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

Dicer knockout – cell Dicer knockout – cell specificspecific

Trigger:endogenousTrigger:endogenousdsRNA derived from endogenous primary

miRNA transcriptprocessed 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 complementary5’

5’

MicroRNAsMicroRNAs

Naturally encoded small (~22nt) non-coding 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: PTGSEffector: PTGSRNA-induced silencing complex (RISC)

targets one of the two strands in the siRNA and induces RNA degradationcytoplasmic functionRNA degradation: the endonuclease is an

argonaute protein (Ago 2 in humans)Translational repression: basis for repression not clear

Rules of the road: effective RxRules 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, dicer-dependent

Target multiple regions of RNA: mixtures of siRNA targets

Focus on function rather than RNA levels

Rules of the roadRules of the road

Get the right strand into the RISC complexControls: scrambled oligos and control for

transfection methodLowest amount of RNAiRescue the targeted gene with either RNAi-

sensitive 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: TGSEffector: TGSThought to only be in lower species: wrongRNA-induced transcriptional silencing (RITS)

targets the DNA helix and induces heterochromatic silencing

nuclear effectcomponents not clearrole of miRNArole of long intergenic small RNAs (lincRNAs)

Regulation of Dicer Regulation of Dicer ExpressionExpression

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 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