Lecture 5- Small RNAs in the Regulation of Biological Processes

Question 1

What is the C-value

Answer 1

Indicates the size of a genome. Constant amount of genetic material in cells from the same species. So use C-value to compare genome size of different species

Question 2

What did ENCODE reveal about the genome?

Answer 2

ENCODE= Encyclopedia of DNA elements

• approx 20% of non-coding DNA in the human DNA is functional
• 60% transcribed with no known function
• non-coding DNA regulates the expression of coding genes
• expression of coding genes controlled by several regulatory sites near and far from the gene.

Question 3

What are non-coding RNAs?

Answer 3

= not translated into protein (not mRNA)

Question 4

Give examples of ncRNAs

Answer 4

1) Housekeeping ncRNAs
- rRNA
- tRNA
- snRNAs
2) Regulatory ncRNAs
- microRNA: control translation of most genes
- siRNA / RNAi: viral defence
- piRNA: germ cell production
- Long ncRNA- Xist X chromosome inactivation (lionisation). Silenced X chromosome packaged into transcriptionally inactive heterochromatin.

Question 5

How is antisense RNA used in gene regulation?

Answer 5

1) Transcribe the reverse of the gene you want to block = antisense mRNA
2) antisense mRNA will H bond to the sense strand mRNA= double stand RNA (dsRNA).
3) dsRNA cannot be translated

Question 6

What is the experimental evidence for dsRNA?

Answer 6

1) dsRNA is used in C.elegans to switch off genes
2) The Flavr Savr tomato contains an artificial gene which is transcribed into antisense RNA, complementary to the mRNA for an enzyme involved in ethylene production. Prevents them from spoiling prematurely

Question 7

How does RNA interference work (RNAi)?

Answer 7

1) dsRNA is derived from a virus
2) DICER (has Rnase III-like endonuclease activity) breaks up RNA into 21-25bp fragments
3) siRNA-guided endonuclease activity along with Argonaute-Piwi proteins remove one of the siRNA strands (the ‘passenger strand’)
4) An RNA-induced silencing complex (‘RISC’) is formed. This can recognise and cleave target mRNA molecules which have complementary sequences to the incorporated single-strand guide siRNA

Question 8

What would happen without DICER?

Answer 8

There’d be lethality in the embryonic stage because embryonic stem cells can’t differentiate –> multipoint stem cell deplete

Question 9

Give a biological use for siRNA?????

Answer 9

Potato Virus X and Y????

Question 10

What is shRNA?

Answer 10

siRNA which has been synthesised artificially as one long piece of RNA which folds back on itself–> small hairpin RNA

Question 11

What are miRNAs?

Answer 11

Micro RNAs- typically 18-26 nucleotides long

1) Transcribed by RNA Pol II/ III into long pri-miRNA with a stem-loop structure several kb long
2) Pri-miRNA processed by RNase III endonuclease, Drosha, in DGCR8 complex–> 60-80bp pre-miRNA
3) Pre-miRNA has a 5’ phosphate and 2 nucleotide overhang at the 3’ end. This is transported to the cytoplasm from the nucleus by Exportin-5.
4) In the cytoplasm, pre-miRNA is processed by DICER into a 22bp duplex

Question 12

How can you sequence someones genome to check if they have a hereditary disease?

Answer 12

• use miRNAs which base pair with mRNA with incomplete complementarity
• You have matching areas separated by a bulge + lower degree of base-pairing in 3’ region which indicates mismatches.
• Seed region formed- its between nucleotide 2 and 8 from 5’ end, flanked by As

Question 13

What else can you use miRNAs for?

Answer 13

Gene-regulation????

Question 14

Give an example of miRNA involvement in human disease…

Answer 14

miRNA prevent mRNA from being translated

• cancer
• fat metabolism
• cell proliferation and differentiation
• development
• epigenetics
• insulin secretion
• apoptosis