RNA interference and its applications Flashcards
what is RNA interference?
RNAi is the knockdown of gene expression by small RNA fragments
Biological roles of RNAi
- antiviral defense
- suppress expression of transposons/repetitive DNA
- gene regulation (silencing)
triggering RNAi
by various sources of ds RNA molecules
triggering RNAi; RNA viruses
some RNA viruses have dsRNA intermediates in their life cycles
triggering RNAi: transposons
DNA transposons that jump into actively transcribed loci (in random orientations) are transcribed into RNA and then hybridise into dsRNA
triggering RNAi; endogenous microRNAs
regulated microRNA gene expression to control development
triggering RNAi; exogenous constructs
- experimentally delivered, synthetic long dsRNA or processed siRNA
- promoter driven expression of a short-hairpin RNA (shRNA)
mechanism of Dicer-mediated dsRNA processing
enzyme recognises the termini of a long dsRNA, cleaves both RNA strands and removes a small RNA duplex upon product release
RISC
RNA induced silencing complex
what are some known component of RISC?
- Dicer
- argonaute proteins
- various RNA binding proteins
what are argonaute proteins?
AGO1 and AGO2 contain PAZ domain (conserved domain present in AGO proteins that binds to small RNA helices) as well as C-term PIWI domain that recognises dsRNA
what does Dicer include?
- endonuclease domain
- dsRNA binding motif
- DEAD box RNA helicase domain responsible for unwinding the dsRNA
what does AGO2 do?
functions as a catalytic enzyme that cleaves the target mRNA at a site that is ~10 nucleotides from the 5’ end of the siRNA guide strand
where have Dicer and AGO homologues been found?
- in drosophila; AGO2 mutants have defects in siRNA-directed RNAi but can undergo miRNA directed RNAi
- however, AGO1 not required for siRNA-directed RNA cleavage but is required for miRNA-directed RNAi
siRNA biogenesis
- long dsRNA molecules are converted to smaller 21-23 nt siRNAs by Dicer which is an RNase III-type endonuclease
- this processing step = RNAs with 2 nucleotide overhangs at the 3’ ends and phosphorylated 5’ ends
what can Dicer use to carry out function?
linear dsRNA or hairpin RNA- DNA vectors that express hairpin constructs are commonly used in experimental RNAi
antisense strand
guide strand, serves as the template for the sequence specific gene silencing
sense strand
passenger strand
what does the guide RNA do?
guide strand of the siRNA assembles into the RISC to form RNA protein complex that binds to the target mRNA and silences gene expression by cleaving the target RNA
what do mobile genetic (transposable) elements have the capacity to do?
integrate into genomes and move from one genomic location to another (often encode proteins enabling them to be mobile)
what are mobile genetic elements?
- ubiquitous components of genomes
- comprise 45% of human chromosomal DNA (most but not all are inactive)
- cause genetic chanfes
- controlling transposition of mobile genetic elements is crucial for maintaining genomic stability
how would a tranposon trigger RNAi?
provides a substrate for Dicer activity and thereby triggers endo-siRNA pathways
transposon triggering RNAi can come from 3 different sites
- actively transcribed region containing transposon
- silenced region containing same transposon
- a second actively transcribed region
what are microRNAs?
- functional miRNA processed from a ~70 nucleotide precursor RNA that= hairpin structure with a few base pair matches at the stem region of the hairpin
