novel regulators of gene expression and genome integrity课件

THE FAMILY OF SMALL NON-CODING RNAs “silencing” Novel regulators of gene expression and genome integrity,Inhibition of translation of mRNA into protein Degradation of messenger RNAs Genome defenders (Virus-Transposons) Chromatine organization,BREAKTHROUGH OF THE YEAR (December 2002),SMALL RNAs: REGULATORS AND GUARDIANS OF THE GENOME,piRNAs,miRNAs,siRNAs,THEY COLLECTIVELY FUNCTION AS SEQUENCE-SPECIFIC GUIDES TO SILENCE OR REGULATE GENES, TRANSPOSONS, AND VIRUSES AND TO MODIFY CHROMATIN AND GENOME STRUCTURE.,(PIWI-ASSOCIATED),(MICRO RNA),(SHORT INTERFERING RNA), 21 nt, 21 nt, 30 nt,Small non-coding RNAs,Long non-coding RNAs,20-35 nt,(Telomerase RNA),500 nt,17 kb 108 kb,(Human Xist) (mouse Air RNAs),(siRNA, piRNA, miRNA),The small non-coding RNA group,THE SMALL INTERFERING RNAs - THE GENOMES IMMUNE SYSTEM -,Nature abhors double-stranded RNA. When confronted with double-stranded RNA (dsRNA), eukaryotic cells respond eliminating their own mRNAs that share sequences with the double strand.,Sources of siRNA are dsRNA that are typically hundreds of base pair long (viral- or transposon-derived replication intermediates).,Fire, A. et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391, 806811 (1998).,THE SMALL INTERFERING RNAs (siRNA),Craig Mello Andrew Fire Nobel Prize 2006 in Physiology and Medicine,RNAi was first discovered in C. elegans as a response to exogenous dsRNA,RNA- INDUCED SILENCING COMPLEX,dsRNA-specific RNase III (Dicer),Guide strand,Biogenesis of Effector siRNAs,Passenger strand,siRNAs,ENDOgenous,EXOgenous,natsiRNAs (natural),tasiRNAs (trans-acting),Transgenes Virus,EXO- AND ENDO- SMALL INTERFERING RNAs,RNAi can be elicited exogenously, by dsRNA supplied from outside the cell, or endogenously, from transcription of coding or noncoding genomic sequences,casiRNAs (cis-acting),Plants worms,Transposons, repetitive elements,Heterochromatin formation by DNA methylation, histone modification,Transcript (mRNA) cleavage,Transcript (mRNA) cleavage (stress reponses),Flies Mammals,Endo-siRNAs,Derived from transposons, heterochromatic sequences, intergenic regions, mRNAs,Transcript cleavage and chromatin organization,FORMATION AND ORIGIN OF ENDO-siRNAS,ARGONAUTE PROTEINS AND SMALL RNA EFFECTORS FUNCTIONS,PIWI domain is a RNAse-H like moiety (Slicer) PAZ domain identify the 3OH end of small RNA,THE ARGONAUTE FAMILY Prime components of small RNA effector complexes Defined by 3 main domains : PAZ (Piwi-Argonaute-Zwile) PIWI (P-element Induced Wimpy Testis) MID (Midle),PIWI-ASSOCIATED SMALL RNAS - piRNAs-,The ARGONAUTE family can be divided into 2 subfamilies: Ago subfamily (Arabidopsis) Expressed ubiquitously and associated in silencing complex of siRNA and miRNA Piwi subfamily (Drosophila) Expression restricted to Germinal and Stem cells (human and mouse orthologs are dubbed Hiwi and Miwi),Piwi proteins associate with 27-31 nt small RNAs which match to genomic repeats and transposable DNA elements,Piwi genes were originally identified as encoding regulatory proteins responsible for maintaining the undifferentiated state and division rates of germinal stem cells in Drosophila. Highly conserved and present in plants, animals and prokaryotes,PIWI-ASSOCIATED SMALL RNAS - piRNAs-,A distinct class of 30 nt RNAs associated with Piwi-like proteins Dicer independent biogenesis Described in flies, fish and mammals Essentials in maintaining germline and stem cell DNA integrity,http:/pirnabank.ibab.ac.in/ piRNAs registred: human 24.000, mouse 40.000 , rat 40.000,PIWI-ASSOCIATED SMALL RNAS - piRNAs-,piRNAs assures the primary control of chromosome structure, chromatin organization, gene transcription, RNA stability and RNA translation,THE MICRORNA (miRNA) FAMILY - a novel class of small non-coding RNA molecules -,5,37,105,211,410,697,1052,1745,1750,2080,Mutations in heterochronic genes cause temporal transformations in cell fates in which stage-specific events are omitted or reiterated.,THE FIRST MICRO-RNAs - Small Temporal RNA (stRNA) -,Heterochronic genes do not encoding proteins but small