Presentation on theme: "Small non-coding RNA with Big Impact in Biology"— Presentation transcript:
1 Small non-coding RNA with Big Impact in Biology microRNA (miRNA)Small non-coding RNA with Big Impact in BiologyHua-Chien Chen Ph.D
2 Type of RNA molecules RNA ncRNA mRNA snRNA snoRNA tRNA RNAi Other rRNA Protein-coding RNAncRNANon-coding RNA. Transcribed RNA with a structural,functional or catalytic rolerRNARibosomal RNAParticipate inprotein synthesistRNATransfer RNAInterface betweenmRNA &amino acidssnRNASmall nuclearRNAIncl. RNA thatform part of thespliceosomesnoRNASmall nucleolarRNAFound in nucleolus,involved inmodificationof rRNARNAiRNA interferenceSmall non-codingRNA involvedin regulationof expressionOtherIncluding large RNAwith roles inchromotin structureand imprintingsiRNASmall interfering RNAActive molecules inRNA interferencemiRNAMicroRNASmall RNA involvedin regulationof expression
3 The Nobel Prize in Physiology or Medicine 2006 Andrew Z. Fire and Craig C. Mellofor their discovery of "RNA interference – gene silencing by double-stranded RNA"
4 Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature (1998)391:806-11Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC.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
6 Major differences between siRNA and microRNA miRNA: microRNA, ntEncoded by endogenous genesssRNA with stem-loop structurePartial complement to the 3’UTR of target genesRecognize multiple targetssiRNA: short-interfering RNA, ntMostly exogenous origindsRNA precursorsMay be target specific
8 C. elegans lin-4 : first identified microRNA lin-4 precursorlin-4 RNAtarget mRNAV. Ambros lab“Translational repression”lin-4 RNAThe story of microRNAs begins almost ten years ago when the Ambros lab made the remarkable discovery that the lin-4 gene, important for controlling development in the nematode C. elegans, did not code for protein, but instead, produced a pair of small RNAs.The longer ~61-nt RNA was predicted to fold into a stem-loop precursor which gave rise to a ~22 nt RNA that was found to function by triggering repression of the translation of the target gene lin-14. The specificity of interaction was thought to derive from complementarity between the lin-4 RNA and the 3’ untranslated region of the target mRNA as shown in this diagram.Not until 2000 did this question begin to be answered when a second gene of this type called let-7 was characterized by the Ruvkun lab. Let-7 also had a strong developmental phenotype when mutated and turned out to encode a 21 nucleotide untranslated RNA. But unlike lin-4… let-7 was found to be conserved in a wide range of other animals including molluscs, worms, flies and vertebrates including human.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 proteinlin-4 small RNA gene product showed sequence complementarity to multiple sites on 3’ UTR of lin-14Lin-4 inhibits lin-14 protein synthesis after the initiation of translation (1999)At the time this mechanism was believed to be exclusive to nematodes
9 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 wormsA homolog of let-7 was identified in humans and DrosophilaLin-4 and let-7 became founding members of a group of endogenous small RNA molecules with regulatory functions
11 microRNAs at a glance miRNA precursor Small, single-stranded forms of RNA (~22 nucleotides in length)generated from endogenous hairpin-shaped transcripts encoded in the genomesNegatively regulate protein-coding genes through translational repression or targeting mRNA for degradationMore than 500 microRNAs encoded in human genenome constitute a largest gene familyIt has been estimate that more than 30% of protein-coding genes can be regulated by miRNAs
12 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 (1)From miRBase Release 10.1 (Dec 2007)
15 microRNA biogenesisMicroRNA (miRNA) genes are generally transcribed by RNA Polymerase II (Pol II) in the nucleus to form large pri-miRNA transcripts, which are capped (7MGpppG) and polyadenylated (AAAAA).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.RAN–GTP and exportin 5 transport the pre-miRNA into the cytoplasmSubsequently, another RNase III enzyme, Dicer, processes the pre-miRNA to generate a transient ~22- nucleotide miRNA:miRNA* duplex.
16 microRNA biogenesisThis duplex is then loaded into the miRNA-associated multiprotein RNA-induced silencing complex (miRISC), which includes the Argonaute proteins, and the mature single-stranded miRNAThe 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 degradationTranslational repression
17 miRNA biogenesis player: Drosha Pro-richRS-richRIIIDaRIIIDbdsRBD1,374 aaProcesses pri-miRNA into pre-miRNALeaves 2 bp 3’ overhangs on pre-miRNANuclear RNAse-III enzyme [Lee at al., 2003]Tandem RNAse-III domainsHow does it identify pri-miRNA?Hairpin terminal loop sizeStem structureHairpin flanking sequencesNot yet found in plantsMaybe Dicer does its job?
