6/7 - DNA demethylation and RNA interference Flashcards
What are the two types of DNA demethylation?
- Passive (replication dependent)
- Active (replication independent, non-dividing cells)
Give the most supported hypothesis for active DNA demethylation
Involving base excision repair (BER)
It is thought that a TET protein (a deoxygenase) has an important role in oxidizing methyl groups, targeting it for excision repair.
Methylated cytosine can be hydroxylated to 5-hydroxymethylcytosine by TTET1. What is the biological function of 5hmC?
- 5hmC levels are reduced in cancer tissue
- Role in transcriptional regulation and contribution to a ‘poised’ chromatin state
- Reduction of 5hmC in undifferentiated cells associated with increased methylation and cellular differentiation
- Compared to DNA methylation, hydroxy-methylation is more highly expressed in the brain as a stable signal
What are three demethylation intermediates that TET proteins can generate from 5mC?
- 5-Hydroxymethylcytosine
- 5-formylcytosine
- 5-caboxylcytosine
Much less of this than methylated cytosine
Give the active model for DNA demethylation that utilizes thymin-DNA glycosylase (TDG)
- 5mC and 5hmC in DNA are oxidized to 5-carboxylcytosine by Tet
- 5caC is recognized and excised by thymine-DNA glycosylase (TDG)
- THe resulting abasic site in turn induces the base excision repair pathway, leading to the incorporation of unmethylated cytosines
Give two ways that ncRNAs can initiate gene silencing
- Covalent modifications of DNA
- Covalent modifications of associated histone proteins
Both interfere with transcription
How does species ‘complexity’ correlate with ncRNA?
The more complex you are, the more non-coding RNA you have. Even if the amount of coding information has not changed much (which is indeed the case).
What are ncRNAs characterized by?
Their origin and biological function.
Eg:
- Epigenetic modifications (PARs)
- Transposon defense (piRNA)
- Transcriptional regulation (miRNA)
- Post transcriptional regulation (snoRNAs, sdRNA)
- Viral defence (endo-siRNA)
What was the hypothesized function of DNA methylation before epigenetics?
Silencing genomic information from a virus
True or false? siRNA is heritable.
True
They are found in sex gametes etc.
What are small RNAs?
These control RNAs that code protein
- 21-24 nt ncRNAs that generally function in gene silencing
- Modify chromatin
- Or modify mRNA translation or stability
What are the three most important classes of small RNAs in humans? Describe each.
- small interfering RNA (siRNA)
21-22 bp, dicer cleavage of dsRNA, complex with argonaute (in RISC), gene regulation, transposon control, viral defense - microRNA (miRNA)
About 22 bp, dicer cleavage of RNA hairpins, argonaute, post-transcriptional gene regulation - PIWI interacting RNA (piRNA)
Dicer independent, 26-30 bp, germline and somatic cells bordering germline, PIWI-clade argonaute, transposon activity and chromatin state.
Describe Argonaute proteins
Bind small RNAs and their targets. Catalytic components of the RNA-induced silencing complex (RISC) cleave RNA to become single stranded.
What is Argonaute named after?
The argonaute1 mutant of Arabidopsis, ago1 has thin radial leaves that resemble the octopus Argonauta
Describe RNAi from siRNA and miRNA
siRNA: Silencing via post-transcriptional and transcriptional gene silencing
miRNA: mediated slicing of mRNA and translational repression. miRNA does not degrade mRNA, but rather binds to complementary mRNA to prevent translation!
