Lecture 11: RNA interference Flashcards
RNA interference is a type of _____
Reverse genetics
Reverse genetic techniques
-Gene ablation/knockout by recombination
-Gene ablation using CRISPR
-Antisense RNA (for transient inhibition of gene expression)
-RNA interference (RNAi)
Gene ablation/knockout by recombination summary
-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
Gene ablation/knockout by recombination drawbacks
-difficult and expensive
-time-consuming
-may not be able to study essential genes cause knock-out organism would die
CRISPR stands for ____
clustered regularly interspaces short palindromic repeats
CRISPR is derived from ____
bacterial antiviral system
Gene ablation using CRISPR summary
-“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
Antisense RNA (for transient inhibition of gene expression) summary
-addition of antisense rna binds to mRNA and prevents translation
-simple but not efficient
-controls: sense RNA (without 5’ cap and 3’ tail)
loss of unc22 gene in C.elegans led to ___
twitching
insertion of dsRNA in C.elegens found that _____
Found that dsRNA was much more potent than
anti-sense RNA for inducing unc22 phenotype
detection of mRNA by in situ hybridization
-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
RNA interference (RNAi) can be applied to ____ and ___ cells
-plants and mammalian cells
RNA interference (RNAi): provided that DNA sequence is known, _____
it is now possible to “knock-down” the activity of any gene rapidly
extreme benefit of RNAi
-can study function of essential genes (not knocking them out completely, just toning down expression)
Dicer (def.)
RNase cleaves dsRNA molecules
Dicer products are ____
21-23 bp RNAs termed small interfering RNAs (siRNAs)
Argonautes (def.)
RNA-binding proteins that interact with siRNAs to form RNA induced silencing complex (RISC)
RISC is targeted to ____ by _____
mRNAs by siRNA or miRNA
microRNAs (miRNAs) (def.)
similar to siRNAs but generally result in translational repression
-miRNAs control expression of ____ of human genes
~70%
siRNA cause ____ while miRNA causes _____ because ____
-RNA cleavage
-translation inhibition
-siRNA base pairing is complementary while miRNA is generally not fully complementary
what encodes for miRNA?
the genome
miRNA blocks translation because of the ___ and can work on ____
-bulge in base-paring
-many targets as a result
long dsRNA are ___
usually not found in euk. cells (sign of danger)
LNP stands for
lipid nanoparticles
LNP is used to ___
deliver siRNA into cells
LNP are
3-4 lipids that bind to RNA + help it cross cell membrane
LNP morphology
-single lipid bilayer
-lipid carrier (+ charged lipid at lower PH), structural lipid (cholesterol + phospholipid) and shielding lipid (PEG-lipid)
protein levels _____ with different kinetics than mrna in response to RNAi
decrease (it can take more than 1 day for protein expression to decrease while mrna decreases in hours)
Northern blot is a laboratory analysis method used to study ____
RNA
P bodies are sites of ___
RNA degradation (processing bodies) + contain mRNA decapping enzymes (Dcp1a,2)
Resolution power of conventional light microscope is ___ nm so objects less than ___ nm apart cannot be resolved
250 nm
Western blot is often used in research to ____
separate and identify proteins in a complex mixture of proteins
western blot + co-immunoprecipitation (co-IP)
IP-Western (immunoblotting); used to analyze specific protein-protein interactions
Co-Immunoprecipitation (co-IP) procedure
-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
western blot procedure
-electrophoresis(SDS)/transfer
-antibody detection (1/2, wash excess)
To confirm interaction between X and Y, ___
reciprocal co-IP Western blot should be
performed.
* i.e. Antibody to protein Y should be able to
co-IP X.
Depending on substrate
(DAB or ECL), IS used, _____
HRP catalyzed oxidation produces a colored
precipitate (from DAB, as shown here) or light (ECL)
on membrane where protein
Y is located
Argonautes ____complexes with Dcp1a
do form
If Ago and Dcp form complex, both
proteins should be ____
co-IP’d by antibodies to either protein
Argonaute proteins are ____
Modular proteins with PAZ and PIWI domains
PAZ domain of argonaute
-si/miRNA binding
PIWI domain of argonaute
-nuclease (slicer and binding to DICER)
when nuclease function of argonaute is knocked out, it goes to ___
P bodies
PAZ mutants of argonaute don’t ____
associate with P-bodies
____ of Ago2 is required for Pbody association
siRNA/miRNA-binding activity