18 miRNA biogenesis player: Dicer DEADHelicasePAZRIIIDaRIIIDbdsRBD1,922 aaCleaves dsRNA or pre-miRNALeaves 3’ overhangs and 5’ phosphate groupsCytoplasmic RNAse-III enzymeFunctional domains in DicerPutative helicasePAZ domainTandem RNAse-III domainsdsRNA binding domainMultiple Dicer genes in Drosophila and plantsFunctional specificity?
19 Working hypothesis of Dicer First contact of dsRNA2 nt overhang on the 3’ end of dsRNABinds to the PAZ binding domain at an oligonucleotide (OB) foldSecond contact at Platform DomainAnti-parallel-beta sheetPositive charged residuesResidues interact with negative charge of RNA backboneA 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 domains2 Mn cation binding sites per RNase domainRNase III domains positioned via bridging domainBind to scissile phosphates of dsRNA backboneA cluster of Acidic residues near the Mn cation binding sites in the RNase III domains is responsible for the hydrolytic cleavage of dsRNAThe small guide RNA is then released and incorporated into the RISC complex by the PAZ-like Argonaut protein
20 Exporting of microRNAThe 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.
22 microRNA-mediated mRNA Degradation Contains a member of the argonaute familyBetween 130 kDa and 500 kDaOther components are being characterizedCleaves RNA complementary to the siRNA, in the middle of the sequenceAssembling the RISC complex requires ATP, while RNA cleavage does not.Novina and Sharp, 2004c
23 microRNA-mediated translational repression Imperfect match between miRNA in RISC and target mRNAsRISC usually binds 3’ UTRMechanism of inhibition... ????He and Hannon, 2004
24 Processing bodiesmicroRNA-mediated mRNA degradation and translational repression are converge in P-body
29 Physiological Roles of miRNAs Organ (or tissues) developmentStem cell differentiation and maturationCell growth and survivalMetabolic homeostasisOncogenic malignancies and tumor formationViral infectionEpigenetic modification
30 Tissue specific expression of microRNA Brain and spine codeMuscleThe 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.The expression of miR-1 is restricted to the muscles and the heart in the mouse.The conserved sequence and expression of miR-1 and miR-124a suggests ancient roles in muscle and brain development.Dev Cell (2006) 11:441
31 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-1Zhao et al. Nature 2005
34 Genomic Localization of EBV-miRNAs BHRF-1-1BHRF-1-2BHRF-1-3BART-3, 4, 1, 15, BART-5, 16, 17, 6BART-2BART-18, 7, 8, 9, 10, 11-12, 19, 20, 13, 14EBV microRNAs were first discovered by Pfeffer et al in hairpins with 6 mature miRNAs were cloned from B95.8, including 3 BHRFs and 2 BARTs (Science 304:734, 2004). All 6 EBV miRNAs were expressed by EBV+ lymphoma cells and the expression seem to be related to the latency of EBV.Cai et al reported at least 17 EBV microRNAs by cloning using BC-1 cells (PLos Pathogens 3:e23, 2006). These miRNAs were encoded by the B95.8-deleted region.Grundoff et al combined a computational and microarray-based approach and reported 18 new EBV microRNAs in Jijoye cells (RNA 12:1-18, 2006). Some of these predicted sequences were confirmed by Northern. Mature region was not fully mapped.Kim et al evaluated the expression of selected EBV microRNAs in EBV-associated gastric carcinoma (J Virol 81:1033, 2007). BART microRNAs but not BHRF miRs were expressed in EBV+ GCs.BHRF1 cluster- span 1.5 kb- 3 precursor- 4 mature miRsBART1 cluster- span 1.0 kb8 precursors12 mature miRsBART7 cluster- span 2.8 kb11 precursors15 mature miRs96 kb5.9 kb3.9 kbBART2 cluster1 precursors1 mature miR
36 Mechanisms that link microRNA to disease Change in miRNA expression levelsChange in miRNA target spectrum
37 miRNA frequently located at chromosome fragile sites
38 Examples of miRNAs located in chromosome fragile sites D : deleted regionA : amplified region
