Gene Silencing (Meena Lecture)

Question 1

What is the key molecular requirement of RNAi tech?

Answer 1

Double Stranded RNA (dsRNA)

Question 2

Provide examples of different silencing RNAs.

Answer 2

siRNA
miRNA
shRNA
CRISPR

Question 3

What is the key enzyme responsible for cleaving long dsRNA into shorter, siRNAs?

Answer 3

Dicer

Question 4

Where is long dsRNA derived from?

Answer 4

-Hairpins
-Complementary RNA
-RNA-Dep RNA Polymerases

Question 5

The shortened dsRNA (once cleaved by Dicer enzyme) contains ___ - ___ nucleotides & two nucleotide overhangs at the __’ end.

Answer 5

21 - 23 nucleotides ; 3’ end

Question 6

Cleavage of long dsRNA via Dicer enzyme happens where in the cell?

Answer 6

Cytoplasm

Question 7

Once Dicer cleaves the long dsRNA into a short siRNA, it interacts with the RISC Complex. What endonuclease protein is associated with the RISC Complex?

Answer 7

Argonaut

Question 8

What causes RISC Complex activation?

Answer 8

Argonaut cleavage of the Passenger Strand.

Question 9

siRNAs are specific to ____ target(s), whereas miRNAs are specific to ____ target(s).

Answer 9

siRNA = One
miRNA = Multiple

Question 10

Through what channel do pre-miRNAs get transported into the Cytoplasm?

Answer 10

Exportin 5

Question 11

Name of microprocessor complex protein that cleaves Primary miRNA (pri-mRNA) into Precursor miRNA (pre-miRNA).

Answer 11

Drosha

Question 12

How does Passenger Strand loss within the Cytoplasm differ between siRNA & miRNA molecules?

Answer 12

siRNA: Cleavage of Passenger Strand via ARGO2 endonuclease.

miRNA: Unwinding of duplex leads to the release / discarding of Passenger Strand… No cleavage whatsoever!

Question 13

Do miRNA molecules still associate with RISC?

Answer 13

Yes! Mechanistically same as siRNA (just no Passenger Strand cleavage).

Question 14

siRNA is usually ____ complementary to coding regions of complementary target mRNAs, whereas miRNA is _____ complementary to target mRNA.

Answer 14

siRNA: Fully Complementary

miRNA: Partially Complementary

Question 15

Complementary binding of miRNA to target mRNA usually occurs at the ___ _____ of miRNA & the 3’ _____ of the target mRNA.

Answer 15

Seed Region ; UTR

Question 16

Why are RNAi therapies better than MABs?

Answer 16

Can enter cell & downregulate expression of virtually any gene & their mRNA transcripts… MABs limited to cell-surface receptor binding &/or circulating proteins (too large to enter cell).

Question 17

Can siRNAs be used as diagnostic / biomarker tools?

Answer 17

Nope… miRNA can though!

Question 18

siRNAs regulate gene expression via endonucleolytic cleavage of mRNA, whereas miRNA regulates gene expression how?

Answer 18

Translational Repression & mRNA Degradation

Question 19

What are the major limitations of RNAi technologies?

Answer 19

-Poor Molecular Stability

-Lack of Membrane Permeability (hydrophilic, negatively charged, large molecular weight)

Question 20

Viral Vectors such as Lentivirus, Adenovirus, & Adeno-Associated Viruses are often used to deliver RNA technologies into the cell. What are some limitations to this delivery system?

Answer 20

-Immunogenic

-Risk Insertional Mutagenesis

-$$$

Question 21

Due to being inexpensive, having positive safety profiles & being very efficient, Polymer / Lipid-Based Delivery Systems are often employed in place of Viral Vectors… Provide an example of a modification that would make these vectors even more efficient in drug delivery.

Answer 21

PEGylation

Question 22

What is the basis of CRISPR-based technologies?

Answer 22

Bacterial recognition of target RNA sites leads to nicks being formed via Cas-9… Nicking target sequence then renders mRNA inactive, or you can replace this nicked segment with a specific sequence of choice that renders self immune to invading viruses.

Question 23

Which of the following nucleotide mutations at Chromosome 11 brings on Sickle Cell Anemia?

AT
TT
AA

Answer 23

AA

Question 24

Which hemoglobin gene is mutated in Sickle Cell Anemia?

Answer 24

Beta

Question 25

How were CRISPR technologies used to treat Sickle Cell Anemia?

Answer 25

Editing of BCL11A gene (transcription factor that represses Gamma globulin expression) led to restoration of fetal hemoglobin production, thus counteracting the Beta gene mutation.