RNAi Tools and Gene Regulation Flashcards
What are the two pathways of RNAi?
The RISC pathway or translation repression pathway
Describe RISC?
RNA-induced silencing complex: recognizes specific sites and cleaves the target– small dsRNA pieces (triggers) are used for targeting complementary sequences within mRNA
What is microRNA?
Small cellular regulatory RNA
How is microRNA produced?
In the nucleus pol 2 produces ssRNA which folds into dsRNA the DROSHA/PASHA complex processes it. The processed pre-miRNA is exported out of the nucleus into the cytoplasm via exportin 5 and processed by DICER to produce a miRNA duplex.
What is cytoplasmic RNAse 3?
DICER
What does DICER do?
Processes both miRNA and exogenous/ longer cellular dsRNA into short dsRNA pieces. Aka siRNA
What are siRNA?
Short inhibitory RNA, approximately 19-21 bp long.
What do the siRNA produced by DICER have?
2 nucleotides long 3’ overhang, 3’ hydroxyl and 5’ phosphate.
What is ATP required for?
DICER translocation and cleavage along long dsRNA. Also important for loading siRNAs into RISC (helicase activity).
What is argonaut?
A ribonuclease 3 at the heart of the RISC complex.
What is a siRNA molecules responsible for?
Many rounds of mRNA degradation by the sub-stoichiometric effect. This could not be achieved by simple anti-sense recognition.
How can RNA interference be exploited?
Temporary knockout using RNAi or RNAi based therapeutics.
What does temporary knockout using RNAis enable?
Function of genes to be mapped and screening for drug targets- genes that are essential for proliferation are often involved in cancer.
How can functions of genes be mapped?
By temporary disruption of function (even if essential) affects cells but does not kill them (unless caused by high, extended dose)
