高级病生第7次课-Noncoding RNA and disease

nullNoncoding RNA and DiseaseNoncoding RNA and DiseaseZhu-Zhong Mei, PhD Department of PathophysiologynullCentral dogma of biologyproteinDNARNARNA and the genome - the end of the central dogmaRNA and the genome - the end of the central dogma~25,000 protein coding genes in humans Protein-coding DNA = 1~2% of genomenullThe number of protein coding genes cannot justify the evolutionary gap between eukaryotesGene numberTranscript number~25k~15k~20k~6.5k~31k>30k<30knullIncrease the complexity by increasing the combinatorial use of exons by alternative splicingprimary transcriptAlternative splicingFunction AFunction Bnull~49k ?~20k~23k~6.5k>30kGene numberTranscript numberStill the number of different transcripts cannot justify the evolutionary gap between eukaryotes~49kThe Old GuardThe Old GuardHolds steadfast to the idea that most of the transcription (like most of the DNA) is “Junk” Key reasoning: Little sequence conservation of non-coding RNA from species to species Explanation: Transcription of leftover retroviruses and RNA Polymerase infidelityThe Old Guard…(holding onto a lack of science)The Old Guard…(holding onto a lack of science)Microarray data of mouse embryonic stem cells identified at least 945 long non-coding RNA’s that are distinctly regulated with development and differentiation. (Dinger, M. E. et al.. Genome Res. 2008, 18:1433–1445 ) Further analysis finds ncRNA to be tissue specific. (Furuno et al. 2006; Kapranov et al. 2002; Nakaya et al. 2007; Ravasi et al. 2006) And, its transcription responds to environmental stimuli. (Cawley et al. 2004; Louro et al. 2007; Ravasi et al. 2006).Continuing…Continuing…Many have upstream transcriptional binding sites for proteins such as Sp1, c-Myc, p53, and Creb. (Cawley et al. 2004; Euskirchen et al. 2004) Long ncRNA with a known function, such as Xist, have very little sequence similarity. The same miRNA regulates different genes in different species (share little sequence homology/ rapid evolution theory). ….these go on and on….Eukaryotic RNA transcriptsEukaryotic RNA transcriptstRNA rRNA snRNA tmRNA Rnase P RNA vRNAs gRNAs MRP RNA SRP RNAs Telomerase RNATranscription/chromatin structure regulators Post-transcriptional regulators Protein function modulators RNA/protein localization regulatorsRNA Transcripts miRNA siRNA piRNA lncRNAProtein-coding mRNA Non-coding RNA Transcripts snoRNAsHousekeeping RNAs Regulatory RNAs micro (mi) RNAmicro (mi) RNASmall RNAs (~ 22 nucleotides in mature form). Encoded within regions of non protein-coding DNA or introns of traditional genes. Seed sequence: nucleotide positions 2~8 from the 5′ end of miRNAs, which generally perfectly base‑pair with target mRNA. Mediate Post-transcriptional regulation of messenger RNAs. Complex set of proteins required for formation and processing of miRNA.General knowledge of miRNAsGeneral knowledge of miRNAsFound in wide range of plants and animal species. 1-5% of predicted genes humans (~1000 total). Individual miRNA’s are present at a level of ~1,000 molecules/cell with some reaching 50,000. Estimated that 1/3 of protein coding genes are regulated by miRNA’s. Cells shorten 3’ UTR to dampen miRNA regulation of important genes at certain times.The discovery of miRNAsThe discovery of miRNAsmiRNA was first discovered in 1993 by Victor Ambros and Gary Ruvkun (published at the same issue of Cell). The second miRNA Let-7 was discovered in 2000 by Frank Slack as a postdoc at Harvard (Ruvkun lab).Victor AmbrosGary RuvkunnullMost genes identified from a mutagenesis screens are protein-coding, but lin-4 encodes a 22-nucleotide non-coding RNA that is partially complementary to 7 conserved sites located in the 3'-untranslated region (UTR) of the lin-14 gene.The first discoered miRNA: lin-4He L et al., Nature Review in Genetics, 2004, 5:522-531nulllin-4 vs lin-14: the first miR-target coupleThought to be an oddity not a general phenomenon.He L et al., Nature Review in Genetics, 2004, 5:522-531nullProcessing and maturation of miRNAPrinciples of miRNA-mRNA interactionPrinciples of miRNA-mRNA interactionWhy all the fun for down-regulation? miRNA mediated TNF-alpha upregulation!Why all the fun for down-regulation? miRNA mediated TNF-alpha upregulation!Vasudevan S. et al. Science 2007, 318:1931-1934nullSince each miRNA may regulate as many as 200 different gene targets, overexpression or loss of function of miRNAs may have widespread oncogenic effects because many genes will be dysregulated. Overexpression of some miRNAs can suppress the expression of tumor-suppressor gene targets, whereas loss of function of other miRNAs may allow overexpression of the oncogenes they regulate. Approximately 10% of miRNAs have been found to be either greatly overexpressed or down-regulated in various tumors, and are referred to as oncomirs.miRNA and diseasenullGarzon R et al., Nature Reviews in Drug Discovery, 2010, 9:775-789miRNAs as oncogenes and tumor suppressorsDysregulated expression of miRNAs in tumorigenesisDysregulated expression of miRNAs in tumorigenesisCho WC et al. Int J Biochem Cell Biol 2010; Cho WC et al.. Mol Cancer. 