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genes and gene expression > RNA interference and its applications > Flashcards

RNA interference and its applications Flashcards

1
Q

what is RNA interference?

A

RNAi is the knockdown of gene expression by small RNA fragments

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2
Q

Biological roles of RNAi

A
  • antiviral defense
  • suppress expression of transposons/repetitive DNA
  • gene regulation (silencing)
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3
Q

triggering RNAi

A

by various sources of ds RNA molecules

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4
Q

triggering RNAi; RNA viruses

A

some RNA viruses have dsRNA intermediates in their life cycles

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5
Q

triggering RNAi: transposons

A

DNA transposons that jump into actively transcribed loci (in random orientations) are transcribed into RNA and then hybridise into dsRNA

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6
Q

triggering RNAi; endogenous microRNAs

A

regulated microRNA gene expression to control development

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7
Q

triggering RNAi; exogenous constructs

A
  • experimentally delivered, synthetic long dsRNA or processed siRNA
  • promoter driven expression of a short-hairpin RNA (shRNA)
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8
Q

mechanism of Dicer-mediated dsRNA processing

A

enzyme recognises the termini of a long dsRNA, cleaves both RNA strands and removes a small RNA duplex upon product release

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9
Q

RISC

A

RNA induced silencing complex

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10
Q

what are some known component of RISC?

A
  • Dicer
  • argonaute proteins
  • various RNA binding proteins
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11
Q

what are argonaute proteins?

A

AGO1 and AGO2 contain PAZ domain (conserved domain present in AGO proteins that binds to small RNA helices) as well as C-term PIWI domain that recognises dsRNA

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12
Q

what does Dicer include?

A
  • endonuclease domain
  • dsRNA binding motif
  • DEAD box RNA helicase domain responsible for unwinding the dsRNA
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13
Q

what does AGO2 do?

A

functions as a catalytic enzyme that cleaves the target mRNA at a site that is ~10 nucleotides from the 5’ end of the siRNA guide strand

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14
Q

where have Dicer and AGO homologues been found?

A
  • in drosophila; AGO2 mutants have defects in siRNA-directed RNAi but can undergo miRNA directed RNAi
  • however, AGO1 not required for siRNA-directed RNA cleavage but is required for miRNA-directed RNAi
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15
Q

siRNA biogenesis

A
  • long dsRNA molecules are converted to smaller 21-23 nt siRNAs by Dicer which is an RNase III-type endonuclease
  • this processing step = RNAs with 2 nucleotide overhangs at the 3’ ends and phosphorylated 5’ ends
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16
Q

what can Dicer use to carry out function?

A

linear dsRNA or hairpin RNA- DNA vectors that express hairpin constructs are commonly used in experimental RNAi

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17
Q

antisense strand

A

guide strand, serves as the template for the sequence specific gene silencing

18
Q

sense strand

A

passenger strand

19
Q

what does the guide RNA do?

A

guide strand of the siRNA assembles into the RISC to form RNA protein complex that binds to the target mRNA and silences gene expression by cleaving the target RNA

20
Q

what do mobile genetic (transposable) elements have the capacity to do?

A

integrate into genomes and move from one genomic location to another (often encode proteins enabling them to be mobile)

21
Q

what are mobile genetic elements?

A
  • ubiquitous components of genomes
  • comprise 45% of human chromosomal DNA (most but not all are inactive)
  • cause genetic chanfes
  • controlling transposition of mobile genetic elements is crucial for maintaining genomic stability
22
Q

how would a tranposon trigger RNAi?

A

provides a substrate for Dicer activity and thereby triggers endo-siRNA pathways

23
Q

transposon triggering RNAi can come from 3 different sites

A
  1. actively transcribed region containing transposon
  2. silenced region containing same transposon
  3. a second actively transcribed region
24
Q

what are microRNAs?

A
  • functional miRNA processed from a ~70 nucleotide precursor RNA that= hairpin structure with a few base pair matches at the stem region of the hairpin
25
Q

what do microRNAs need?

A

Dicer (cleaves ds RNA) needed for production of the mature miRNAs from precursor

26
Q

target cleavage

A

achieved by perfect base pairing

27
Q

differences in target recognition between miRNA and siRNA

A
  • miRNA> translation inhibition
  • siRNA > RNA cleavage
  • nucleotides 2-7 of a miRNA are critical for targeting
  • siRNAs hybridise perfectly and cause cleavage of mRNA
28
Q

what is microRNA-21?

A

miR-21 is an oncogene
-frequently upregulated in cancer and majority of its reported targets are tumor suppressors

29
Q

mechanism of RNAi-mediated heterochromatin formation

A
  • generation of small interfering RNAs
  • loading of siRNAs onto argonaute proteins
  • targeting of histone modifying enzymes
  • propagation of heterochromatin
  • maintenance of heterochromatin
30
Q

piRNAs (Piwi-associated)

A

size; ~26-33 nt
protein partners; Piwi proteins (germ cells), Tudor family proteins and helicases

30
Q

biogenesis of Piwi

A
  • derived from transcription of specific gene clusters as well as from transposon mRNA (amplification)
  • Dicer- independent processing from (putative) single stranded transcripts
  • abundantly expressed in germ cells
31
Q

function of Piwi

A
  • germ cell development
  • transposon control in germ cells (preserving germline integrity)
32
Q

MOA of Piwi

A

unclear
- rna destruction, heterochromatin function?

33
Q

practical aspect of RNAi ; biological research

A
  • defining gene function (gene lockdowns)
  • definining biochemical pathways (microarray screening of RNAi knockouts)
  • identifying novel components of signalling pathways
34
Q

practical aspect of RNAi; therapeutic treatment-

A
  • cancer
  • viral infection
  • parasitic infection
35
Q

experimental siRNA

A

induced apoptosis of cervical cancer cells infected with HPV
- E7 gene of HPV encodes a protein that inhibits pRB activity and thereby promotes cell cycle progression and block apoptosis

36
Q

siRNAs and HIV

A
  • RNAi has been demonstrated to stop spread of HIV
  • high mutation rate = problems
37
Q

siRNAs and hep C

A

blocking has been demonstrated in monkeys but it is temporary due to virus mutation

38
Q

siRNAs and cancer

A

alnylam compound in clinical trials for treatment of liver cancer contains 2 siRNAs in lipid nanopracticle (tekmira) targeting KSP and VEGF

39
Q

siRNAs and genetic disease

A

amyotrophic lateral sclerosis treatment targeting superoxide dismutase 1 mutation (which kills motor neurons)

genes and gene expression (14 decks)

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