  • Epstein Barr virus (23)

  • Human cytomegalovirus (11)

  • Kaposi sarcoma-associated herpesvirus (13)

  • Simian virus 40(1)

From miRBase Release 10.1 (Dec 2007)


Gene regulation by transcription factors and micrornas l.jpg

Gene regulation by transcription factors and microRNAs

Transcription factors

microRNAs


Microrna biogenesis l.jpg

microRNA Biogenesis


Microrna biogenesis13 l.jpg

microRNA biogenesis

 Transcription

 Processing

 Maturation

 Execution

Nature Rev. Immunology (2008) 8: 120-130


Pri mirnas are processed by drosha rnase iii enzyme l.jpg

Pri-miRNAs are processed by Drosha RNase III enzyme

Pro-rich

RS-rich

RIIIDa

RIIIDb

dsRBD

  • Processes pri-miRNA into pre-miRNA

    • Leaves 2 bp 3’ overhangs on pre-miRNA

  • Nuclear RNAse-III enzyme [Lee at al., 2003]

    • Tandem RNAse-III domains

  • How does it identify pri-miRNA?

    • Hairpin terminal loop size

    • Stem structure

    • Hairpin flanking sequences

  • Not yet found in plants

    • Maybe Dicer does its job?

1,374 aa


Mature mirnas are generated by dicer l.jpg

Mature miRNAs are generated by Dicer

DEAD

Helicase

RIIIDa

RIIIDb

dsRBD

PAZ

  • Cleaves dsRNA or pre-miRNA

    • Leaves 3’ overhangs and 5’ phosphate groups

  • Cytoplasmic RNAse-III enzyme

  • Functional domains in Dicer

    • Putative helicase

    • PAZ domain

    • Tandem RNAse-III domains

    • dsRNA binding domain

  • Multiple Dicer genes in Drosophila and plants

    • Functional specificity?

1,922 aa


Working hypothesis of dicer l.jpg

Working hypothesis of Dicer

  • First contact of dsRNA

    • 2 nt overhang on the 3’ end of dsRNA

  • Binds to the PAZ binding domain at an oligonucleotide (OB) fold

  • Second contact at Platform Domain

    • Anti-parallel-beta sheet

    • Positive charged residues

  • Residues interact with negative charge of RNA backbone

  • A connector helix forms 65 Angstrom (24nt) distance between the PAZ holding and the RNase III cleaving domains – “ruler”

  • Third contact at the 2 RNase III domains

    • 2 Mn cation binding sites per RNase domain

    • RNase III domains positioned via bridging domain

    • Bind to scissile phosphates of dsRNA backbone

  • A cluster of Acidic residues near the Mn cation binding sites in the RNase III domains is responsible for the hydrolytic cleavage of dsRNA

  • The small guide RNA is then released and incorporated into the RISC complex by the PAZ-like Argonaut protein


Mechanisms of mirna mediated gene silencing ago2 mediated rna degradation l.jpg

Mechanisms of miRNA-mediated gene silencingAGO2-mediated RNA degradation


Slide18 l.jpg

From base pairing to gene silencing

Plant miRNA

Animal miRNA


Current model for mirna mediated translational repression l.jpg

Current model for miRNA-mediated translational repression


Majority of mirnas are binding to the 3 utr of mrna genes l.jpg

Majority of miRNAs are binding to the 3’UTR of mRNA genes

3’UTR: regulates mRNA stability and translational efficacy


Slide23 l.jpg

From base pairing to gene silencing

Plant miRNA

Animal miRNA


Prediction of mirna targets l.jpg

Prediction of miRNA targets


Target prediction by targetscan l.jpg

Target Prediction by TargetScan

  • Seed region : TargetScan defines a seed as positions 2-7 of a mature miRNA.

  • miRNA family : A miRNA family is comprised of miRNAs with the same seed region (positions 2-8 of the mature miRNA, also called seed+m8).

