microRNA (miRNA)

Hua-Chien Chen Ph.D

Small non-coding RNA with Big Impact in Biology

RNA

mRNAProtein-coding RNA

ncRNANon-coding RNA. Transcribed RNA with a structural,

functional or catalytic role

rRNARibosomal RNA

Participate in protein synthesis

tRNATransfer RNA

Interface betweenmRNA &

amino acids

snRNASmall nuclear

RNA Incl. RNA that

form part of the spliceosome

snoRNASmall nucleolar

RNAFound in nucleolus,

involved in modification

of rRNA

RNAiRNA interferenceSmall non-coding

RNA involvedin regulation of expression

OtherIncluding large RNA

with roles in chromotin structure

and imprinting

siRNASmall interfering RNAActive molecules in

RNA interference

miRNAMicroRNA

Small RNA involvedin regulation of expression

Type of RNA molecules

The Nobel Prize in Physiology or Medicine 2006

Andrew Z. Fire and Craig C. Mello

for their discovery of "RNA interference – gene silencing by double-stranded RNA"

Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.

Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression. Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process

Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC.

Nature (1998)391:806-11

RNA interference pathway

RISC

siRNAs

Dicer

mRNA cleavage, degradation

Exogenous dsRNA, transposon, viral products, etc.

Target genes

dsRNA

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

• siRNA: short-interfering RNA, 21-25 nt– Mostly exogenous origin– dsRNA precursors– May be target specific

miRNA and RNAi pathways

RISC

Dicerprecursor

miRNA siRNAs

Dicer

“translational repression”and/or mRNA degradation mRNA cleavage, degradation

RNAi pathwaymicroRNA pathway

MicroRNA primary transcript Exogenous dsRNA, transposon, etc.

target mRNA

Drosha

RISCRISC

C. elegans lin-4 : first identified microRNA

lin-4 precursor

lin-4 RNA

“Translational repression”

V. Ambros lab lin-4 RNA

target mRNA

• 1993 Victor Ambros (Dartmouth) and colleagues showed that lin-4, a gene that controls developmental timing in C. elegans encodes two small RNA molecules and not protein

• lin-4 small RNA gene product showed sequence complementarity to multiple sites on 3’ UTR of lin-14

• Lin-4 inhibits lin-14 protein synthesis after the initiation of translation (1999)• At the time this mechanism was believed to be exclusive to nematodes

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

Nature (2000)

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 miRNAs

miRNA precursor

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)

MicroRNA Biogenesisand Mechanism of Action

Summary of microRNA biogenesis

Dicer

microRNA biogenesis1. MicroRNA (miRNA) genes are generall

y transcribed by RNA Polymerase II (Pol II) in the nucleus to form large pri-miRNA transcripts, which are capped (7MGpppG) and polyadenylated (AAAAA).

2. These pri-miRNA transcripts are processed by the RNase III enzyme Drosha and its co-factor, Pasha, to release the ~70-nucleotide pre-miRNA precursor product.

3. RAN–GTP and exportin 5 transport the pre-miRNA into the cytoplasm

4. Subsequently, another RNase III enzyme, Dicer, processes the pre-miRNA to generate a transient ~22- nucleotide miRNA:miRNA* duplex.

microRNA biogenesis

5. This duplex is then loaded into the miRNA-associated multiprotein RNA-induced silencing complex (miRISC), which includes the Argonaute proteins, and the mature single-stranded miRNA

6. The mature miRNA then binds to complementary sites in the mRNA target to negatively regulate gene expression in one of two ways that depend on the degree of complementarity between the miRNA and its target.

– mRNA degradation– Translational repression

miRNA biogenesis player: Drosha

• 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

• 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

miRNA biogenesis player: 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

Exporting of microRNA

The pre-miRNA with its typical ~2 nucleotide overhang at its 3′end is specifically recognized by exportin‑5 and is transported to the cytoplasm, where it dissociates from its receptor after RanGTP hydrolysis.

microRNA mediated gene silencingmiRNA

miRNA

mRNA degradation

Translational repression

microRNA-mediated mRNA Degradation

• Contains a member of the argonaute family

• Between 130 kDa and 500 kDa

• Other components are being characterized

• Cleaves RNA complementary to the siRNA, in the middle of the sequence

• Assembling the RISC complex requires ATP, while RNA cleavage does not.

Novina and Sharp, 2004c

microRNA-mediated translational repression

• Imperfect match between miRNA in RISC and target mRNAs

• RISC usually binds 3’ UTR• Mechanism of inhibition... ????