RNA transcripts of 21nt and 70nt.,lin-4 (Horvitz et al. 1980; Lee et al. 1993) let-7 (Reinhard et al, 2000),The first microRNA,The let-7 RNA is conserved across all the bilaterian phylogeny of metazoans (Pasquinelli et al., 2000),BLASTN searches reveal one gene for D. melanogaster or C. elegans, and three segments from the human genome sequence on chromosomes 9, 11 and 22 bearing exact sequence matches.,Small Temporal RNAs,- Conservation of let-7 sequence and expression in humans -,THE MICRORNA (miRNA) FAMILY - a novel class of small non-coding RNA molecules -,To date there are more than 1000 human microRNA genes (http:/microrna.sanger.ac.uk/ sequences/index.shtml) miRNAs induce degradation or translational inhibition of target mRNAs after binding to the untranslated 3UTRs. miRNAs are estimated to comprise 2% of animal genes and regulate more than 20% of genes,miRNAs TRANSCRIPTION AND MATURATION,SHARED BIOSYNTHETIC PATHWAYS OF siRNAS AND miRNAS,THE HUMAN miRNAs GENES,Approx. 30% of miRNA genes are in intergenic regions Approx. 70% of miRNA genes are located in defined transcription units,75%,25%,1%,GENOMIC LOCALIZATION OF miRNAs GENES,Canonical miRNA genes,Non-canonical miRNA genes (mirtrons),MECHANISMS OF miRNA-MEDIATED TRANSLATIONAL DOWN-REGULATION,Inhibition of translational initiation by interfering with eIF4E binding,1. INTERFERENCE WITH THE TRANSLATIONAL MACHINERY,Post-initiation blockage of (polysome associated),Sheth, U. & Parker, R. (2003) Decapping and decay of messenger RNA occur in cytoplasmic processing bodies. Science 300, 805808.,From yeast to mammals a major route for mRNA degradation proceeds in cytoplasmic P-bodies:,Main Components of P-bodies Decapping and desadenylating enzymes and cofactors Exonucleases Ago proteins Untranslated mRNA,2. microRNA SEQUESTRATION OR DEGRADATION INTO P-BODIES,MECHANISMS OF miRNA-MEDIATED TRANSLATIONAL DOWN-REGULATION,GENE-EXPRESSION PROFILES (cDNA microarrays) can only be used to interpret cellular changes that affect mRNA synthesis but not real protein levels,MicroRNA PROFILING microRNA profiling expression provides information about translation efficiency of transcripts detected by cDNA microarrays,PROTEOMICS It should identify the real profile of proteins including their post-translational modifications,PRINCIPLES OF miRNA-mRNA RECOGNITION,Brennecke, J. et al. (2005) PLoS Biol 3(3): e85.,Complementarity of seven or more bases to the 5 end miRNA is sufficient to confer regulation Sites with weaker 5 complementarity require compensatory pairing to the 3 end to be functional Extensive pairing to the 3 end of without 5 pairing is not functional,GENOME-WIDE ANALYSIS OF miRNA CONTROL,miRNAs are estimated to comprise 1-2% of animal genes (most abundant classes of regulators of gene expression) 20 to 30% of all human genes are targets of miRNA regulation (100-200 mRNA per miRNA),“ANTITARGETS” Involved in basic processes common to all cells,“TARGETS” Involved in developmental timing, cell proliferation, apoptosis, metabolism, cell differentiation and morphogenesis,Selective pressure to avoid miRNA regulation segregates mRNAs into,VIRUS-ENCODED miRNAs AND VIRAL PATHOGENESIS,All three Herpesvirus subfamilies have been found to encode miRNAs,-herpesvirus Epstein Barr virus (EBV) 23 miRNAs Kaposis sarcoma-associated herpesvirus (KSHV) 12 miRNAs -herpesvirus Human cytomegalovirus (HCMV) 11 miRNAs -herpesvirus Herpes simplex virus 1 (HSV-1) 6 miRNAs Adeno/polioma viruses 1-2 miRNAs,miRNA-producing DNA viruses,Biosynthetic cellular pathways of virus-derived miRNAs,RNA viruses have been reported to lack miRNAs,The controversy of HIV The HIV transactivating response (TAR) transcript has secondary structure features that suggesting that it may act as a pre-miRNA Another miRNA derived from the HIV-1 nef gene,VALIDATED TARGETS OF VIRAL miRNAs,VIRUS-ENCODED miRNAs AND VIRAL PATHOGENESIS,Several viruses have now been shown to reshape the cellular environment by reprogramming the hosts RNA-interference machinery Viral microRNAs are particularly important for regulating the transition from latent to lytic