MOLBIOL 3B03 Chapter Notes - Chapter 6: P53, Neural Development, Ribonuclease

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3B03: Small Non-Coding RNA
RNAi, RNA Interference
- Post-transcriptional gene silencing technique
- Recruits RISC (RNA induced silencing complex) and miRNA + siRNA to
silence a transcript via cleavage or degrade the transcript before
Ex. Petunia studied by Jorgensena: added an extra copy of chalcone synthase
gene to create darker flowers, but observed the repression of the phemotype
- White sections: RNAi silenced the gene
siRNA degrade mRNA
- Source: exogenous form of RNAi (ie. transposons, viruses), that integrate into human genome
- Mutations are lethal and recessively inherited in diploid organisms
o Can use antibody neutralization and pharmacological perturbation, but that requires preparation and
characterization of the target transcript and inhibitor
- Studied unc-2: involved in muscle formation and maintaining shape of the worm, and its movement
o When they injected sense and antisense RNA separately, they got WT
o When they injected both mixed => dsRNA = 100% twitch.
Mechanism: post-transcriptional gene silencing (PTGS)
1) Viral infection directly into host, transcription of sense and antisense strands form long dsDNA
2) dsRNA moves from nucleus cytoplasm, where it interacts with Dicer
a. dsRNA-specific RNAase III enzyme
3) Dicer cleaves dsRNA siRNA 21-23 bps, with 2nt 3’ overhangs
4) siRNA is incorporated into RISC
a. RNA induced silencing complex: Ago-2 (argonaute-2), Dicer, and TRBP (TAR-RNA binding protein)
5) ds-siRNA is separated, and RISC carries only one strand to target mRNA
6) ss-siRNA complementary binds to mRNA, causing Ago-2 to cleave target mRNA
a. Cleave: phosphate background at centre of the siRNA:mRNA duplex via RNase-H like activity
b. Non-perfect base pairing => off-target effects
7) mRNA halves are further degraded
Gene Knockdown:
- Can identify the physiological function of a gene- knock them down and study the effects
Ex. C. elegans: RNAi feeding; Mammalian cells transfection or lentiviral infection: via shRNA
- Introduce shRNA plasmid DNA and shRNA lentiviral particles into a cell. Plasmid is integrated into the genome and
undergoes transcription shRNA hairpin loop; the loop is cleaved by Dicer siRNA, that enters RISC and
eventually binds to lentiviral mRNA and cleaves = gene knock out
shRNA Gene Knockout:
- Source: nucleus of transfected/transduced cells
- Structure: sense:anti-sense duplex of 19-22 bps, separated by a loop
of 4-11 nts
1) Introduced into cell by bacterial/viral vectors, and vectors are
incorporated into the genome
2) shRNA is transcribed by RNAP2 or RNAP3 (depends on promoter)
long dsDNA
a. Structure: slight overhang, ds binding, loop, ds binding, slight
3) Drosha and DGCR8 cleaves the over hangs pre-shRNA
4) Pre-shRNA exported nucleus cytoplasm via exportin-5
5) Dicer and TRBP/PACT cleaves pre-shRNA hairpin siRNA 21-23
bps, with 2nt 3’ overhangs
a. dsRNA-specific RNAase III enzyme
6) Active siRNA is incorporated into RISC etc…
Results: gene-knockdown cell line
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