Examples of Homologydependent Gene Silencing TGS Pairing of

Examples of Homology-dependent Gene Silencing TGS – Pairing of tightly linked homologous loci induces methylation Transcriptional Gene Silencing PTGS – Transcript-specific degradation Post-transcriptional Gene Silencing SAS – Spread of PTGS Systemic Acquired Silencing RIP – Induction of C-T transitions Repeat-induced Point Mutation RNAi RNA interference from Wu and Morris, Curr. Opin. Genet. Dev. 9, 237 (1999)

Models for 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 Wu and Morris, Curr. Opin. Genet. Dev. 9, 237 (1999)

Response of Mammalian Cells to Long ds. RNA induces interferon response in vertebrates PKR phosphorylates e. IF 2 a to inhibit translation 2’-5 -oligoadenylate synthase is induced, which activates RNase. L and leads to nonspecific m. RNA degradation si. RNA does not invoke the interferon response from Mc. Manus and Sharp, Nature Rev. Genet. 3, 737 (2002)

The lin-14 Mutant has an Altered Pattern of Cell Division The PNDB neuroblast is generated prematurely The LIN-14 protein prevents L 2 -type cell divisions from Lodish et al. , Molecular Cell Biology, 6 th ed. Fig 21 -6

mi. RNAs Regulate Development in C. elegans The LIN-14 protein prevents L 2 -type cell divisions During L 2, lin-4 mi. RNA prevents translation of lin-14 m. RNA 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 from Lodish et al. , Molecular Cell Biology, 6 th ed. Fig 21 -6

lin-4 Inhibits Translation of lin-14 m. RNA 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 micro. RNA that is partially complementary to sites in the 3’UTR of lin-14 m. RNA Annealing of lin-4 to lin-14 m. RNA inhibits translation from Li and Hannon, Nature Rev. Genet. 5, 522 (2004)

Biogenesis of mi. RNAs and si. RNAs mi. RNAs are genomically encoded si. RNAs are produced exogenously or from bidirectionally transcribed RNAs Drosha processes pri-mi. RNA to pre-mi. RNA in the nucleus mi. RNA is selectively incorporated into the RISC for target recognition Guide strand of si. RNA is incorporated into the RISC for target recognition mi. RNAs have imperfect complementarity to their target m. RNA and inhibit translation si. RNAs form perfect duplex with their target m. RNA and trigger m. RNA degradation from Li and Hannon, Nature Rev. Genet. 5, 522 (2004)

Triggers of RNAi-Mediated Gene Silencing in Mammals from Mittal, Nature Rev. Genet. 5, 355 (2004)

Generation of mi. RNAs in Plants and Animals In plants, mi. RNA maturation occurs in the nucleus In animals, pre-mi. RNA is formed in the nucleus and mature mi. RNA occurs in the cytoplasm from Chen and Rajewsky, Nature Rev. Genet. 8, 93 (2007)

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)

Domain Structures of Dicer Enzymes Dicer generates mature mi. RNA and si. RNA in the cytoplasm In Arabidopsis, DCL-1 contains NLS and processes pri-mi. RNA from Li and Hannon, Nature Rev. Genet. 5, 522 (2004)

Strand Selection of Processed si. RNA into the RISC The PAZ domain of Dicer binds to the pre-existing ds. RNA end The strand that has its 3’-end bound to the PAZ domain preferentially assembles into the RISC from Sontheimer, Nature Rev. Mol. Cell Biol. 6, 127 (2005)

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 si. RNA from Parker and Barford, Trends Biochem. Sci. 31, 622 (2006)

The Fate of m. RNA Loaded With the mi. RISC Targeted m. RNA accumulates in P bodies m. RNA 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 si. RNA triggers endonucleolytic cleavage of perfectly-matched complementary targets Cleavage is catalyzed by Argonaute proteins The resulting m. RNA fragments are degraded mi. RNA triggers accelerated deadenylation and decapping of partially-complementary targets and requires Argonaute proteins and a P-body component mi. RNA represses translation from Eulalio et al. , Nature Rev. Mol. Cell Biol. 8, 9 (2007)

