RNA Interference Flashcards
Goal of RNAi in cells
Repress and silence gene expression
Goal of RNAi in the lab
Knock down gene expression (especially relevant in cancer research for knocking down oncogenes)
Ways to perform RNAi in C. elegans
- Clone trigger-encoding DNA into vector and then transform into E. coli, and the worms eat the E. coli (60% efficient)
- Bathe worms in trigger
- Bathe worms’ ovaries in trigger
Techniques used to confirm that RNA has been knocked down
Western blot
In situ (in situ probe is triggered against exon-exon junction of target, which ensures that the probe only hybridizes with mRNA)
RT-PCR
RT-PCR steps
Extract RNA from cells
Reverse transcribe into cDNA
Run amplification reaction on gel: see target
Scrambled control in RT-PCR
Determines that RNAi is specific for RNA and that it is actually causing gene silencing
dsRNA
Double-stranded RNA
Expressed in cells or invades cells (triggers)
shRNA
Short hairpin RNA
Expressed from plasmid or injected (genetically engineered)
siRNA
Short interfering RNA
Made from dsRNA or shRNA
miRNA
Micro RNA Encoded in class 2 genes (transcribed by RNA pol II)
piRNA
Piwi-interacting RNA
Defends against transposons
Nomenclature of miRNA
Organism abbreviation, mir (miRNA), number that signifies the order in which it was discovered
Effects of RNAi in cells
Destroy the mRNA before it is translated
Directly inhibit translational machinery
Transcriptional silencing of the promoter that directs expression of that mRNA by triggering chromatin modifications
RNAi steps
- siRNA and miRNA are processed by Dicer
Dicer and R2D2 deliver miRNA to RISC (RNA-induced silencing complex) loading complex - RISC binds to miRNA
- miRNA is denatured by Armitage to form guide RNA (gRNA, which remains part of RISC)
- Mature complex is directed towards the target by complementary base pairing between gRNA and target
a) gRNA and target are bound and translation is inhibited
b) gRNA and target are bound and target is cleaved - Chromatin remodeling: RITS recruits remodelers to part of the chromosomes
- Amplification of RNAi by RNA-dependent polymerase (RdRP), generating more dsRNA
Likelihood that target is cleaved when gRNA and target bind
The greater the complementarity between the gRNA and the target, the greater the likelihood that target will be cleaved
Protein that cuts the 3’ end of the target
Argonaute (has a PAZ domain hat recognizes the 3’ end of the RNA)
How RdRP works
RdRP uses gRNA to make a copy of the target (uses target as template)
How mature miRNA is made
- Primary miRNA (pri-miRNA) is processed by Drosha to form pre-miRNA
- Pre-miRNA is transported out of the nucleus and processed by Dicer to form mature miRNA
Shape of pre-miRNA
Hairpin shape
Has 3’ overhang (created by Drosha, recognized by Dicer)
What Drosha and Dicer recognize
A specific-sized hairpin, not a sequence
Dicer components
Shaped like a hatchet
Has “blade,” which cuts the target
“Handle” has ruler helix, which measures pre-miRNA hairpin (has to be about 22 bp)
PAZ domain: has positively-charged amino acids which associate with the backbone of the 3’ overhang
Results of losing poly A tail of target
Target is degraded or its translation is inhibited
P-bodies
Where mRNA transcripts are sequestered by the RNAi machinery
Non-membrane bound, cytosolic, and enriched with regulatory RNAs
RNA-induced transcriptional silencing (RITS)
Binds complementarily to sequences
Is still bound to the transcriptional bubble
Brings in histone modifiers
Involved in H3K9 methylation to silence gene expression