He and Hannon, 2004

Processing bodies

microRNA-mediated mRNA degradation and translational repression are converge in P-body

From base pairing to gene silencing

Seed sequence hypothesis

The 5’ region, and particularly seed positions 2-8, is the most conserved region of miRNAs and has been shown to play a key role in the target recognition

Two classes of microRNA binding sites in animal

Biological Functions

Physiological Roles of miRNAs

• 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

microRNAs and cardiogenesis

• microRNA-1-1 (miR-1-1) and miR-1-2 are specifically expressed in cardiac and skeletal muscle precursor cells.

• miR-1 genes are direct transcriptional targets of muscle differentiation regulators including serum response factor, MyoD and Mef2.

• Hand2, a transcription factor that promotes ventricular cardiomyocyte expansion, is a target of miR-1

Zhao et al. Nature 2005

microRNA promotes photoreceptor differentiation

miR-7 promotes photoreceptor development.

Genomic Localization of EBV-miRNAs

BHRF-1-1 BHRF-1-2 BHRF-1-3 BART-3, 4, 1, 15, BART-5, 16, 17, 6 BART-2BART-18, 7, 8, 9, 10, 11 -12, 19, 20, 13, 14

BHRF1 cluster- span 1.5 kb- 3 precursor- 4 mature miRs

BART1 cluster- span 1.0 kb- 8 precursors- 12 mature miRs

BART7 cluster- span 2.8 kb- 11 precursors- 15 mature miRs

BART2 cluster- 1 precursors- 1 mature miR

96 kb 5.9 kb 3.9 kb

microRNA and Cancer

Mechanisms that link microRNA to disease

Change in miRNA expression levels

Change in miRNA target spectrum

miRNA frequently located at chromosome fragile sites

Examples of miRNAs located in chromosome fragile sites

D : deleted regionA : amplified region

• miR-17-92 cluster (containing miR-19a and miR-20) is markedly overexpressed in lung cancer cell lines

Cancer Research (2005) 65 : 9628

miR-17-92 cluster is over-expressed in human lung cancer

A microRNA polycistron as a potential human oncogene

Nature (2005) 435 : 828

• 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 and p53 network

1. miR‑34 is a direct transcriptional target of p53, which in turn downregulates genes required for proliferation and survival.

2. Along with other p53 targets, such as p21 and BAX, miR‑34-family miRNAs promote growth arrest and cell death in response to cancer related stress.

TS : tumor suppressorOG : oncogene

microRNAs associated with human cancer

microRNAs are oncogenes or tumor suppressors

microRNAs down-regulated in tumor

microRNAs up-regulated in tumor

Hierarchical clustering analysis of microRNA expression profiles in 59 tumor-derived cell lines

Expression levels of majority microRNAs are down-regulated in tumor cells

Comparison of dendrograms derived from hierarchical clustering of miRNA and mRNA expression profiles in NCI60 cell lines

miRNA

mRNA

Hierarchical clustering of miRNA expression

• Samples from colon, liver, pancreas and stomach all clustered together in 214 miRNA profiling, reflecting their common derivation from tissues of embryonic endoderm

• A 16,000 mRNA profiling of the same samples failed to observe the coherence of gut derived sample in clustering

Nature (2005) 435 : 834 - 838

Global miRNA change during tumorigenesis

• Most of the miRNAs (129 out of 217) had lower expression levels in human tumors compared with normal tissues, irrespective of cell type

• Cancer cell lines also have lower miRNA levels• A tumor/normal classifier constructed using human sample had 100% acc

uracy when tested in the mouseNature (2005) 435 : 834 - 838

Human samples K-ras mice

A MicroRNA Signature Associated with Prognosis and Progression in Chronic Lymphocytic Leukemia

Expression profile of 13 miRNA represents the patient’s prognosis

N Engl J Med (2005) 353 : 1793

Detection of microRNA

Technologies for microRNA detection

• Solution phase hybridization– RNase protection assay– Splint ligation method

• Solid phase hybridization– Northern hybridization– Microarray technology– Bead-base technology

• Real-time PCR amplification– Precursor detection– Primer extension– Stem-loop RT primer

Solution phase hybridization : Splint ligation

RNA (2007) 13: 1-7

RNA (2007) 13: 1-7

Solution phase hybridization : Splint ligation

Array-based miRNA detection

RNA (2007) 13: 151-159

Total RNA vs purified small RNA

Specificity Sensitivity & dynamic range

RNA (2007) 13: 151-159

Array-based miRNA detection

qPCR-based miRNA detection

Nucleic Acids Research (2005) 33: e179

microRNA-related Database

miRNA target prediction programs

microRNA database: miRBase

microRNA database: miRBase

(Precursor and mature miRNA sequence)

(Chromosome)(Transcript)

(Cluster)

miRBase: target prediction