RNA Interference

Question 1

Goal of RNAi in cells

Answer 1

Repress and silence gene expression

Question 2

Goal of RNAi in the lab

Answer 2

Knock down gene expression (especially relevant in cancer research for knocking down oncogenes)

Question 3

Ways to perform RNAi in C. elegans

Answer 3

1. Clone trigger-encoding DNA into vector and then transform into E. coli, and the worms eat the E. coli (60% efficient)
2. Bathe worms in trigger
3. Bathe worms’ ovaries in trigger

Question 4

Techniques used to confirm that RNA has been knocked down

Answer 4

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

Question 5

RT-PCR steps

Answer 5

Extract RNA from cells
Reverse transcribe into cDNA
Run amplification reaction on gel: see target

Question 6

Scrambled control in RT-PCR

Answer 6

Determines that RNAi is specific for RNA and that it is actually causing gene silencing

Question 7

dsRNA

Answer 7

Double-stranded RNA

Expressed in cells or invades cells (triggers)

Question 8

shRNA

Answer 8

Short hairpin RNA

Expressed from plasmid or injected (genetically engineered)

Question 9

siRNA

Answer 9

Short interfering RNA

Made from dsRNA or shRNA

Question 10

miRNA

Answer 10

Micro RNA
Encoded in class 2 genes (transcribed by RNA pol II)

Question 11

piRNA

Answer 11

Piwi-interacting RNA

Defends against transposons

Question 12

Nomenclature of miRNA

Answer 12

Organism abbreviation, mir (miRNA), number that signifies the order in which it was discovered

Question 13

Effects of RNAi in cells

Answer 13

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

Question 14

RNAi steps

Answer 14

1. siRNA and miRNA are processed by Dicer
   Dicer and R2D2 deliver miRNA to RISC (RNA-induced silencing complex) loading complex
2. RISC binds to miRNA
3. miRNA is denatured by Armitage to form guide RNA (gRNA, which remains part of RISC)
4. 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
5. Chromatin remodeling: RITS recruits remodelers to part of the chromosomes
6. Amplification of RNAi by RNA-dependent polymerase (RdRP), generating more dsRNA

Question 15

Likelihood that target is cleaved when gRNA and target bind

Answer 15

The greater the complementarity between the gRNA and the target, the greater the likelihood that target will be cleaved

Question 16

Protein that cuts the 3’ end of the target

Answer 16

Argonaute (has a PAZ domain hat recognizes the 3’ end of the RNA)

Question 17

How RdRP works

Answer 17

RdRP uses gRNA to make a copy of the target (uses target as template)

Question 18

How mature miRNA is made

Answer 18

1. Primary miRNA (pri-miRNA) is processed by Drosha to form pre-miRNA
2. Pre-miRNA is transported out of the nucleus and processed by Dicer to form mature miRNA

Question 19

Shape of pre-miRNA

Answer 19

Hairpin shape

Has 3’ overhang (created by Drosha, recognized by Dicer)

Question 20

What Drosha and Dicer recognize

Answer 20

A specific-sized hairpin, not a sequence

Question 21

Dicer components

Answer 21

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

Question 22

Results of losing poly A tail of target

Answer 22

Target is degraded or its translation is inhibited

Question 23

P-bodies

Answer 23

Where mRNA transcripts are sequestered by the RNAi machinery

Non-membrane bound, cytosolic, and enriched with regulatory RNAs

Question 24

RNA-induced transcriptional silencing (RITS)

Answer 24

Binds complementarily to sequences
Is still bound to the transcriptional bubble
Brings in histone modifiers
Involved in H3K9 methylation to silence gene expression

Question 25

RNAi regulation

Answer 25

Control protein levels
Signaling proteins: feedback loop
Control export of miRNA
Reduce half-life of miRNA transcript
Control miRNA through promoter

Question 26

Circular RNAs

Answer 26

Sequester miRNAs that are bound to RISC
Primarily function in the cytosol
Results of backsplicing of mRNA transcripts