Small RNAs in the regulation of biological processes

Question 1

C value

Answer 1

Genome size

Question 2

What is the C-value paradox?

Answer 2

Size of genome is not necessarily related to its complexity.

Question 3

What did ENCODE show?

Answer 3

20% non coding DNA is functionally active

60% non coding DNA is transcribed with no known function

Question 4

What are non-coding RNAs?

Answer 4

Any RNA that isn’t translated into a protein (not including mRNA)

Question 5

Examples of non-coding RNAs

Answer 5

tRNA
rRNA
siRNA

Question 6

How does microRNA act as a regulatory ncRNA?

Answer 6

Controls translation of most genes

Question 7

How does siRNA/RNAi act as a regulatory ncRNA?

Answer 7

Viral defence

Experimental tool

Question 8

How does piRNA act as a regulatory ncRNA?

Answer 8

Important for germ cell production

Question 9

Give an example of long ncRNA

Answer 9

Xist: Important for X chromosome inactivation

Question 10

What can Antisense RNA be used as a tool for?

Answer 10

To block mRNA function

Question 11

Describe how RNA can be used to block mRNA function.

Answer 11

Antisense RNA can be synthesised

This binds to the complementary sense RNA forming double stranded RNA (dsRNA) thus preventing it from being translated

Question 12

What is RNA silencing used by, why and how?

Answer 12

Used by plants as a defence mechanism against viruses

Generate small single stranded RNAs (siRNA) that anneal to viral RNA and cause these to degrade

Question 13

Describe the formation of siRNA.

Answer 13

Viral dsRNA DICER (RNA III like endonuclease) cuts the dsRNA into smaller pieces (21-25bp)
siRNA guided endonuclease activity removes 1 “passenger” strand (requires AGO proteins), only leaving the strand that is anti-sense to the target RNA
RISC complexes are formed: recognise and cleave target mRNA with complementary sequences to incorporate single stranded guide siRNA

Question 14

What are AGO proteins?

Answer 14

Agronaute-Piwi proteins

Question 15

What is the consequence of having no DICER gene?

Answer 15

Lethality in early embryonic stages as stem cells unable to differentiate
Leads to depletion of multipoint stem cells, causing limb morphogenesis defects

Question 16

What feature of siRNA allows it to be used as a viable therapy?

Answer 16

It’s small enough to enter the cell without invoking a detrimental cytokine responses
Therefore can be used as therapy to turn off genes we don’t want expressed

Question 17

How can siRNA be synthesised artificially?

Answer 17

As small hairpin RNA (shRNA)

Question 18

What did the Lin-14/Lin-4 experiment show?

Answer 18

There is genomically encoded miRNA that is involved in gene regulation.
RNA production for Lin-14 disappears in early development. Lin 4 is a microRNA which can bind to Lin-14 mRNA and stop it making proteins

Question 19

Describe the production of miRNA

Answer 19

miRNA is made from chopping long RNAs (pri-miRNAs) into smaller fragments, then exporting to cytoplasm as pre-miRNA
Processing allows the pre-miRNA to be passed into the RISC complex
Once pre-miRNAs become miRNAs they can target genes at an RNA level and silence genes

Question 20

Gene knockdown

Answer 20

Decreasing amount of protein you get from a gene

Question 21

What is the classic arrangement of miRNA within the genome?

Answer 21

Usually 2 matching areas separated by a bulge
match-bulge-match arrangement in the genome.
One of the matching regions is usually a 3’ UTR and the other is a seed region

Question 22

What is the seed region?

Answer 22

Most important region of miRNA for targeting
Lies between nucleotide 2-8 from 5’ end
Often flanked by adenosines

Question 23

What forms the bulge in a match-bulge-match arrangement?

Answer 23

Mismatches

Question 24

List 3 diseases where alteration in ncRNA or miRNA expression is seen

Answer 24

Leukaemia
Carcinoma
Heart disease

Question 25

Describe Chronic Lymphoid Leukaemia (CLL)

Answer 25

Deletion of part of gene on chromosome 14 leads to loss of miRNA and promotes CLL