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GBB > RNA Interference > Flashcards

RNA Interference Flashcards

1
Q

Passive demethylation

A

-DNA demethylation (replication dependent) occurs due to lack of maintenance methylation during several cycles of DNA replication

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2
Q

Active demethylation

A

-DNA demethylation (replication independent) occurs in the absence of DNA replication

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3
Q

Proposed pathways for active DNA demethylation

A
  1. Hydrolytic demethylation
  2. Di-nucleotide excision
  3. 5meC excision
  4. Deamination and thymine excision

-these demethylation pathways may involve signification overlap among DNA repair proteins and pathways used

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4
Q

Active DNA demethylation in neurons

A

-removal of 5-mC via sequential modification of cytosine base that have been converted by TET enzyme-mediated oxidation

  • TET is a family of 5mC hydroxylates
  • TET proteins convert 5mC to 5hmC to 5fC to 5caC through hydroxylase activity
  • 5caC is recognized and excised by enzyme TDG
  • the resulting abasic site in turn initiates base excision repair (repairs DNA through cell cycle)
  • BER processing leads to incorporation of an unmethylated cytosine
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5
Q

Biological functions of 5hmC

A
  • 5hmC levels reduced in cancer tissues compared to normal tissues
  • 5hmC containing DNA shows strong enrichment within exons and near transcriptional start sites
    • likely role in transcriptional regulation and could contribute to a poised chromatin state
  • in undifferentiated embryonic stem cells a reduction of 5hmC is associates with increase methylation and cellular differentiation
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6
Q

RNA interference in nucleus

A
  • only 2% of mammalian genome encodes mRNA
  • majority of genome encodes functional long and short non-protein coding RNAs (ncRNAs)
  • ncRNAs contribute to transcriptional and post transcriptional gene silencing, chromosome dosage compensation and allelic exclusion, germ cell reprogramming and paramutation
    • all involve epigenetic processes

-ncRNAs can initiation gene silencing through covalent modifications of DNA or its associated histone proteins interfering with transcription

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7
Q

NcRNAs characterized by origin and function

A
  • PARs = promoter associated RNA
  • lncRNA = long coding RNA
  • miRNA = microRNA
  • snoRNA = small nucleolar RNA
  • sdRNA = SNP-derived RNA
  • endo-siRNA = endogenous siRNA
  • piRNA = PIWI-interacting RNA
  • tiRNA = transcription initiation RNA
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8
Q

MiRNA function

A
  • CNS processes
  • molecular functions
  • life cycle
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9
Q

Small RNAs control RNAs that code proteins

A
  • small RNA are a pool of 21-24nt ncRNAs that generally function in gene silencing
  • contribute to transcriptional gene silencing through epigenetic modifications to chromatin
  • contribute to post-transcriptional gene silencing by affecting mRNA translation or stability
  • important classes in humans include si-RNAs (700nt), miRNA (100nt) and piRNA
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10
Q

Dicers and argonauts

A
  • RNA silencing uses a set of core reactions in which double stranded RNA is processed by diver or diver-like proteins into short RNA duplexes
  • small RNAs subsequently associate with argonaute proteins (catalytic components of the RNA induced silencing complex - RISC - to confer silencing)
  • dicer proteins cleaves long dsRNA or foldback (hairpin) RNA into 21-25nt fragments
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11
Q

Argonaute proteins

A
  • bind small RNAs and targets
  • named after argonaute1 mutant of arabidopsis

-it is the catalytic component of RISC

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12
Q

RNAi

A
  • siRNA mediated silencing via post-transcriptional and transcriptional gene silencing
  • miRNA mediated silencing of mRNA and translational repression
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13
Q

Endogenous and exogenous RNAi

A

-important pathways that is used in many different organisms to regulate gene expression involving siRNAs and miRNAs to silence specific mRNAs in cytoplasm

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14
Q

siRNA vs miRNA

A
  • both are processed inside cell by enzyme Dicer and incorperated into RISC complex and play a role in epigenetics
  • siRNA are exogenous ds-RNA that are transfection INTO cells and bind perfectly to the mRNA target
  • miRNA are endogenous single strand RNA made inside the cell from ncRNA found within introns and can inhibit translation of many different mRNA sequences because their pairing is imperfect in mammals
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15
Q

Conserved seed sequence of miRNA

A
  • miRNAs contain a short conserved seed sequence which is essential for binding of miRNAs to their mRNA target
  • situated at position 2-7 of miRNA. 5’-end
  • even though base pairing dont match perfectly, the short, conserved sequence is perfectly complementary
  • while siRNAs are specific, miRNAs have many targets
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16
Q

Coupling transcriptional and post-transcriptional regulation

A
  • control of gene expression involves control of transcription initiation
  • gene expression can be controlled after transcription by
    • RNE interference
    • alternative splicing
    • RNA editing
    • mRNA degradation
    • protein degradation
17
Q

Post transcriptional regulation

A
  • RNAi involves the use of small RNA molecule
  • Dicer chops dsRNA into small RNA
    • miRNA bind to complementary RNA to prevent translation
    • siRNA degrade particular mRNA before translation
18
Q

Post-transcriptional regulation cont

A
  • introns are spliced out of pre-mRNA to produce the mature mRNA
  • alternative splicing recognizes different splice sites in different tissue types
  • mature mRNA in each tissue possesses different exons resulting in different polypeptide products from same gene
19
Q

RNA editing

A
  • creates mature mRNA that are not truly encoded by genome
    • apolipoprotein B exists in 2 isoforms
    • one is produced by editing mRNA to create stop codon
    • this RNA editing is tissue specific
  • mature mRNA molecules have various half lives depending on gene and location of expression
    • amount of polypeptide produced from a particular gene can be influences by the half-life of the mRNA molecules
20
Q

Protein degradation

A
  • proteins are produced and degrades continually in cell
  • proteins to be degraded are tagged with ubiquitin
  • degradation of proteins marked occurs at the proteasome, which are free enzyme complexes in the cytoplasm
  • lysosomes are membrane bounds vesicle space and also degrade protein by endocytosis, phagocytosis and autophagy

GBB (9 decks)

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