mRNA’s and RNA interference

Question 1

What is the Central Dogma of molecular biology?

Answer 1

DNA–>mRNA–>Protein
But mRNA does not always correlate with rpotein levels due to post-transcriptional regulation

Question 2

How is mRNA regulated?

Answer 2

Qualitative control - Splicing allows multiple proteins from one mRNA
Quantitative control - mRNA levels refulate protein translation (e..g, decapping, deadenylation, RNAi)

Question 3

What are the two classes of non-coidng RNA?

Answer 3

1 - Long (>200 bp)
2 - Short (20-30 bp)

Question 4

What happened in CHS-expressing petunias?

Answer 4

Extra CHS gene caused gene silencing, leading to loss of mRNA and white flowers instead of purple

Question 5

What are the key features of TIGS?

Answer 5

Acts in trans, meaning it spreads beyond the modified gene
Hereditary, affecting future generations

Question 6

What triggers RNAi?

Answer 6

Double-stranded RNA (dsRNA), not single-stranded RNA

Question 7

What does RNAi do to mRNA?

Answer 7

Degrades targete mRNA, making it disappear

Question 8

What is RNA interference (RNAi)?

Answer 8

SiRNAs from longer dsRNA modulate mRNA expression via degradation or translational inhibition

Question 9

What are microRNAs (miRNAs)?

Answer 9

Endogenously expressed small RNAs, processed from pri-miRNAs, that regulate mRNA expression

Question 10

What are the four main steps in RNAi?

Answer 10

1 - Processing by Drosha - Cuts pri-miRNAs into 70 pre-miRNAs fro Dicer processing
2 - Processing into siRNA fragments by Dicer - Dicer cleaves precursor RNA into 21-25 nt siRNA molecules - Measures RNA length, ensuring uniform fragment sizes
3 - Incorporation into RISC - Ago proteins eleminate one strand of dsRNA -> single-stranded siRNA binds to target mRNA
4 - RISC binding and mRNA inactivation - Degradation or translational repression via ribosome inhibition, deadenylation or decapping

Question 11

How do Ago proteins function?

Answer 11

Process dsRNA into single-stranded siRNA/miRNA
Ago2 = endonuclease that cuts targeted mRNA (RNAi-dependednt silencing)
Ago 1, 3, 4 = bind siRNA/miRNA but do not cleave mRNA

Question 12

How widespread are miRNAs in eukaryotes?

Answer 12

1,500 miRNAs in humans, controlling 90% of genes
Each miRNA targets hundred of mRNAs

Question 13

How is RNAi used to study gene function?

Answer 13

siRNA libariers - Screen gene knockdowns
shRNA in plasmids/viruses - Express siRNA in cells
RNAi in C.elegans - Extremely efficient, spreading through the body