microRNAs

Small Non-coding RNAs with Big Impact in Biology

Hua-Chien Chen Ph.D

Lecture Content

• Non-coding RNAs and discovery of microRNAs

• microRNA biogenesis– Tanscription of miRNA primary transcripts– microRNA processing and maturation– microRNA action

• microRNA and target interaction

• microRNA function– Normal physiological function– miRNA and human diseases

The genetic basis of developmental complexity

• Humans (and other vertebrates) have approximately the same number of protein-coding genes (~20,000) as C. elegans, and less than those of plants (Arabidopsis ~28,000, rice ~40,000) and protozoa (30,000).

• Most of the proteins are orthologous and have similar functions from nematodes to humans, and many are common with yeast.

• Where is the information that programs our complexity?

Genome: 15Mb

Genes: 6,000

Cell: 1

Genome: 100Mb

Genes: 18,500

Cell: 1,100

Genome: 3,200Mb

Genes: 25,000

Cell: 1X1012

Yeast Warm Human

The proportion of noncoding DNA broadly increases with developmental complexity

Nature Rev. Genetics (2004) 5: 316-323

RNA

mRNAProtein-coding RNA

ncRNA: non-coding RNAsTranscribed RNA with a structural,

functional or catalytic role

rRNARibosomal RNAParticipate in

protein synthesis

tRNATransfer RNA

Interface betweenmRNA &

amino acids

snRNASmall nuclear

RNA RNA that form

part of the spliceosome

snoRNASmall nucleolar

RNA Found in nucleolus,

involved in modification

of rRNA

RNAiRNA interferenceSmall non-coding

RNA involvedin regulation

of gene expression

OtherIncluding large RNA

with roles in chromotin structure

and imprinting

siRNASmall interfering RNAActive molecules in

RNA interference

miRNAMicroRNA

Small RNA involvedin regulation

of protein-coding gene

Type of RNA molecules

Modified from Dr Morten Lindow slide

C. elegans lin-4 : first identified microRNA

V. Ambros lab

lin-4 RNA

lin-4 precursor

lin-4 RNA

“Translational repression”

target mRNA

• 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

• 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 stageLet-7: regulates heterochronic development at L4 to adult stage

Nature (2000) 403: 901-906

Let-7 sequence and gene regulation

Nature (2000) 403: 901-906

microRNAs at a glance

• 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

microRNA precursor

More than 5,000 miRNAs in public databases

• Homo sapiens (706) • Mus musculus (547)• Rattus norvegicus (286)• Drosophila melanogaster (152)• Caenorhabditis elegans (155)• Arabidopsis thaliana (187)• Epstein Barr virus (25)• Human cytomegalovirus (11)• Kaposi sarcoma-associated herpesvirus (13)• Simian virus 40 (1)

From miRBase Release 13.0 (Mar 2008)

Gene regulation by transcription factors and microRNAs

Transcription factors microRNAs

microRNA Biogenesis

microRNA biogenesis

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

Transcription

Processing

Maturation

Execution

Majority of miRNAs are transcribed by RNA polymerase II• Intronic miRNAs

– Located in the intron region of protein coding or non-coding transcripts

– Transcribed by RNA polymerase II• Extronic miRNAs

– Located in the exon region of protein-coding or non-coding transcripts

– Transcribed by RNA polymerase II• Intergenin miRNAs

– Located in the intergenic region of chromosome– Transcribed by RNA polymerase II or polymerase III

Genomic location of microRNAs

Pri-miRNAs are processed by Drosha RNase III enzyme

• 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

Pro-rich RS-rich RIIIDa RIIIDb dsRBD

Multiple mechanisms for miRNA processinga) The microprocessor complex

Drosha–DGCR8 cleaves the pri-miRNA, releasing the pre-miRNA

b) Some miRNAs require additional specificity factors (for example p68 and p72) for efficient cleavage

c) Interaction of pri-miR-18a with hnRNP A1 facilitates cleavage of this specific miRNA by Drosha

d) TGF-β signalling induces SMAD binding to the miR-21 precursor and enhances its efficient processing by Drosha.

e) Splicing can replace Drosha processing if the released and debranched intron (mirtron) has the length and hairpin structure of a pre-miRNA.

Ran/Expotin 5 system for pre-miRNA export

RanGAPRan GDP

Cytoplasm

Nucleoplasm

Export cargo

Exportin

Ran GTP

NES

Export cargo

Exportin

Ran GTP

NES

Export cargo

Exportin

Ran GTP

NES

PI

Export cargo

Exportin

NES

Ran-GTP: predominantly in the nucleoplasmRan-GDP: predominantly in the cytoplasm

Exportin-5: transport pre-miRNA

• Cleaves dsRNA or pre-miRNA– Leaves 3’ overhangs and 5’ phosphat

e 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?

DEAD Helicase RIIIDa RIIIDb dsRBDPAZ

1,922 aa

Mature miRNAs are generated by Dicer

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 silencingAGO2-mediated RNA degradation

From base pairing to gene silencing

Plant miRNA Animal miRNA

Current model for miRNA-mediated translational repression

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

3’UTR: regulates mRNA stability and translational efficacy

Prediction of miRNA targets

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

1. Number of miRNA binding sites in 3’UTR

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 3’UTR, but Not too close to the stop codon

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

Pathophysiological Function of microRNA

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

Brain and spine code

Muscle

Tissue specific expression of microRNA

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) 11:441

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

Trend in Genetics (2008)

Tissue specific expression of miRNA

Nature Rev Genetics 2004)

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

• 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

Examples of miRNAs located in chromosome fragile sites

D : deleted regionA : amplified region

microRNAs are commonly down regulated in tumor biopsies

Nature (2005) 435 : 834-838

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

Nature (2005) 435 : 839-843

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

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

Nature (2005) 435 : 828-833

• 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

miR-34 family function as tumor suppressors

Cancer Research (2007) 67: 11099-11101

• 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

Expression of miR-378 Promotes Tumorigenesis and Angiogenesis

Capillary formation

Tumor growth

PNAS (2007) 104: 20350-20355

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

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

Nature Cell Biol (2007)

Migration and invasion assays

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

Nature Cell Biol (2007)

Ref: 1789_8713

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

miR-10b induced tumor metastasis in vivo

Ref: 1789_8713

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

• Genomic alteration of microRNA– Chromosome deletion, amplification, and translocation– Single nucleotide polymorphism of miRNA or miRNA targe

ts

• Alteration on the expression of levels of miRNA– Transcriptional control: transcription factor, enhancer, re

pressor– Epigenetic modification: DNA methylation, histone acetyl

ation

• Alteration on the processes of microRNA biogenesis

Potential mechanisms that link microRNA to diseases

Mechanisms that link microRNA to diseases

Change in miRNA expression levels

Change in miRNA target spectrum

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

Cancer Research (2008) 68:8191-8194

Nature Rev Cancer (2007)

Regulation of miRNA expression: DNA methylation in CpG Islands

CpG Island= CpG

Promoter

• 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

Exon 1 Exon 2

Coding region

microRNAs regulated by promoter methylation

Expression of miRNA also regulated by other epigenetic mechanisms