Regulation of si. RNA Levels in C. elegans RNA-dependent RNA polymerase amplifies si. RNA RRF-3 prevents si. RNA amplification ERI-1 is an si. RNA-specific RNase from Timmons, Bio. Essays 26, 715 (2004)

Prevalence of and Regulation by mi. RNAs At least 800 mi. RNA-encoding genes in humans At least 5000 genes are regulated by mi. RNAs

Organismal Complexity May Be Due to Differences in Regulation of Gene Expression Number of protein-coding genes are similar in animals mi. RNA complexity correlates with an increase in morphological complexity from Technau, Nature 455, 1184 (2008)

Human Accelerated Regions HAR 1 -HAR 49 are sequences that are highly conserved among mammals, but have exhibited rapid, recent sequence divergence Most HARs occur outside protein coding regions HAR 1 F RNA can fold into a stable stem-loop secondary structure HAR 1 F is expressed in the developing neocortex, a region important in directing brain development and neuronal migration from Pollard et al. , Nature 443, 167 (2006)

A Micro. RNA Regulates Neuronal Differentiation by Controlling Alternative Splicing mi. R-124 targets a component of a repressor of neuron-specific genes mi. R-124 results in reduced expression of PTBP 1 leading to the accumulation of PTBP 2 results in a global switch to neuronspecific alternative splicing patterns from Makeyev et al. , Mol. Cell 27, 435 (2007)

The Role of mi. RNA in Cancer mi. RNA profiles define the cancer type better than expression data from 16, 000 m. RNAs mi. RNA expression is lower in cancers than in most normal tissues Down-regulation of all mi. RNAs enhanced tumor growth The undifferentiated state of malignant cells is correlated with a decrease in mi. RNA expression c 13 orf 25 mi. RNA is the first non-coding oncogene, is upregulated by c-Myc, and is involved in leukemia development c 13 orf 25 inhibits expression of E 2 F 1, a cell cycle regulator from He et al. , Nature 435, 828 (2005) Lu et al. , Nature 435, 834 (2005)

mi. RNAs Suppress Breast Cancer Metastasis Loss of mi. R-126 and mi. R-355 when human breast cancer cells develop metastatic potential Restoring expression of these mi. RNAs in malignant cells suppresses metastasis in vivo mi. R-355 targets the progenitor cell transcription factor, SOX 4, and the ECM component, tenascin C from Tavasoie et al. , Nature 451, 147 (2008)

A Micro. RNA is Involved in Metastasis Twist induces mi. R-10 b transcription mi. R-10 b inhibits HOXD 10 translation which increases RHOC expression mi. R-10 b levels are elevated in metastasis-positive patients from Steeg, Nature 449, 671 (2007)

Role of Micro. RNAs and Epigenetics in Cancer EZH 2 overexpression promotes cell proliferation Expression of EXH 2 is inhibited by mi. R-101 expression decreases during prostate cancer progression from Varambally et al. , Science 322, 1695 (2008)

Immunostimulatory Effects of ds. RNA Long ds. RNA induces PKR Toll-like receptors in endosomes recognize ds. RNA and activate the interferon response Blunt-ended ds. RNA are recognized by RIG-1 helicase and activates the immune response from Kim and Rossi, Nature Rev. Genet. 8, 173 (2007)

DNA Vector-based RNAi from Shi, Trends Genet. 19, 9 (2003)

The Design of Optimal si. RNAs 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 m. RNA from Mittal, Nature Rev. Genet. 5, 355 (2004)

Microarray-based Screening for Effective si. RNA Transfer a mixture with si. RNA, target m. RNA fused with EGFP, and control RFP construct to a glass slide Overlay with a cell monolayer and transfect Effective si. RNA suppresses EGFP expression, but not RFP expression from Mittal, Nature Rev. Genet. 5, 355 (2004)

