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L3 Epigenetics > Epigenetic regulation by non-coding RNAs > Flashcards

Epigenetic regulation by non-coding RNAs Flashcards

1
Q

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

A
  • Short (22-28 nt)
  • Long (70 nt-118 kb)
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2
Q

What are examples of short ncRNAs? (3)

A
  • miRNA
  • siRNA
  • piRNA
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2
Q

What are examples of long ncRNAs? (4)

A
  • incRNA
  • snRNA
  • tRNA
  • rRNA
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3
Q

What is the general function of short ncRNAs?

A

Silencing expression (RNA interference)

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4
Q

What is the function of siRNA?

A

Cleaves mRNA (research tool)

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4
Q

What is the purpose of RNAi?

A

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

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5
Q

What is the function of miRNA?

A

Inhibits mRNA translation (in most cases)

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6
Q

What is the function of piRNA?

A

Cleaves products of transposons in germ cells

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7
Q

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

A
  • 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
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8
Q

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

A
  • 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
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9
Q

What happens when siRNA binds to its target mRNA?

A

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

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10
Q

What happens when miRNA binds to its target mRNA?

A

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

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11
Q

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

A
  • Drosha
  • Dicer
  • RISC
  • AGO1/2
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12
Q

What is drosha?

A

RNA ribonuclease which cleaves primary miRNA to form pre-miRNA

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13
Q

What is dicer?

A

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

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14
Q

What is RISC?

A

RNA-induced silencing complex which interacts with short dsRNAs

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15
Q

What is AGO1/2?

A

Endonuclease and a component of RISC which cleaves the passenger strand

16
Q

What causes fragile X syndrome? (3)

A
  • 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
17
Q

What is the nature of FMRP?

A

miRNA binding protein

18
Q

What is the impact of overexpression of FMR1 in drosophila?

A

Rough eyes (ommatidia are damaged and misaligned)

19
Q

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)

A
  • RISC generates miRNAs
  • Suppression of the rough eye phenotype
  • Illustrates that FMR1 and AGO1 function in the same miRNA-dependent pathway
20
Q

How is FMRP involved in axon outgrowth? (2)

A
  • FMRP interacts with miR-125a-bound RISC complex which translationally inhibits Map1 and Calm1
  • Causes inhibition of axon outgrowth
21
Q

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

A
  • Required for neurite outgrowth
  • Map1 is a microtubule binding protein
  • Calm1 is a calcium signalling protein
22
Q

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

A

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

23
Q

How is FMRP involved in synapse stabilisation? (4)

A
  • 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
24
Q

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

A
  • Gene regulation in cis
  • Scaffolding of nucleoprotein complexes
  • Decoys
  • Silencing of mRNA by forming double stranded complexes with mRNAs (antisense like miRNAs)
25
Q

What does in cis mean?

A

On the same chromosome

26
Q

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

A
  • Transcription through a promoter blocks its function: transcriptional interference
  • Recruiting histone-modifying enzymes in cis
27
Q

What are examples of scaffolding long ncRNAs?

A

roX1 and roX2

28
Q

Why are long ncRNAs used as scaffolds instead of proteins?

A

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

29
Q

What is HOTAIR?

A

Antisense RNA complex within the Hox C complex

30
Q

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

A
  • Forms a multi stem loop structure
  • 5’ portion binds PRC2 (methylates H3K27)
  • 3’ portion binds LSD1 (DEmethylates H3K4me3)
31
Q

What is the action of PRC2 complex?

A

Methylates H3K27

32
Q

What is LSD1? (2)

A
  • Histone demethylase
  • Demethylates H3K4me3
33
Q

What is the function of HOTAIR? (2)

A
  • Binds PRC2 and LSD1
  • Switches target loci from transcriptionally active (H3K4 methylated) to inactive (H3K27 methylated)
34
Q

What is the impact of HOTAIR overexpression? (3)

A
  • 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
35
Q

What is WIF-1?

A

Wnt inhibitory factor 1

36
Q

What is EMT?

A

Epithelial to mesenchymal transition

37
Q

What are decoy long ncRNAs? (2)

A
  • Bind to proteins to inhibit their action
  • E.g. growth arrest-specific transcript 5 (Gas5)
38
Q

What is the cellular response to stress? (4)

A
  • 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
39
Q

What is the action of Gas5? (3)

A
  • 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
40
Q

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

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

L3 Epigenetics (15 decks)

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