Epigenetic regulation by non-coding RNAs

Question 1

What are the 2 groups of non-coding RNAs (ncRNAs)?

Answer 1

• Short (22-28 nt)
• Long (70 nt-118 kb)

Question 2

What are examples of short ncRNAs? (3)

Answer 2

• miRNA
• siRNA
• piRNA

Question 2

What are examples of long ncRNAs? (4)

Answer 2

• incRNA
• snRNA
• tRNA
• rRNA

Question 3

What is the general function of short ncRNAs?

Answer 3

Silencing expression (RNA interference)

Question 4

What is the function of siRNA?

Answer 4

Cleaves mRNA (research tool)

Question 4

What is the purpose of RNAi?

Answer 4

Natural cellular process for silencing gene expression, plays roles in gene regulation and innate defence against invading viruses

Question 5

What is the function of miRNA?

Answer 5

Inhibits mRNA translation (in most cases)

Question 6

What is the function of piRNA?

Answer 6

Cleaves products of transposons in germ cells

Question 7

How does gene silencing by siRNA and miRNA work? (6)

Answer 7

• siRNAs produced in vitro
• miRNAs transcribed by RNA polymerase II to make primary miRNA with some sequence complementarity so forms a stem loop
• Primary miRNA cleaved by Drosha to become pre-mRNA which can be exported into the cytoplasm
• Dicer cleaves pre-mRNA to produce double stranded miRNA product
• miRNA/siRNA are substrates for AGO1/2/RISC complex, removes the passenger strand so left only with the strand which is complementary to the target mRNA
• RISC bound to the single stranded miRNA/siRNA binds the target mRNAs

Question 8

What is the difference between siRNA and miRNA? (4)

Answer 8

• miRNA is longer than siRNA
• siRNA is fully complementary to target mRNA but miRNA has less than 100% sequence complementarity and typically targets 3’UTR
• miRNAs can target multiple mRNAs but siRNAs have one specific target
• siRNA causes mRNA cleavage but miRNA causes translational repression

Question 9

What happens when siRNA binds to its target mRNA?

Answer 9

Binds with 100% sequence complementarity and causes target mRNA cleavage and degradation

Question 10

What happens when miRNA binds to its target mRNA?

Answer 10

Binds with less than 100% sequence complementarity and mainly causes translational repression, sometimes mRNA cleavage

Question 11

What are the components of the gene silencing mechanism by siRNA and miRNA? (4)

Answer 11

• Drosha
• Dicer
• RISC
• AGO1/2

Question 12

What is drosha?

Answer 12

RNA ribonuclease which cleaves primary miRNA to form pre-miRNA

Question 13

What is dicer?

Answer 13

Ribonuclease III-like enzyme which processes double stranded RNA (dsRNA) into smaller dsRNA fragments (pre-miRNA to miRNA)

Question 14

What is RISC?

Answer 14

RNA-induced silencing complex which interacts with short dsRNAs

Question 15

What is AGO1/2?

Answer 15

Endonuclease and a component of RISC which cleaves the passenger strand

Question 16

What causes fragile X syndrome? (3)

Answer 16

• CGG trinucleotide expansion in the FMR1 gene suppresses its expression and reduces abundance of the fragile X mental retardation protein (FMRP)
• FMRP regulates neuronal connectivity and plasticity
• Causes mental impairment and autism in humans

Question 17

What is the nature of FMRP?

Answer 17

miRNA binding protein

Question 18

What is the impact of overexpression of FMR1 in drosophila?

Answer 18

Rough eyes (ommatidia are damaged and misaligned)

Question 19

What is the impact of overexpression of FMR1 in combination with a mutation which reduces expression of AGO1 complex (part of RISC) in drosophila? (3)

Answer 19

• RISC generates miRNAs
• Suppression of the rough eye phenotype
• Illustrates that FMR1 and AGO1 function in the same miRNA-dependent pathway

Question 20

How is FMRP involved in axon outgrowth? (2)

Answer 20

• FMRP interacts with miR-125a-bound RISC complex which translationally inhibits Map1 and Calm1
• Causes inhibition of axon outgrowth

Question 21

What is the function of Map1 and Calm1? (3)

Answer 21

• Required for neurite outgrowth
• Map1 is a microtubule binding protein
• Calm1 is a calcium signalling protein

Question 22

How does nerve growth factor (NGF) promote axon outgrowth?