Delivery of si. RNA for Therapy si. RNA is not taken up by most mammalian cells Cholesterol-conjugated si. RNA is taken up by the LDL receptor si. RNA bound to targeted antibody linked to protamine can achieve cell-specific si. RNA delivery from Dykxhoorn and Lieberman, Cell 126, 231 (2006)

Cell-Specific Delivery of si. RNA Fuse Fab targeting antibody with protamine si. RNA binds noncovalently with protamine Complex is endocytosed into cells expressing the epitope si. RNA is released from the endosome and enters the RISC from Rossi et al. , Nature Biotechnol. 23, 682 (2005)

Non-specific si. RNA Inhibition of Angiogenesis si. RNAs targeting VEGF and VERGR 1 are effective treatments for macular degeneration Non-specific si. RNAs are also effective si. RNA inhibits angiogenesis by activating the TLR 3 signalling cascade from Kalluri and Kanasaki, Nature 452, 543 (2008)

Alphavirus-mediated RNAi DNA inserted into an infectious c. DNA silences genes homologous to the insert Dengue virus-resistant mosquitoes are produced by inoculation of Aedes aegypti with ds. SIN 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 ds. RNA 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 si. RNA activates or recruits Clr 3 methyltransferase that methylates H 3 on K 9 Deletion of RNAi pathway genes cause loss of silencing at centromeres and reduced H 3 K 9 methylation at centromeric regions from Allshire, Science 297, 1818 (2002)

Small RNAs Modulate Viral Infection Viral-encoded mi. RNA facilitate viral infection and persistence Host cell-encoded mi. RNAs inhibit or facilitate viral replication Viral suppressors of RNA silencing (VSR) inhibit the RNAi pathway

Function of SV 40 mi. RNA is synthesized late in the viral life cycle and targets TAg m. RNA SV 40 mi. RNA aids immune invasion by reducing susceptibility to lysis by CTLs from Sarnow et al. , Nature Reviews Microbiol. 4, 651 (2006)

Effects of Adenovirus VA 1 Micro. RNA VA 1 binds to and prevents PKR activation to inhibit the innate immune response VA 1 competes with exportin-5 and inhibits Dicer to inhibit the RNAi pathway from Sarnow et al. , Nature Reviews Microbiol. 4, 651 (2006)

A Micro. RNA Protects HSV-1 -infected Neurons from Apoptosis LAT is the only viral gene expressed during latent infection in neurons mi. R-LAT is generated from the LAT gene mi. R-LAT downregulates TGF-b and SMAD 3 and contributes to the persistence of HSV-1 in neurons in a latent form from Gupta et al. , Nature 442, 82 (2006) Paper retracted – 2008. Repeatedly unable to detect mi. RNA

Cellular mi. RNAs Modulates Viral Infection PFV-1 replication is stimulated by a plant VSR implicating the role of small RNAs in the viral life cycle mi. R-32 inhibits viral replication Tas is a PFV-1 -encoded protein that inhibits RNAi from Sarnow et al. , Nature Reviews Microbiol. 4, 651 (2006) mi. R-122 increases HCV replication in the liver

Features of pi. RNAs Piwi and Aubergine complexes contain pi. RNAs antisense to transposon m. RNAs Argonaute 3 complexes contain pi. RNAs biased to the sense strand of transposon m. RNAs from Aravin et al. , Science 318, 761 (2007) pi. RNAs display 10 nt complementarity at their 5’-ends

Model for Biogenesis of pi. RNAs that Target Mobile Elements Pool of pi. RNAs bound to Piwi or Aubergine anneals to transposon m. RNA target Cleave transposon m. RNA 10 nt from 5’-end of associated pi. RNA to create 5’-end of Ago 3 pi. RNA Ago 3 -associated pi. RNA anneals to pi. RNA cluster transcript to create additional copies of antisense pi. RNA Transposon is silenced from Aravin et al. , Science 318, 761 (2007)