转录后基因沉默posttranscriptional gene

from Wu and Morris, Curr.Opin.Genet.Dev. 9, 237 (1999) Examples of Homology-dependent Gene Silencing TGS Pairing of tightly linked homologous loci induces methylation PTGS Transcript-specific degradation SAS Spread of PTGS RIP Induction of C-T transitions RNAi from Wu and Morris, Curr.Opin.Genet.Dev. 9, 237 (1999) Models for Transvection Transvection is an alteration of gene function by homologous pairing trans action of an element on a paired homolog Propagation of chromatin structure to a paired homolog Pairing-sensitive silencing that acts at the level of chromatin RNA triggers silencing at paired homologs Pairing of dissimilar homologs causes a topological change from McManus and Sharp, Nature Rev.Genet. 3, 737 (2002) Response of Mammalian Cells to Long dsRNA Long dsRNA induces interferon response in vertebrates PKR phosphorylates eIF2a to inhibit translation 2-5-oligoadenylate synthase is induced, which activates RNaseL and leads to nonspecific mRNA degradation siRNA does not invoke the interferon response The lin-14 Mutant has an Altered Pattern of Cell Division The PNDB neuroblast is generated prematurely The LIN-14 protein prevents L2-type cell divisions from Lodish et al., Molecular Cell Biology, 6th ed. Fig 21-6 from Lodish et al., Molecular Cell Biology, 6th ed. Fig 21-6 The LIN-14 protein prevents L2-type cell divisions During L2, lin-4 miRNA prevents translation of lin-14 mRNA In the adult, let-7 inhibits lin-14 and lin-41 translation Absence of LIN-41 permits lin-29 translation and generation of adult cell lineages miRNAs Regulate Development in C. elegans from Li and Hannon, Nature Rev.Genet. 5, 522 (2004) lin-4 Inhibits Translation of lin-14 mRNA Mutations in lin-4 disrupt regulation of larval development in C. elegans lin-4 antagonizes lin-14 function lin-4 encodes a 22 nt-long microRNA that is partially complementary to sites in the 3UTR of lin-14 mRNA Annealing of lin-4 to lin-14 mRNA inhibits translation from Li and Hannon, Nature Rev.Genet. 5, 522 (2004) Biogenesis of miRNAs and siRNAs miRNAs are genomically encoded siRNAs are produced exogenously or from bidirectionally transcribed RNAs miRNAs have imperfect complementarity to their target mRNA and inhibit translation siRNAs form perfect duplex with their target mRNA and trigger mRNA degradation Drosha processes pri-miRNA to pre-miRNA in the nucleus miRNA is selectively incorporated into the RISC for target recognition Guide strand of siRNA is incorporated into the RISC for target recognition Triggers of RNAi-Mediated Gene Silencing in Mammals from Mittal, Nature Rev.Genet. 5, 355 (2004) from Chen and Rajewsky, Nature Rev.Genet. 8, 93 (2007) Generation of miRNAs in Plants and Animals In plants, miRNA maturation occurs in the nucleus In animals, pre-miRNA is formed in the nucleus and mature miRNA occurs in the cytoplasm Strand Selection Into the RISC The strand with its 5-terminus at the less stable end of the duplex is incorporated into the RISC from Sontheimer, Nature Rev.Mol.Cell Biol. 6, 127 (2005) from Li and Hannon, Nature Rev.Genet. 5, 522 (2004) Domain Structures of Dicer Enzymes Dicer generates mature miRNA and siRNA in the cytoplasm In Arabidopsis, DCL-1 contains NLS and processes pri-miRNA Strand Selection of Processed siRNA into the RISC from Sontheimer, Nature Rev.Mol.Cell Biol. 6, 127 (2005) The PAZ domain of Dicer binds to the pre-existing dsRNA end The strand that has its 3-end bound to the PAZ domain preferentially assembles into the RISC Guide RNA Loading Onto Argonaute PAZ domain binds 3-overhang 5-end of guide RNA is anchored in a conserved pocket of the PIWI domain Argonaute slices passenger strand of siRNA from Parker and Barford, Trends Biochem.Sci. 31, 622 (2006) The Fate of mRNA Loaded With the miRISC Targeted mRNA accumulates in P bodies mRNA is stored in P bodies, undergoes degradation, or reenters the translation pathway from Rana, Nature Rev.Mol.Cell Biol. 8, 23 (2007) Overview of RNA-Mediated Gene Silencing from Eulalio et al., Nature Rev.Mol.Cell Biol. 8, 9 (2007) siRNA triggers endonucleolytic cleavage of perfectly-matched complementary targets The resulting mRNA fragments are degraded miRNA triggers accelerated deadenylation and decapping of partially-complementary targets and requires Argonaute proteins and a P-body component Cleavage is catalyzes by Argonaute proteins miRNA represses translation siRNA miRNA Regulation of siRNA Levels in C. elegans from Timmons, BioEssays 26, 715 (2004) RNA-dependent RNA polymerase amplifies siRNA RRF-3 prevents siRNA amplification ERI-1 is an siRNA-specific RNase At least 800 miRNA-encoding genes in humans At least 5000 genes are regulated by miRNAs Prevalence of and Regulation by miRNAs Human Accelerated Regions from Pollard et al., Nature 443, 167 (2006) HAR1F RNA can fold into a stable stem-loop secondary structure HAR1F is expressed in the developing neocortex, a region important in directing brain development and neuronal migration HAR1-HAR49 are sequences that are highly conserved among mammals, but have exhibited rapid, recent sequence divergence Most HARs occur outside protein coding regions The Role of miRNA in Cancer miRNA profiles define the cancer type better than expression data from 