microrna (mirna) hua-chien chen ph.d small non-coding rna with big impact in biology
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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
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