L6 - MicroRNAs

Question 1

What do the double-stranded siRNAs bind to?

Answer 1

Incorporates with the RISC complex

Question 2

What is the next step for mRNA degradation?

Answer 2

siRNA is complementary to the mRNA target by base pairing (has to be highly specific). This enables the RISC complex to cleave and degrade the mRNA

Question 3

Short hairpin RNAs are similar in function to siRNAs. How do they differ in their formation?

Answer 3

siRNAs have to be synthetically produced whereas shRNA can b encoded by a normal RNA transcript within the cell from DNA vectors for a long-term, stable expression of shRNA within the cell for sustainable knockdown of the target gene.

Question 4

miRNAs share a similarity in the use of the RISC complex and binding to the mRNA, but how does it differ in its degradation ?

Answer 4

They typically bind to the 3’ UTR, leading to inhibition of translation and reduction in protein expression

Question 5

What is the role of the dicer enzyme?

Answer 5

Double-stranded RNA is recognised by the dicer enzyme which cleaves it into shorter, dsRNA. They can then be loaded into the RISC.

Question 6

miRNAs have a role in embryo development. Dicer knockouts have lethal phenotypes in zebrafish and mouse. Why is this?

Answer 6

microRNAs cannot be produced in the cell by the dicer. It also shows that miRNAs are required for the development and survival of the early embryo.

Question 7

What is the first step of Northern blotting to quantitate RNA from a sample of tissues or cells?

Answer 7

1. Extract RNA from cells/tissues

Question 8

Second step?

Answer 8

2. RNA size fractionation. Gel electrophoresis allows the RNA sample to be loaded onto a gel matrix and an electrical current is applied, causing the RNA molecules to migrate through based on their size towards the positive pole with smaller RNAs migrating faster.

Question 9

Third step?

Answer 9

RNA detection. RNA is immobilised on the membrane and can be detected using a labelled probe that is specific to the RNA of interest.

Question 10

Fourth step?

Answer 10

Exposure to X-ray film - the hybridised probes will cause the film to react. The amount of RNA on the blot is proportional to the strength of the signal.

Question 11

C2C12 cells are a line of mouse myoblasts. What are myoblasts?

Answer 11

Immature muscle cells that have not differentiated into mature muscle fibres

Question 12

What is the role of miR-1/206 in C2C12 cells?

Answer 12

Enhances DIFFERENTIATION into myotubes (mature muscle fibres)

Question 13

What is the role of miR-133

Answer 13

They enhance PROLIFERATION of myoblasts