from Wu and Morris, Curr.Opin.Genet.Dev. 9, 237 1999

1
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)
2
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 Wu and Morris, Curr.Opin.Genet.Dev. 9, 237
(1999)
3
dsRNA Activates Protein Kinase R in Vertebrates
Long dsRNA induces interferon response in
vertebrates
PKR prosphorylates 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
from Everett and McFadden, Trends Microbiol. 7,
160 (1999)
4
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)
5
Biogenesis of miRNAs and siRNAs
miRNAs are genomically encoded
siRNAs are produced exogenously or from
bidirectionally transcribed RNAs
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
miRNAs have imperfect complementarity to their
target mRNA and inhibit translation
siRNAs form perfect duplex with their target
mRNA and trigger mRNA degradation
from Li and Hannon, Nature Rev.Genet. 5, 522
(2004)
6
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)
7
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
from Chen and Rajewsky, Nature Rev.Genet. 8, 93
(2007)
8
Domain Structures of Dicer Enzymes
Dicer generates mature miRNA and siRNA in the
cytoplasm
In Arabidopsis, DCL-1 contains NLS and processes
pri-miRNA
from Li and Hannon, Nature Rev.Genet. 5, 522
(2004)
9
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
The undifferentiated state of malignant cells is
correlated with a decrease in miRNA expression
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
from He et al., Nature 435, 828 (2005)
10
Does This Make Us Human?
HAR1-HAR49 are sequences that are highly
conserved among mammals, but have exhibited
rapid, recent sequence divergence
Most HARs occur outside protein coding regions
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
from Pollard et al., Nature 443, 167 (2006)
11
Regulation of siRNA Levels in C. elegans
RNA-dependent RNA polymerase amplifies siRNA
RRF-3 prevents siRNA amplification
ERI-1 is an siRNA-specific RNase
from Timmons, BioEssays 26, 715 (2004)
12
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)
13
DNA Vector-based RNAi
from Shi, Trends Genet. 19, 9 (2003)
14
Alphavirus-mediated RNAi
DNA inserted into an infectious cDNA silences
genes homologous to the insert
Production of 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