1
Q
Post transcriptional gene silencing (PTGS)
A
- silenced genes are still transcribed, but the resultant mRNAs are degraded before they can be translated into proteins
- was first identified as co-suppression
- increased gene lead to increased mRNA lead to decreased protein
2
Q
Micro RNA expression and processing
A
- miRNAs first transcribed as primary miRNA (pri-miRNA) with one or more sets of internally complementary sequences that can fold to form hairpin-like structures
- pri-miRNAs cleaved by nuclear endonucelase (Drosha) to produce shortened hairpins (60-70 nucleotides)
- have a 5’ phosphate and 2-nucleotide 3’ overhang
- now called precursor miRNAs (pre-miRNA) - pre-miRNAs bind to export receptor proteins, transported from nucleus to cytoplasm
3
Q
siRNA and miRNA gene silencing
A
- Once in cytoplasm, pre-miRNAs cleaved by Dicer ribonuclease to produce 20-22 nucleotide ds-miRNA
- miRNAs incorporated into RNA-induced silencing complex (RISC), includes AGO protein - After cleavage, miRNA is unwound, the unneeded passenger strand is discarded
- the strand complementary to the target is delivered to a particular mRNA by AGO protein
- once mRNA has been targeted, degree of miRNA-mRNA base pairing leads to either AGO-mediated degradation (high complementarity) or translational repression (lower complementarity), followed by degradation
4
Q
miRNA binding
A
- generally bind to 3’ UTR of an mRNA in animals
- can make reporter plasmid with coding for 3’UTR of interest downstream of a gene that codes for a fluorescent protein (luciferase)
5
Q
Regulation of gene expression by Non-coding RNAs
A
- long non-coding (lncRNA) and circular (circRNA) RNAs have multiple functions, one is to compete as targets for miRNA binding
- contain miRNA binding regions, miRNA response elements (MREs)
- when miRNAs bind non-coding RNAs instead of target mRNA, results in increased translation of the gene of interest
- known as ‘competing endogenous model’
BCHM 218 Part 2
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