DNA Demethylation and RNA Interference (RNAi)

passive DNA demethylation

replication dependent

occurs due to lack of maintenance methylation during several cycles of DNA replication (e.g. zygotic maternal genome, after fertilization; dividing cells, such as glial cells)

active DNA demethylation

replication independent

occurs in the absence of DNA replication (e.g. zygotic paternal genome, after fertilization; non-dividing cells, such as neurons)

demethylation pathways may involve significant overlap among

DNA repair proteins and pathways used

active DNA demethylation in neurons general overview

the removal of 5-methylcytosine (5mC) via the sequential modification of cytosine bases that have been converted by TET enzyme-mediated oxidation

active DNA demethylation in neurons, detailed

the ten-eleven translocation (TET) is a family of 5mC hydroxylates (e.g. TET1, TET2, and TET3)

TET proteins convert 5mC to 5-hmC, 5-hmC to 5-fC, and 5-fC to 5-caC through hydroxylase activity

5caC is recognized and excised by the enzyme thymine-DNA glycosylase (TDG)

resulting abasic site in turn initiates base excision repair (BER), a cellular mechanism that repairs damaged DNA throughout the cell cycle

BER processing leads to the incorporation of an unmethylated cytosine

biological functions of 5hmC

levels are reduced in cancer tissues compared to normal tissues

this-containing DNA shows strong enrichment within exons and near transcriptional start sites. this indicates that it has a likely role in transcriptional regulation and could contribute to a poised chromatin state

in undifferentiated, embryonic stem cells a reduction of this is associated with increased methylation and cellular differentiation

only _____ of the mammalian genome encodes mRNA (protein-coding transcripts)

2%

vast majority of genome (long regarded as junk) encodes

functional long and short non-protein encoding RNAs (ncRNAs) species

ncRNAs

contribute to transcriptional and post-transcriptional gene silencing, chromosome dosage compensation and allelic exclusion, germ cell reprogramming and para-mutation, all of which involved epigenetic processes

can initiate gene silencing through covalent modifications of the DNA or its associated histone proteins, interfering with transcription

ncRNAs are characterized by

their origin and biological function

small RNAs

control RNAs that code protein

are a pool of 21-24 nt 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 endogenous siRNAs, miRNAs, and PIWI-interacting RNAS (piRNAs)

RNA silencing uses a set of core reactions in which

dsRNA is processed by Dicer or Dicer-like proteins into short RNA duplexes

short RNA duplexes subsequently associate with

ARGONAUTE proteins to confer silencing

Dicer or Dicer-like (DCL) proteins

cleave long dsRNA or foldback (hairpin) RNA into ~21-25 nt fragments

structure allows it to measure the RNA it is cleaving, chops into uniformly-sized pieces

argonaute proteins

bind small RNAs and their targets

catalytic components of the RNA-induced silencing complex (RISC)

siRNA interference

mediated silencing via post-transcriptional and transcriptional gene silencing

exogenous dsRNA that are transfected into cells (e.g. through vectors, viruses, etc) and bind perfectly to their mRNA target

miRNA interference

mediated slicing of mRNA and translational repression

endogenous ssRNA—made inside the cell from ncRNA found within the introns of larger RNA molecules—and can inhibit translation of many different mRNA sequences because their pairing is imperfect in mammals

RNA interference

an important pathway that is used in many different organisms to regulate gene expression, involving siRNAs and miRNAs to silence specific mRNAs in the cytoplasm

similarities with siRNA and miRNA

both are processed inside the cell by the enzyme Dicer and incorporated into a RISC complex and play a role in epigenetics through RNA-induced transcriptional silencing (RITS)

conserved seed sequences of miRNAs

contain a short, this sequence which is essential for the binding of miRNAs to their mRNA targets

conserved heptametrical sequence which is mostly situated at positions 2-7 from the miRNA 5’ end

even though base pairing of miRNAs and their target mRNAs do not match perfectly, the short, this sequence is perfectly complementary

so have multiple targets

control of gene expression involves the control of

transcription initiation

gene expression can be controlled after transcription by mechanisms such as

RNA interference, alternative splicing, RNA editing, mRNA degradation, protein degradation

alternative splicing

introns are spliced out of pre-mRNAs to produce the mature mRNA that is translated

this recognizes different splice sites in different tissue types

mature mRNAs in each tissue possess different exons, resulting in different polypeptide products from the same gene

RNA editing

creates mature mRNA that are not truly encoded by the genome

RNA editing example

apolipoprotein B exists in 2 isoforms

one isoform is produced by editing the mRNA to create a stop codon

this is tissue specific (apoB-100 in liver, B48 in intestine)

mature mRNA molecules

have various half-lives depending on gene and location (tissue) of expression

amount of polypeptide produced from a particular gene can be influenced by the half-life of this

protein degradation

proteins are produced and degraded continually in the cell

proteins to be degraded are tagged with ubiquitin

degradation of proteins marked with ubiquitin occurs at the proteasome

lysosomes also degrade protein

proteasome

free enzyme complexes in the cytoplasm

lysosomes

membrane-bound vesicle sacs (bound by a single-membrane)

degrade protein by endocytosis, phagocytosis, and autophagy