microRNAs Small Non-coding RNAs with Big Impact in Biology

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microRNAsSmall Non-coding RNAs with Big Impact
in Biology

• Hua-Chien Chen Ph.D

Molecular Medicine Research Center Chang Gung
University
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Numbers of protein coding genes do not scale
strongly with complexity
yeast Worm Fly Human Genes 6,000 18,500 1
3,500 30,000 Genome 15 Mb 100 Mb 120
Mb 3,300 Mb Cell types 1 1,100 2,000 1 X
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Biological complexity
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Biological complex may come from non-coding region
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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 that form 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
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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-4s 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).

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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
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Let-7 sequence and gene regulation
Nature (2000) 403 901-906
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Major differences between siRNA and microRNA

• miRNA microRNA, 21-25 nt
• Encoded by endogenous genes
• ssRNA with stem-loop structure
• Partial complement to the 3UTR 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

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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

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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)
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Gene regulation by transcription factors and
microRNAs
Transcription factors
microRNAs
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microRNA Biogenesis
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microRNA biogenesis
? Transcription
? Processing
? Maturation
? Execution
Nature Rev. Immunology (2008) 8 120-130
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Pri-miRNAs are processed by Drosha RNase III
enzyme
Pro-rich
RS-rich
RIIIDa
RIIIDb
dsRBD
1,374 aa

• 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?

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Mature miRNAs are generated by Dicer
DEAD
Helicase
RIIIDa
RIIIDb
dsRBD
PAZ
1,922 aa

• 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?

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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

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Mechanisms of miRNA-mediated gene
silencingAGO2-mediated RNA degradation
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From base pairing to gene silencing
Plant miRNA
Animal miRNA
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Current model for miRNA-mediated translational
repression
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Majority of miRNAs are binding to the 3UTR of
mRNA genes
3UTR regulates mRNA stability and translational
efficacy
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From base pairing to gene silencing
Plant miRNA
Animal miRNA
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Prediction of miRNA targets
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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 seedm8).
• 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

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Additional factors impact miRNA efficacy

1. Number of miRNA binding sites in 3UTR
2. Closely Spaced Sites Often Act Synergistically
3. Additional Watson-Crick Pairing at Nt 12-17
   Enhances miRNA Targeting
4. Effective Sites Preferentially Reside within a
   Locally AU-rich Context
5. Effective Sites Preferentially Reside in the
   3UTR, but Not too close to the stop codon
6. Effective Sites preferentially reside near both
   ends of the 3UTR

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Pathophysiological Function of microRNA
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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

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Tissue specific expression of microRNA
Brain and spine code
Muscle

1. 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.
2. The expression of miR-1 is restricted to the
   muscles and the heart in the mouse.
3. The conserved sequence and expression of miR-1
   and miR-124a suggests ancient roles in muscle and
   brain development.

Dev Cell (2006) 11441
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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
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Muscle-specific microRNAs and their targets
Trend in Genetics (2008)
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Tissue specific expression of miRNA
Nature Rev Genetics 2004)
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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
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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)

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miRNA frequently located at chromosome fragile
sites
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Examples of miRNAs located in chromosome fragile
sites
D deleted region A amplified region
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microRNAs are commonly down regulated in tumor
biopsies
Nature (2005) 435 834-838
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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
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miR-17/92 cluster showed increase expression in B
lymphoma and colon cancers
Nature (2005) 435 828-833
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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
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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
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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
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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

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Identification of miRNA involved in cell
migration and invasion
Migration and invasion assays
Nature Cell Biol (2007)
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miR-373 and miR-520c promote tumor metastasis in
vivo
Nature Cell Biol (2007)
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miR-10b is highly expressed in metastatic breast
cancer cells
Ref 1789_8713
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miR-10b induced tumor metastasis in vivo
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HOXD10 transcription factor is a down-stream
target of miR-10b
Ref 1789_8713
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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

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Mechanisms that link microRNA to diseases
Change in miRNA expression levels
Change in miRNA target spectrum
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Regulation of microRNA expression
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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

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Myc regulates the expression of miR-17-92 cluster
Cancer Research (2008) 688191-8194
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Nature Rev Cancer (2007)
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Regulation of miRNA expression DNA methylation
in CpG Islands
Coding region
Promoter
Exon 1
Exon 2
CpG Island

• Definition
• At least 200 bases long
• GC content gt 55
• observed CpG/expected CpG ratio gt 0.65
• There are about 29,000 such regions in the human
  genome
• 65 protein-coding genes contain CpG islands in
  promoter region

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microRNAs regulated by promoter methylation
Expression of miRNA also regulated by other
epigenetic mechanisms
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microRNA-related Databases
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microRNA database miRBase
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microRNA database miRBase
(Precursor and mature miRNA sequence)
(Transcript)
(Chromosome)
(Cluster)
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miRBase target prediction
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UCSC Genome browser