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MBP1007/1008 - Nucleic Acids
A functional mRNA: The cytoplasmic story
Objectives
(1) To discuss the iNUTS and iBOLTS of how mRNAs
function in the cytoplasm
(2) To review older and introduce newer methods of
studying mRNA biology in the cytoplasm
(3) Understand the basics of RNAi-induced posttranscriptional gene silencing
Breakthrough
of the Year in
2002
Nobel prize in
2006
RNAi: RNA Interference
Mechanism of post-transcriptional gene
silencing mediated by double-stranded
RNA molecules
Small RNA molecules target mRNAs for
degradation or translational
repression based upon sequence
specificity
A classic paper
A classic paper
In 1998, Fire and Mello reported that
dsRNA was a potent trigger for gene
silencing in the nematode worm C.
elegans (Nature 1998, 391:806)
Effect was specific and not generalized
Revelation because dsRNA is inherently very
stable and incapable of further WatsonCrick base-pairing
Passed to neighbouring and daughter cells:
mitosis and meiosis
RNAi in C. elegans
Wild-type
RNAi-deficient
Wild type and RNAi-deficient worms
fed on bacteria expressing GFP dsRNA
DNA versus RNA!
Structure of RNA versus DNA
Life of dsRNA
Life of ssRNA
Sense and antisense genes
Antisense approaches to inhibit gene
expression
RNA - G:U
RNA: Primary, secondary and tertiary structure
RNAi:
Dicer and
RISC
Trigger
dsRNA
length of base pairing segment can be
long or short
PKR as an anti-viral response
Two strands can be derived from two genes
(intermolecular interaction), which can be in
cis or trans
Two strands can be derived from one gene, via
an intramolecular interaction
Trigger:exogenous
dsRNA
diced up by Dicer into small interfering
RNAs (siRNAs)
characteristic structure of 21 to 23 nt
complementary RNA hybrid: 5’-phosphate
and two nt 3’-overhang
5’
5’
Dicer
Highly evolutionarily conserved
Disruption of the Dicer gene in many
species leads to lethality or severe
developmental phenotypes
– Related to lack of miRNA processing
Dicer knockout – cell specific
Trigger:endogenous
dsRNA derived from endogenous primary
miRNA transcript
processed by Drosha (nuclear) and then Dicer
(cytoplasmic) into micro RNAs (miRNAs)
same characteristic structure of 21 to 23 nt
complementary RNA hybrid: 5’-phosphate
and two nt 3’-overhang yet not always
perfectly complementary
5’
5’
MicroRNAs
Naturally encoded small (~22nt) noncoding RNA molecules found in
eukaryotes
Participate in gene regulation,
especially translation
Found to regulate a wide array of
cellular activities in eukaryotes
Many are evolutionarily conserved
across distantly related species
Effector: PTGS
RNA-induced silencing complex (RISC)
targets one of the two strands in the siRNA
and induces RNA degradation
cytoplasmic function
RNA degradation: the endonuclease is an
argonaute protein (Ago 2 in humans)
Translational repression: basis for
repression not clear
Rules of the road: effective Rx
siRNAs versus DNA vectors that express shRNAs
siRNAs: efficient transfections, transient,
dicer-independent
shRNAs: plasmids with RNA pol II or pol III
promoters, viral gene vectors, stable, dicerdependent
Target multiple regions of RNA: mixtures of siRNA
targets
Focus on function rather than RNA levels
Rules of the road
Get the right strand into the RISC complex
Controls: scrambled oligos and control for
transfection method
Lowest amount of RNAi
Rescue the targeted gene with either RNAisensitive or RNAi-insensitive expression
vectors
Ensure the processing of dsRNA templates is equal
if there is a control and treatment group
Grimm et al
Nature 2006
Off target effects of
RNAi -How?
Effector: TGS
Thought to only be in lower species: wrong
RNA-induced transcriptional silencing (RITS)
targets the DNA helix and induces
heterochromatic silencing
nuclear effect
components not clear
role of miRNA
role of long intergenic small RNAs
(lincRNAs)
Regulation of Dicer
Expression
Regulation of Dicer will have downstream
effects on miRNA production and, in turn, on
gene expression
Potentially involved in RNA silencing-mediated
antiviral response in both plants and animals
MBP1007/1008 - Nucleic Acids
A functional mRNA: The cytoplasmic story
Objectives
(1) To discuss the iNUTS and iBOLTS of how mRNAs
function in the cytoplasm
(2) To review older and introduce newer methods of
studying mRNA biology in the cytoplasm
(3) Understand the basics of RNAi-induced posttranscriptional gene silencing