39 miR-17-92 cluster is over-expressed in human lung cancer miR cluster (containing miR-19a and miR-20) is markedly overexpressed in lung cancer cell linesFigure 1. Search for miRNAs with altered expression in lung cancers.A, Northern blot analysis of miRNAs in lung cancer cell lines. Note markedoverexpression of miR-19a, miR-20, miR-106a, and miR-106b. Normal Lung,a mixture of RNAs from 11 normal lung tissues; BEAS-2B and HPL1D, twoimmortalized human epithelial cell lines representing proximal and distal airwaycells, respectively. B, unsupervised hierarchical clustering analysis highlightingoverexpression of miR-19a, miR-20, miR-106a, and miR-106b mainly insmall-cell lung cancer cell lines (red). Blue, normal lung tissues and the twoimmortalized human epithelial cell lines, BEAS-2B and HPL1D.Cancer Research (2005) 65 : 9628
40 A microRNA polycistron as a potential human oncogene Overexpression of the mir-17-19b cluster accelerates c-myc-induced lymphomagenesis in miceEm-myc/mir-17-19b tumors show a more disseminated phenotype compared with control tumorFigure 2 | Overexpression of the mir-17–19b cluster acceleratesc-myc-induced lymphomagenesis in mice. a, Schematic representation ofthe adoptive transfer protocol using Em-myc HSCs. b, Mice reconstitutedwith HSCs expressing mir-17–19b in an MSCV retroviral vector (MSCVmir-17–19b) or infected with a control MSCV virus were monitored byblood smear analysis starting 5 weeks after transplantation. The Kaplan-Meier curves represent the percentage of leukaemia-free survival or overallsurvival. c, External GFP imaging of tumour-bearing mice with Em-myc/mir-17–19b or Em-myc/MSCV shows the overall distribution of tumour cells.Em-myc/mir-17–19b tumours show a more disseminated phenotypecompared with control tumours. These animals are representative of theirgenotype.Nature (2005) 435 : 828
41 miR-34 and p53 networkmiR‑34 is a direct transcriptional target of p53, which in turn downregulates genes required for proliferation and survival.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.
42 microRNAs associated with human cancer TS : tumor suppressorOG : oncogene
43 microRNAs are oncogenes or tumor suppressors microRNAs down-regulated in tumormicroRNAs up-regulated in tumor
44 Hierarchical clustering analysis of microRNA expression profiles in 59 tumor-derived cell lines Expression levels of majority microRNAs are down-regulated in tumor cells
45 Comparison of dendrograms derived from hierarchical clustering of miRNA and mRNA expression profiles in NCI60 cell linesmiRNAmRNA
46 Hierarchical clustering of miRNA expression Clustering of 73 bone marrow samples from patients with acute lymphoblastic leukaemia(ALL). Coloured bars indicate the different ALL subtypes.Samples from colon, liver, pancreas and stomach all clustered together in 214 miRNA profiling, reflecting their common derivation from tissues of embryonic endodermA 16,000 mRNA profiling of the same samples failed to observe the coherence of gut derived sample in clusteringNature (2005) 435 :
47 Global miRNA change during tumorigenesis Human samplesK-ras miceFigure 3 | Comparison between normal and tumour samples reveals globalchanges in miRNA expression. a, Markers were selected to correlate withthe normal versus tumour distinction. A heatmap of miRNA expression isshown, with miRNAs sorted according to the variance-thresholded t-testscore. b, miRNA markers of normal versus tumour distinction in humantissues from a, applied to normal lungs and lung adenocarcinomas ofK-RasLA1 mice. A k-nearest neighbour (kNN) classifier based on humansample-derived markers yielded a perfect classification of the mouse samples(euclidean distance, k ¼ 3). Mouse tumour T_MLUNG_5 (third columnfrom right) was occasionally classified as normal using other kNNparameters (see Supplementary Information).Most of the miRNAs (129 out of 217) had lower expression levels in human tumors compared with normal tissues, irrespective of cell typeCancer cell lines also have lower miRNA levelsA tumor/normal classifier constructed using human sample had 100% accuracy when tested in the mouseNature (2005) 435 :
48 A MicroRNA Signature Associated with Prognosis and Progression in Chronic Lymphocytic Leukemia Expression profile of 13 miRNA represents the patient’s prognosisN Engl J Med (2005) 353 : 1793