Transcript lecture24_RnaInterfe.. - University of Alberta

revisions to the “central
dogma” of molecular biology
genomic
DNA
mRNA1
no introns
protein
isoform1
mRNA2
no introns
protein
isoform2
pre-mRNA
with introns
non-coding
RNA genes
over the last 10 years, scientists have discovered an entirely new category of
non-coding RNA genes whose existence had never even been suspected; they
are distinguished by their small sizes relative to traditional RNA genes (i.e. 21
to 25 bp versus 96 bp) and are therefore commonly referred to as “microRNAs”
The Nobel Prize in Physiology or Medicine 2006
Andrew Z. Fire
Stanford University School
of Medicine
b. 1959
Craig C. Mello
University of Massachusetts
Medical School
b. 1960
for their discovery of RNA interference gene silencing by double-stranded RNA
microRNA biogenesis and action
gene silencing is mediated by short double-stranded RNAs (dsRNAs)
microRNA-mediated gene
silencing is observed in
plants, animals, and many
fungi; it plays essential
roles in development and
virus resistance; it has
already provided powerful
tools for studying gene
function and spawned new
therapeutic industries
Saumet A, Lecellier CH. 2006. Retrovirology 3: 3
biologists working with transgenic plants had long observed a bewildering array of unanticipated
gene silencing phenomena; in 1998, Fire and Mello compared the silencing activity of singlestranded RNAs (ssRNAs) (sense or antisense) with double-stranded (dsRNAs) hybrids; marginal
silencing was achieved by injecting C. elegans with ssRNAs, but potent and specific silencing
was achieved by injecting a sense-antisense mixture; in other words it is the dsRNAs that matter
functional genomics of C. elegans
chromosome 1 by RNA interference
ingestion of bacteria
expressing dsRNAs is
as effective for gene
silencing as the direct
injection of dsRNAs
C. elegans scientists
can silence nearly any
desired gene just by
feeding the worm with
one of these custommodified bacteria they
sell on the Internet
Fraser AG, …, Ahringer J. 2000. Nature 408: 325-330
(notice how few genes have an identifiable phenotype)
evolutionarily conserved genes
are more likely to have a phenotype
percentages for all genes (blue bars) or all genes with RNAi phenotype (red
bars) that have matches in S. cerevisiae (SC), D. melanogaster (DM), human
(HS), all three combined (ALL), or have no matches in any organism (NO M)
commercial libraries are widely
available for most popular organisms
RNA interference identifies over
250 host factors required by HIV-1
Brass AL, …, Elledge SJ. 2008. Science 319: 921-926
genetic defect in CCR5 chemokine receptor
provides immunity against HIV-1 and is/was a
model for development of drug therapeutics
HIV virus
HIV virus
HIV virus
normal
receptor
mutant
receptor
human
cell
DRUG
normal
receptor
human
cell
human
cell
susceptible
non-susceptible
susceptible
PLUS drug

DISEASED

HEALTHY

HEALTHY
RNAi exists because eukaryotic
cells make microRNAs of their own
Griffiths-Jones S, et al. 2008. Nucleic Acids Res 36: D154-D158
the miR-124 regulatory network
gene batteries to global regulators of transcription and alternative splicing
active interactions are solid
black; inactive interactions are
solid gray; weak interactions
are thinner lines; gene battery
expression inputs to cellular
transcriptome are dashed lines;
non-neuronal elements have
dark blue coloring if present or
light blue if absent; neuronal
specific elements are likewise
colored in shades of reds
Makeyev EV, Maniatis T. 2008. Science 319: 1789-1790
Amaral PP, …, Mattick JS. 2008. The eukaryotic
genome as an RNA machine. Science 319: 1787
how many other
RNA mechanisms
are remaining to
be discovered?!?