  • 8mer : An exact match to positions 2-8 of the mature miRNA (the seed + position 8) with a downstream 'A' across from position 1 of the miRNA

  • 7mer-m8 : An exact match to positions 2-8 of the mature miRNA (the seed + position 8)

  • 7mer-1A : An exact match to positions 2-7 of the mature miRNA (the seed) with a downstream 'A' across from position 1 of the miRNA


Additional factors impact mirna efficacy l.jpg

Additional factors impact miRNA efficacy

  • Number of miRNA binding sites in 3’UTR

  • Closely Spaced Sites Often Act Synergistically

  • Additional Watson-Crick Pairing at Nt 12-17 Enhances miRNA Targeting

  • Effective Sites Preferentially Reside within a Locally AU-rich Context

  • Effective Sites Preferentially Reside in the 3’UTR, but Not too close to the stop codon

  • Effective Sites preferentially reside near both ends of the 3’UTR


Pathophysiological function of microrna l.jpg

Pathophysiological Function of microRNA


Physiological roles of microrna l.jpg

Physiological Roles of microRNA

  • Organ (or tissues) development

  • Stem cell differentiation and maturation

  • Cell growth and survival

  • Metabolic homeostasis

  • Oncogenic malignancies and tumor formation

  • Viral infection

  • Epigenetic modification


Slide29 l.jpg

Tissue specific expression of microRNA

Brain and spine code

Muscle

  • The expression of miR-124a is restricted to the brain and the spinal cord in fish and mouse or to the ventral nerve cord in the fly.

  • The expression of miR-1 is restricted to the muscles and the heart in the mouse.

  • The conserved sequence and expression of miR-1 and miR-124a suggests ancient roles in muscle and brain development.

Dev Cell (2006) 11:441


Control of skeletal muscle proliferation and differentiation by mir 1 and mir 133 l.jpg

Control of skeletal muscle proliferation and differentiation by miR-1 and miR-133

MEF2: myocyte enhancer factor 2

HDAC4: histone deacetylase 4

SRF: serum response factor

No miR-1/miR-133a expression in MEF2 knockout mice

E11.5 transgenic mouse embryos


Muscle specific micrornas and their targets l.jpg

Muscle-specific microRNAs and their targets

Trend in Genetics (2008)


Tissue specific expression of mirna l.jpg

Tissue specific expression of miRNA

Nature Rev Genetics 2004)


Microrna networks and diseases l.jpg

microRNA networks and diseases

  • The number of microRNA in human genome may over 1,000 genes (currently 570 miRNAs in miRBase database)

  • Tens to hundreds of protein-coding genes are regulated by single miRNA

  • Estimated that around 30% of genes are regulated by microRNA

  • Almost every cellular processes are regulated by microRNA

Mutation or dysregulation of microRNA  Diseases formation


Several evidences suggest that micrornas may play an important role in tumor development l.jpg

Several evidences suggest that microRNAs may play an important role in tumor development

  • More than 50% of microRNAs are located within the chromosome fragile sites

  • Expression levels of microRNA in tumor biopsies are commonly altered

  • Several microRNAs have been shown to regulate the proliferation and differentiation of cells

  • micorRNAs also control the pathways of cell death (apoptosis)


Mirna frequently located at chromosome fragile sites l.jpg

miRNA frequently located at chromosome fragile sites


Examples of mirnas located in chromosome fragile sites l.jpg

Examples of miRNAs located in chromosome fragile sites

D : deleted region

A : amplified region


Micrornas are commonly down regulated in tumor biopsies l.jpg

microRNAs are commonly down regulated in tumor biopsies

Nature (2005) 435 : 834-838


C myc induces expression of the mir 17 92 cluster l.jpg

C-myc induces expression of the miR-17/92 cluster

Tet-off system to induce c-myc expression in P493 cells

Nature (2005) 435 : 839-843


Mir 17 92 cluster showed increase expression in b lymphoma and colon cancers l.jpg

miR-17/92 cluster showed increase expression in B lymphoma and colon cancers

Nature (2005) 435 : 828-833


Mir 17 92 clusters function as oncogenes l.jpg

miR-17/92 clusters function as oncogenes

  • Overexpression of the mir-17-19b cluster accelerates c-myc-induced lymphomagenesis in mice

  • Em-myc/mir-17-19b tumors show a more disseminated phenotype compared with control tumor

Nature (2005) 435 : 828-833


Mir 34 family function as tumor suppressors l.jpg

miR-34 family function as tumor suppressors

  • miR-34 family members are highly conserved during evolution

  • miR-34a is located within chromosome 1p36 region, which is commonly deleted in human neuroblastoma

