Gene Silencing (Meena Lecture) Flashcards
What is the key molecular requirement of RNAi tech?
Double Stranded RNA (dsRNA)
Provide examples of different silencing RNAs.
siRNA
miRNA
shRNA
CRISPR
What is the key enzyme responsible for cleaving long dsRNA into shorter, siRNAs?
Dicer
Where is long dsRNA derived from?
-Hairpins
-Complementary RNA
-RNA-Dep RNA Polymerases
The shortened dsRNA (once cleaved by Dicer enzyme) contains ___ - ___ nucleotides & two nucleotide overhangs at the __’ end.
21 - 23 nucleotides ; 3’ end
Cleavage of long dsRNA via Dicer enzyme happens where in the cell?
Cytoplasm
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?
Argonaut
What causes RISC Complex activation?
Argonaut cleavage of the Passenger Strand.
siRNAs are specific to ____ target(s), whereas miRNAs are specific to ____ target(s).
siRNA = One
miRNA = Multiple
Through what channel do pre-miRNAs get transported into the Cytoplasm?
Exportin 5
Name of microprocessor complex protein that cleaves Primary miRNA (pri-mRNA) into Precursor miRNA (pre-miRNA).
Drosha
How does Passenger Strand loss within the Cytoplasm differ between siRNA & miRNA molecules?
siRNA: Cleavage of Passenger Strand via ARGO2 endonuclease.
miRNA: Unwinding of duplex leads to the release / discarding of Passenger Strand… No cleavage whatsoever!
Do miRNA molecules still associate with RISC?
Yes! Mechanistically same as siRNA (just no Passenger Strand cleavage).
siRNA is usually ____ complementary to coding regions of complementary target mRNAs, whereas miRNA is _____ complementary to target mRNA.
siRNA: Fully Complementary
miRNA: Partially Complementary
Complementary binding of miRNA to target mRNA usually occurs at the ___ _____ of miRNA & the 3’ _____ of the target mRNA.
Seed Region ; UTR
Why are RNAi therapies better than MABs?
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).
Can siRNAs be used as diagnostic / biomarker tools?
Nope… miRNA can though!
siRNAs regulate gene expression via endonucleolytic cleavage of mRNA, whereas miRNA regulates gene expression how?
Translational Repression & mRNA Degradation
What are the major limitations of RNAi technologies?
-Poor Molecular Stability
-Lack of Membrane Permeability (hydrophilic, negatively charged, large molecular weight)
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?
-Immunogenic
-Risk Insertional Mutagenesis
-$$$
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.
PEGylation
What is the basis of CRISPR-based technologies?
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.
Which of the following nucleotide mutations at Chromosome 11 brings on Sickle Cell Anemia?
AT
TT
AA
AA
Which hemoglobin gene is mutated in Sickle Cell Anemia?
Beta
How were CRISPR technologies used to treat Sickle Cell Anemia?
Editing of BCL11A gene (transcription factor that represses Gamma globulin expression) led to restoration of fetal hemoglobin production, thus counteracting the Beta gene mutation.
