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 Genes6,00018,50013,50030,000 Genome15 Mb100 Mb120 Mb3,300 Mb Cell types11,1002,0001 X Biological complexity

Biological complex may come from non- coding region

RNA mRNA Protein-coding RNA ncRNA: non-coding RNAs Transcribed RNA with a structural, functional or catalytic role rRNA Ribosomal RNA Participate in protein synthesis 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 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 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 stage Let-7: regulates heterochronic development at L4 to adult stage Nature (2000) 403:

Let-7 sequence and gene regulation Nature (2000) 403:

Major differences between siRNA and microRNA miRNA: microRNA, 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, nt –Mostly exogenous origin –dsRNA precursors –May be target specific –Regulate mRNA stability

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 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 Transcription factorsmicroRNAs

microRNA Biogenesis

microRNA biogenesis Nature Rev. Immunology (2008) 8:  Transcription  Processing  Maturation  Execution

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-richRIIIDaRIIIDbdsRBD

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? DEAD HelicaseRIIIDaRIIIDbdsRBD PAZ 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 silencing AGO2-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

From base pairing to gene silencing Plant miRNA Animal miRNA

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 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 region A : amplified region

microRNAs are commonly down regulated in tumor biopsies Nature (2005) 435 :

C-myc induces expression of the miR-17/92 cluster Nature (2005) 435 : 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 :

miR-17/92 clusters function as oncogenes Nature (2005) 435 : 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: 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: U87 cells  transfect with miR-378 exp. Vector  tumor xenograft model

microRNAs regulate tumor angiogenesis Pro-angiogenic microRNAs –miR 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 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 Potential mechanisms that link microRNA to diseases

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

Regulation of microRNA expression

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 cluster Cancer Research (2008) 68:

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 1Exon 2 Coding region

microRNAs regulated by promoter methylation Expression of miRNA also regulated by other epigenetic mechanisms

microRNA-related Databases

microRNA database: miRBase

(Precursor and mature miRNA sequence) (Chromosome) (Transcript) (Cluster)

miRBase: target prediction

UCSC Genome browser