2007;6:60.Dysregulated expression of miRNAs in tumorigenesisDysregulated expression of miRNAs in tumorigenesisCho WC et al. Int J Biochem Cell Biol 2010; Cho WC et al.. Mol Cancer. 2007;6:60.nullmiRNA expression pattern changes during oncogenesis, and is unique for each cancernullmiR-34 family as mediator of tumor suppression by p53Hermeking H et al., Cancer Cell, 2007, 12:414-418nullMa L et al., Nature cell biology, 2010, 247miR-9, a MYC/MYCN activated microRNA, regulates E-cadherin and cancer metastasisCellular mRNA targets of viral miRNAsCellular mRNA targets of viral miRNAsCullen BR, Nature, 2009, 457:421-425Some viruses encode miRNAsnullNobel Prize in Physiology or Medicine 2006Andrew Z. Fire Craig C. MelloC. elegansmiRNAmiRNAExogenously delivered 21-23mer dsRNA Acts through RISC Induces homologous target cleavage Perfect sequence match Results in target degradation Endogenously produced 21-23mer dsRNA Acts through RISC Induces homologous target cleavage Imperfect sequence match Results in target degradation or translation arrestsiRNAA comparison between miRNA and siRNAnullA comparison between miRNA and siRNAHe L, Nature Review in Genetics, 2004, 5:522-531nulllncRNA: long non-coding RNATranscripts longer than 200 nucleotides that have little or no protein-coding capacity. Long ncRNAs can regulate gene expression through a diversity of mechanisms.Genomic organiztion of coding and non-coding transcriptsMercer TR, Nature Reviews in Genetics, 2009, 10:155-159nullFunctions of lnRNAsMercer TR, Nature Reviews in Genetics, 2009, 10:155-159nullncRNAs mediated X chromosome inactivationnulllnRNA HOTAIR reprograms chromatin state to promote cancer metastasisGupta RA, Nature, 2010, 464:1071-1076nullRate limiting stepamyloid precursor proteinBACE1-AS antisense RNA and Alzheimer’s DiseasenullChr.11(104 nts complete match)BACE1-AS upregulates the expression of BACE1Faghihi MA, Nature Medicine, 2008: 14, 723-730Dysregulation of BACE1 and Aβ production in Alzheimer's diseaseDysregulation of BACE1 and Aβ production in Alzheimer's diseaseSt George-Hyslop P, Nature Medicine, 2008: 14, 711 - 712nullPosttranscriptional regulation of PTEN dosage by noncoding RNAHe L, Science Signaling, 2010, 3: pe39p53 mRNA controls p53 activity by managing Mdm2 functionsp53 mRNA controls p53 activity by managing Mdm2 functionsCandeinas MM et al. Nature cell biology, 2008, 10:1098Clinical application of non-coding RNAsClinical application of non-coding RNAsAlnylam Pharmaceuticals recently passed phase II human clinical trials showing statistically significant treatment of human RSV with siRNA Many other early clinical trials beginning Many siRNA’s have worked in animal models such as recently, at Harvard’s Immune Disease Institute, Ab and polyarginine conjugated siRNA’s blocked HIV replication in humanized mice.Strategies for miRNA based therapiesStrategies for miRNA based therapiesGarzon R, Nature Reviews in Drug Discovery, 2010, 9:775-789nullTherapeutic silencing of miR-10b inhibits metastasis in a mouse mammary tumor modelMa L, Nature Biotechnology, 2010, 28:341-347Future Directions – RNA therapyFuture Directions – RNA therapySMALL (small enough to use as a therapeutic?) Would function as an entirely new class of therapy Goal: To selectively inhibit viral or oncogenic protein transcription/translation Goal 1a: antisense small RNA to soak up detrimental RNA (such as miR-155 in lymphomas) Goal 1b: Could be used to regulate metabolic or inflammatory disorders with more understanding Summary – RNA’s canSummary – RNA’s canDown-regulate transcription/translation Up-regulate transcription/translation Protect from viruses/transposons Serve in epigenetic inheritance (via histone patterns) Signaling Molecule?Perhaps the rabbit hole is deeper than what was originally imagined?nullRNA worldnullThank youEmail: mzhuong@QQ.com