Answer 22

Stimulates translation of Map1 and Calm1 by displacing FMRP-RISC to promote axon outgrowth

Question 23

How is FMRP involved in synapse stabilisation? (4)

Answer 23

- FMRP-miR125a RISC binds to PSD-95 mRNA to inhibit translation - PSD-95 is expressed in post-synaptic neurons which stabilises glutamate receptors - Glutamate stimulation causes disassembly of FMRP-miR125a RISC on PSD-95 mRNA via phosphorylation of FMRP - Allows PSD-95 expression and synapse stabilisation

Question 24

What are the main functions of long ncRNAs? (4)

Answer 24

- Gene regulation in cis - Scaffolding of nucleoprotein complexes - Decoys - Silencing of mRNA by forming double stranded complexes with mRNAs (antisense like miRNAs)

Question 25

What does in cis mean?

Answer 25

On the same chromosome

Question 26

How are long ncRNAs involved with gene regulation in cis? (2)

Answer 26

- Transcription through a promoter blocks its function: transcriptional interference - Recruiting histone-modifying enzymes in cis

Question 27

What are examples of scaffolding long ncRNAs?

Answer 27

roX1 and roX2

Question 28

Why are long ncRNAs used as scaffolds instead of proteins?

Answer 28

RNA tethers can be long and varied in sequence creating many binding sites for proteins that can extend for hundreds/thousands of base pairs which allows distinct complexes to be brought together

Question 29

What is HOTAIR?

Answer 29

Antisense RNA complex within the Hox C complex

Question 30

What are the features of HOTAIR antisense RNA? (3)

Answer 30

- Forms a multi stem loop structure - 5' portion binds PRC2 (methylates H3K27) - 3' portion binds LSD1 (DEmethylates H3K4me3)

Question 31

What is the action of PRC2 complex?

Answer 31

Methylates H3K27

Question 32

What is LSD1? (2)

Answer 32

- Histone demethylase - Demethylates H3K4me3

Question 33

What is the function of HOTAIR? (2)

Answer 33

- Binds PRC2 and LSD1 - Switches target loci from transcriptionally active (H3K4 methylated) to inactive (H3K27 methylated)

Question 34

What is the impact of HOTAIR overexpression? (3)

Answer 34

- Functions as an oncogene and promotes tumorigenesis - Suppresses inhibitors of Wnt signalling - Recruits PRC2 to the promoter region of WIF-1 causing silencing so Wnt pathway is uninhibited, allowing proliferation, migration and EMT

Question 35

What is WIF-1?

Answer 35

Wnt inhibitory factor 1

Question 36

What is EMT?

Answer 36

Epithelial to mesenchymal transition

Question 37

What are decoy long ncRNAs? (2)

Answer 37

- Bind to proteins to inhibit their action - E.g. growth arrest-specific transcript 5 (Gas5)

Question 38

What is the cellular response to stress? (4)

Answer 38

- Cortisol binds to glucocorticoid receptor (GR) - Cortisol-bound GR translocates into the nucleus - GR binds to glucocorticoid response elements (GREs) in target genes - Expression of genes involved in increased metabolism

Question 39

What is the action of Gas5? (3)

Answer 39

- Gas5 binds to and inhibits GR so it can no longer enter the nucleus and bind to target genes to cause upregulation of energy metabolism - When cells enter growth arrest they need to conserve energy rather than utilise - Gas5 decoy causes energy conservation as an adaptive response to starvation/metabolic restriction

Question 40

What happens when you remove serum from cells growing in culture? (2)

Answer 40

- Enter growth arrest which can be reversed by adding back the serum - Accompanied by induction of gene expression including Gas5