Lecture 11: RNA interference

Question 1

RNA interference is a type of _____

Answer 1

Reverse genetics

Question 2

Reverse genetic techniques

Answer 2

-Gene ablation/knockout by recombination
-Gene ablation using CRISPR
-Antisense RNA (for transient inhibition of gene expression)
-RNA interference (RNAi)

Question 3

Gene ablation/knockout by recombination summary

Answer 3

-to figure out what a gene is for, you take it out and see what doesn’t work
-permanent insertion of marker (usually drug resistance) into chromosome to create knockout organism

Question 4

Gene ablation/knockout by recombination drawbacks

Answer 4

-difficult and expensive
-time-consuming
-may not be able to study essential genes cause knock-out organism would die

Question 5

CRISPR stands for ____

Answer 5

clustered regularly interspaces short palindromic repeats

Question 6

CRISPR is derived from ____

Answer 6

bacterial antiviral system

Question 7

Gene ablation using CRISPR summary

Answer 7

-“Guide” RNAs target the nuclease Cas9 to homologous DNA sequences after which cuts are
made in the DNA.
-Can be used to delete, modify or activate genes
-permanent

Question 8

Antisense RNA (for transient inhibition of gene expression) summary

Answer 8

-addition of antisense rna binds to mRNA and prevents translation
-simple but not efficient
-controls: sense RNA (without 5’ cap and 3’ tail)

Question 9

loss of unc22 gene in C.elegans led to ___

Answer 9

twitching

Question 10

insertion of dsRNA in C.elegens found that _____

Answer 10

Found that dsRNA was much more potent than
anti-sense RNA for inducing unc22 phenotype

Question 11

detection of mRNA by in situ hybridization

Answer 11

-Digoxigenin is a small molecule to which there are good antibodies available to it (a way to mark
the cDNA probe for detection with secondary antibody
-Digoxigenin label on uridine + alkaline phosphate and colourless chemical –> purple dye

Question 12

RNA interference (RNAi) can be applied to ____ and ___ cells

Answer 12

-plants and mammalian cells

Question 13

RNA interference (RNAi): provided that DNA sequence is known, _____

Answer 13

it is now possible to “knock-down” the activity of any gene rapidly

Question 14

extreme benefit of RNAi

Answer 14

-can study function of essential genes (not knocking them out completely, just toning down expression)

Question 15

Dicer (def.)

Answer 15

RNase cleaves dsRNA molecules

Question 16

Dicer products are ____

Answer 16

21-23 bp RNAs termed small interfering RNAs (siRNAs)

Question 17

Argonautes (def.)

Answer 17

RNA-binding proteins that interact with siRNAs to form RNA induced silencing complex (RISC)

Question 18

RISC is targeted to ____ by _____

Answer 18

mRNAs by siRNA or miRNA

Question 19

microRNAs (miRNAs) (def.)

Answer 19

similar to siRNAs but generally result in translational repression

Question 20

-miRNAs control expression of ____ of human genes

Answer 20

~70%

Question 21

siRNA cause ____ while miRNA causes _____ because ____

Answer 21

-RNA cleavage
-translation inhibition
-siRNA base pairing is complementary while miRNA is generally not fully complementary

Question 22

what encodes for miRNA?

Answer 22

the genome

Question 23

miRNA blocks translation because of the ___ and can work on ____

Answer 23

-bulge in base-paring
-many targets as a result

Question 24

long dsRNA are ___

Answer 24

usually not found in euk. cells (sign of danger)

Question 25

LNP stands for

Answer 25

lipid nanoparticles

Question 26

LNP is used to ___

Answer 26

deliver siRNA into cells

Question 27

LNP are

Answer 27

3-4 lipids that bind to RNA + help it cross cell membrane

Question 28

LNP morphology

Answer 28

-single lipid bilayer -lipid carrier (+ charged lipid at lower PH), structural lipid (cholesterol + phospholipid) and shielding lipid (PEG-lipid)

Question 29

protein levels _____ with different kinetics than mrna in response to RNAi

Answer 29

decrease (it can take more than 1 day for protein expression to decrease while mrna decreases in hours)

Question 30

Northern blot is a laboratory analysis method used to study ____

Answer 30

RNA

Question 31

P bodies are sites of ___

Answer 31

RNA degradation (processing bodies) + contain mRNA decapping enzymes (Dcp1a,2)

Question 32

Resolution power of conventional light microscope is ___ nm so objects less than ___ nm apart cannot be resolved

Answer 32

250 nm

Question 33

Western blot is often used in research to ____

Answer 33

separate and identify proteins in a complex mixture of proteins

Question 34

western blot + co-immunoprecipitation (co-IP)

Answer 34

IP-Western (immunoblotting); used to analyze specific protein-protein interactions

Question 35

Co-Immunoprecipitation (co-IP) procedure

Answer 35

-Cells lysed with nondenaturing detergent (does not disrupt protein-protein interactions) -Incubate with antibody to protein X and Protein A agarose -Wash away unbound proteins -Analyze complex by Western blotting

Question 36

western blot procedure

Answer 36

-electrophoresis(SDS)/transfer -antibody detection (1/2, wash excess)

Question 37

To confirm interaction between X and Y, ___

Answer 37

reciprocal co-IP Western blot should be performed. * i.e. Antibody to protein Y should be able to co-IP X.

Question 38

Depending on substrate (DAB or ECL), IS used, _____

Answer 38

HRP catalyzed oxidation produces a colored precipitate (from DAB, as shown here) or light (ECL) on membrane where protein Y is located

Question 39

Argonautes ____complexes with Dcp1a

Answer 39

do form

Question 40

If Ago and Dcp form complex, both proteins should be ____

Answer 40

co-IP’d by antibodies to either protein

Question 41

Argonaute proteins are ____

Answer 41

Modular proteins with PAZ and PIWI domains

Question 42

PAZ domain of argonaute

Answer 42

-si/miRNA binding

Question 43

PIWI domain of argonaute

Answer 43

-nuclease (slicer and binding to DICER)

Question 44

when nuclease function of argonaute is knocked out, it goes to ___

Answer 44

P bodies

Question 45

PAZ mutants of argonaute don't ____

Answer 45

associate with P-bodies

Question 46

____ of Ago2 is required for Pbody association

Answer 46

siRNA/miRNA-binding activity