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 post-transcriptional gene silencing
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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.
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MBP1007/1008 - Nucleic AcidsA 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 post-
transcriptional gene silencing
Breakthrough Breakthrough of the Year in of the Year in
20022002
Nobel prize in Nobel prize in 20062006
RNAi: RNA InterferenceRNAi: 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 paperA classic paper
A classic paperA classic paperIn 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 generalizedRevelation because dsRNA is inherently very
stable and incapable of further Watson-Crick base-pairing
Passed to neighbouring and daughter cells: mitosis and meiosis
RNAi in RNAi in C. elegansC. elegans
Wild type and RNAi-deficient worms fed on bacteria expressing GFP dsRNA
Wild-type RNAi-deficient
DNA versus RNA!DNA versus RNA!
Structure of RNA versus DNALife of dsRNALife of ssRNASense and antisense genes
Antisense approaches to inhibit gene expression
RNA: Primary, secondary and tertiary structure
RNA - G:U
RNAiRNAi: Dicer and
RISC
TriggerTriggerdsRNA
length of base pairing segment can be long or shortPKR 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:exogenousTrigger:exogenousdsRNA
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’
DicerDicer
Highly evolutionarily conservedDisruption of the Dicer gene in many
species leads to lethality or severe developmental phenotypes– Related to lack of miRNA processing