replication and for attenuating antiviral immune responses,tRNA-DERIVED SMALL RNAs - A new family of regulatory small RNAs? -,Filtering of deep sequencing data reveals the existence of abundant Dicer-dependent small RNAs derived from tRNAs COLE C., et al. RNA (2009),Deep sequencing revealed that 5 halves from tRNAs was the most abundant small RNA population,The tRNAs are almost exclusively processed from the 5 end with cleavage by Dicer at the D-loop resulting in the generation of 19 nt-long fragments Increased levels of tRNAs might force Dicer to accept tRNAs as substrate (modulation of miRNA biogenesis?),In HeLa cells the most abundant small tRNAs were derived from tRNALys, tRNAVal, tRNAGln and tRNAArg,Deep sequencing revealed that 5 halves from tRNAs was the most abundant small RNA population,Hidden layers of human small RNAs Kawaji, H. et al. BMC Genomics 2008, 9:157, 21 nt, 32 nt,Dicer,Angiogenin,Are miRNA and small tRNA pathways connected? - Small tRNAs vs microRNAs -,tRNA-DERIVED SMALL RNAs - A new family of regulators small RNAs? -,THE tRNase Z FAMILY OF PROTEINS,Precise trimming and processing of precursor tRNA molecules requires: removal of the 5 leader sequences by the ubiquitous enzyme RNase P removal of the 3 trailer sequences by tRNase Z the modification of tRNA nucleotides intron removal,EXTERNAL GUIDE SEQUENCES (EGS) TARGET TrzL CLEAVAGE,tRNase ZL can cleave any target RNA at any desired site by recognizing a pre-tRNA-like or micro-pre-tRNA-like complex formed between the target RNA and small guide or external guide sequence RNA (sgRNA).,piRNA-like ncRNAs co-immunoprecipitate with human tRNase ZL 5-half-tRNAGlu was predominant,Genuine targets of tRNase ZL-guided by 5-half-tRNAGlu revealed by microarrays,p53 SIGNALING PATHWAYS,Genuine targets of tRNase ZL-guided by 5-half-tRNAGlu revealed by microarrays,APOPTOTIC PATHWAYS,miRNAs CAN BE USED BY tRNase Z AS GUIDES TO TARGET mRNAS,Human cytosolic tRNase ZL can downregulate gene expression through miRNA Elbarbary R.A., et al. FEBS Letters (2009) 583:3241,A subset of free miRNAs forming hook structures can guide tRNase Z to target mRNAs,Many off-target mRNA reductions induced by siRNA may be attributable to tRNase Z (Ago2-independent processes) The 5 seven or eight nt of miRNA (seed) are important for target recognition in both mechanisms using tRNase ZL and RISC.,Asp 900.000 reads,Asp 900.000 reads,TUMOR,NORMAL,DEREGULATED PATTERN OF stRNA IN CANCER ?,Deep sequencing of small RNAs in breast cancer samples,EXOSOME-MEDIATED INTERCELLULAR TRANSFER OF mRNAs AND microRNAs EXOSOMAL SHUTTLE RNA (ESRNA),Novel mechanisms of genetic exchange between cells and intercellular comunication,Valadi, H., et al. (2007) Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654-659.,“exosomes contain both mRNA and microRNA, (no DNA) which can be delivered to another cell, and can be functional in this new location”,Different types of secreted membrane vesicles,Microvesicles Exosome-like Exosomes,Circulating plasma microvesicles containing miRNAs, mRNA and proteins were demonstrated in humans and other mammals They contribute to biological homeostasis by an unknown mechanism The actual composition of plasma microvesicles is shaped by the contribution of several kind of cells dispersed through the body.,A novel mode of intercellular communication, where the units of information are microvesicles which could contribute to the equilibrium Health vs. Disease,Plasma circulating microvesicles in cancer , diabetes and other human diseases revealed the presence of oncogenes and cancer-related miRNAs and mRNAs.,Potential of circulating microRNAs as prognostic and predictive biomarkers in human disease.,BIOMEDICAL RELEVANCE,microRNA THERAPEUTICS Antagomirs and Promirs: a new niche for nucleic acid-mediated therapies,Delivery of sRNA for therapy,miRNA-1 EXCEPTIONAL MUSCULARITY OF THE BELGIAN TEXEL SHEEP,A point mutation that creates an illegitimate microRNA target site in the 3 UTR of myostatin Inhibits its expression and contributes to the muscular hypertrophy of Texel sheep.,MicroARNs Y CNCER,The microRNA-10