Control of gene expression

Question 1

RNAi

Answer 1

RNA interference. mechanism by which gene expression is regulated in eukaryotes.

Question 2

What can happen when injecting double stranded RNA into model?

Answer 2

Andrew Fire and Craig Mello - NP in 2006.

Potent silencing.

Question 3

Down-regulating

Answer 3

Gene silencing

Question 4

What are the two RNAi pathways?

Answer 4

1. Small RNAs - From transposons (little parts of DNA from historic viral infection and move around genome and cells). Defense against viral RNA and mobilisation.
2. microRNAs - Widely present through eukaryotes - around 3000 characterised. Recently in unicellular organisms. 1/3 of genes modulated by micro RNAs. Generally expressed from intergenic (between gene) DNA sequence. Dont code for proteins - but for a small RNA. Key system for translational repression in cells. Regulate development. Active when don’t want a gene switched on. Also important in mature neurons.

Question 5

Do micro RNA target only one mRNA?

Answer 5

No. One microRNA can target many mRNAs

Question 6

What is the microRNA pathway?

Answer 6

In nucleus, pri-mRNA single strand but loops from complementary hybridisation. PolyA tail. Enzymes (drosha and pasha) cleaves ends and exports (at exportin 5). Then cleaved again by Dicer and co-factors. The more mature miRNA is cleaved and double stranded. One is guide strand and one is passenger strand (not active). Happen in p body in cytoplasm. Guide strand finds its complementary mRNA target. Requires assembly of proteins to make up RISC complex. Typically miRNA will target untranslated region for more specificity - part doesnt code protein. DCP1 DCP2 now degrade or prevent transcription. Inhibition of translation and degradation of mRNA. Aprox 21nt

Question 7

What is the small RNA pathway?

Answer 7

Synthetic RNA of viral RNA also uses dicer, trimmed with slightly larger double stranded silencing RNA. Also has guise strand and passenger strand but complete hybridisation. Also uses RISC but one strand can be continuously reused. mRNA cleaving or inhibit transcription.

Question 8

What are the implications in nervous system disease due to miRNAs?

Answer 8

Fragile X syndrome - CGG repeat in UTR FMR1.
Cognitive impairment and intellectual disability.
Supresses normal activity in gene - developmental disorder.

Tourette’s syndrome - SLITRK1 involved in neuronal growth, guidance and branching. Single bp that is different in some people. SNP inhibits binding of has-miR-189. Gene is not silenced.

Question 9

RNAi used in research?

Answer 9

Reverse genetics in mice, flies, plants, worms.
Gene knockouts.
Cell culture - Make synthetic SRNA and put into permeable membranes.