16,000 mRNAs miRNA expression is lower in cancers than in most normal tissues c13orf25 miRNA is the first non-coding oncogene, is upregulated by c-Myc, and is involved in leukemia development c13orf25 inhibits expression of E2F1, a cell cycle regulator The undifferentiated state of malignant cells is correlated with a decrease in miRNA expression from He et al., Nature 435, 828 (2005) Lu et al., Nature 435, 834 (2005) Down-regulation of all miRNAs enhanced tumor growth Loss of miR-126 and miR-355 when human breast cancer cells develop metastatic potential Restoring expression of these miRNAs in malignant cells suppresses metastasis in vivo miR-355 targets the progenitor cell transcription factor, SOX4, and the ECM component, tenascin C miRNAs Suppress Breast Cancer Metastasis from Tavasoie et al., Nature 451, 147 (2008) from Steeg, Nature 449, 671 (2007) A MicroRNA is Involved in Metastasis Twist induces miR-10b transcription miR-10b inhibits HOXD10 translation which increases RHOC expression miR-10b levels are elevated in metastasis-positive patients Immunostimulatory Effects of dsRNA from Kim and Rossi, Nature Rev.Genet. 8, 173 (2007) Long dsRNA induces PKR Toll-like receptors in endosomes recognize dsRNA and activate the interferon response Blunt-ended dsRNA are recognized by RIG-1 helicase and activates the immune response DNA Vector-based RNAi from Shi, Trends Genet. 19, 9 (2003) from Mittal, Nature Rev.Genet. 5, 355 (2004) The Design of Optimal siRNAs 21 nt RNA that contains 2 nt 3- overhangs and phosphorylated 5-ends Lower stability at the 5-end of the antisense terminus Low stability in the RISC cleavage site Low secondary structure in the targeted region of the mRNA from Mittal, Nature Rev.Genet. 5, 355 (2004) Microarray-based Screening for Effective siRNA Transfer a mixture with siRNA, target mRNA fused with EGFP, and control RFP construct to a glass slide Overlay with a cell monolayer and transfect Effective siRNA suppresses EGFP expression, but not RFP expression from Dykxhoorn and Lieberman, Cell 126, 231 (2006) Delivery of siRNA for Therapy siRNA is not taken up by most mammalian cells Cholesterol-conjugated siRNA is taken up by the LDL receptor siRNA bound to targeted antibody linked to protamine can achieve cell-specific siRNA delivery from Rossi et al., Nature Biotechnol. 23, 682 (2005) Fuse Fab targeting antibody with protamine siRNA binds noncovalently with protamine Complex is endocytosed into cells expressing the epitope siRNA is released from the endosome and enters the RISC Cell-Specific Delivery of siRNA Alphavirus-mediated RNAi DNA inserted into an infectious cDNA silences genes homologous to the insert Dengue virus-resistant mosquitoes are produced by inoculation of Aedes aegypti with dsSIN viruses with Dengue virus inserts Hairpin Dengue virus-specific RNA transcribed from a plasmid generated virus-resistant cells Induction of RNAi pathway in the midgut prior to viral infection Production of genetically modified mosquitoes that transcribe virus-specific dsRNA in response to a blood meal Potential to change vector competence RNAi-dependent Chromatin Silencing in S. pombe Overlapping RNAs from centromeric region is processed into siRNA siRNA activates or recruits Clr3 methyltransferase that methylates H3 on K9 Deletion of RNAi pathway genes cause loss of silencing at centromeres and reduced H3 K9 methylation at centromeric regions from Allshire, Science 297, 1818 (2002) Small RNAs Modulate Viral Infection Viral-encoded miRNA facilitate viral infection and persistence Viral suppressors of RNA silencing (VSR) inhibit the RNAi pathway Host cell-encoded miRNAs inhibit or facilitate viral replication from Sarnow et al., Nature Reviews Microbiol. 4, 651 (2006) SV40 miRNA is synthesized late in the viral life cycle and targets TAg mRNA SV40 miRNA aids immune invasion by reducing susceptibility to lysis by CTLs Function of SV40 miRNA from Sarnow et al., Nature Reviews Microbiol. 4, 651 (2006) Effects of Adenovirus VA1 MicroRNA VA1 binds to and prevents PKR activation to inhibit the innate immune response VA1 competes with exportin-5 and inhibits Dicer to inhibit the RNAi pathway from Gupta et al., Nature 442, 82 (2006) LAT is the only viral gene expressed during latent infection in neurons miR-LAT is generated from the LAT gene A MicroRNA Protects HSV-1-infected Neurons from Apoptosis miR-LAT downregulates TGF-b and SMAD3 and contributes to the persistence of HSV-1 in neurons in a latent form Paper retracted 2008. Repeatedly unable to detect miRNA from Sarnow et al., Nature Reviews Microbiol. 4, 651 (2006) Cellular miRNAs Modulates Viral Infection Tas is a PFV-1-encoded protein that prevents RNAi miR-32 inhibits viral replication PFV-1 replication is stimulated by a plant VSR implicating the role of small RNAs in the viral life cycle miR-122 increases HCV replication in the liver Features of piRNAs Piwi and Aubergine complexes contain piRNAs antisense to transposon mRNAs Argonaute3 complexes contain piRNAs biased to the sense strand of transposon mRNAs piRNAs display 10 nt complementarity at their 5-endsfrom Aravin et al., Science 318, 761 (2007) Model for Biogenesis of piRNAs that Target Mobile Elements Pool of piRNAs bind to Piwi or Aubergine anneals t