  • Primary neuroblastomas and cell lines often showed low levels of miR-34a expression

  • Forced expression of miR-34a in these cells inhibited proliferation and activated cell death pathways

Cancer Research (2007) 67: 11099-11101


Expression of mir 378 promotes tumorigenesis and angiogenesis l.jpg

Expression of miR-378 Promotes Tumorigenesis and Angiogenesis

Tumor growth

Capillary formation

U87 cells  transfect with miR-378 exp. Vector  tumor xenograft model

PNAS (2007) 104: 20350-20355


Micrornas regulate tumor angiogenesis l.jpg

microRNAs regulate tumor angiogenesis

  • Pro-angiogenic microRNAs

    • miR-17-92 cluster: TSP-1, CTGF

    • miR-378: Sufu (suppressor of fused)

    • Let-7f

  • Anti-angiogenic microRNAs

    • miR-221 and miR-222: c-Kit and eNOS

    • miR-15 and miR-16: VEGF and Bcl-2

    • miR-20a and -20b: VEGF and Bcl-2


Identification of mirna involved in cell migration and invasion l.jpg

Identification of miRNA involved in cell migration and invasion

Migration and invasion assays

Nature Cell Biol (2007)


Mir 373 and mir 520c promote tumor metastasis in vivo l.jpg

miR-373 and miR-520c promote tumor metastasis in vivo

Nature Cell Biol (2007)


Mir 10b is highly expressed in metastatic breast cancer cells l.jpg

miR-10b is highly expressed in metastatic breast cancer cells

Ref: 1789_8713


Mir 10b induced tumor metastasis in vivo l.jpg

miR-10b induced tumor metastasis in vivo


Hoxd10 transcription factor is a down stream target of mir 10b l.jpg

HOXD10 transcription factor is a down-stream target of miR-10b

Ref: 1789_8713


Slide49 l.jpg

Potential mechanisms that link microRNA to diseases

  • Genomic alteration of microRNA

    • Chromosome deletion, amplification, and translocation

    • Single nucleotide polymorphism of miRNA or miRNA targets

  • Alteration on the expression of levels of miRNA

    • Transcriptional control: transcription factor, enhancer, repressor

    • Epigenetic modification: DNA methylation, histone acetylation

  • Alteration on the processes of microRNA biogenesis


Slide50 l.jpg

Mechanisms that link microRNA to diseases

Change in miRNA expression levels

Change in miRNA target spectrum


Regulation of microrna expression l.jpg

Regulation of microRNA expression


Regulatory mechanism in mirna expression l.jpg

Regulatory mechanism in miRNA expression

  • Regulate by transcription factors

    • P53, c-myc, AP1, NFkB pathway ---

  • Regulate epigenetic mechanisms

    • DNA methylation

    • Histone modification

  • Regulate at miRNA processing


Myc regulates the expression of mir 17 92 cluster l.jpg

Myc regulates the expression of miR-17-92 cluster

Cancer Research (2008) 68:8191-8194


Slide54 l.jpg

Nature Rev Cancer (2007)


Regulation of mirna expression dna methylation in cpg islands l.jpg

= CpG

Regulation of miRNA expression: DNA methylation in CpG Islands

Coding region

Promoter

Exon 1

Exon 2

CpG Island

  • Definition

    • At least 200 bases long

    • G+C content: > 55%

    • observed CpG/expected CpG ratio: >= 0.65

  • There are about 29,000 such regions in the human genome

  • 65% protein-coding genes contain CpG islands in promoter region


Micrornas regulated by promoter methylation l.jpg

microRNAs regulated by promoter methylation

Expression of miRNA also regulated by other epigenetic mechanisms


Microrna related databases l.jpg

microRNA-related Databases


Microrna database mirbase l.jpg

microRNA database: miRBase


Microrna database mirbase59 l.jpg

microRNA database: miRBase

(Precursor and mature miRNA sequence)

(Transcript)

(Chromosome)

(Cluster)


Slide61 l.jpg

miRBase: target prediction


Ucsc genome browser l.jpg

UCSC Genome browser


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