Antisense RNA Flashcards
Antisense Definition
shorter fragments of nucleic acid complimentary to the sense strand
Antisense function
blocks mRNA (sense strand) blocks translation and expression --> reduction protein also alter mRNA production/turnover
Ribozymes
Catalytic RNAs/enzymes
endonucleases
anti-sense elements dictate
Antisense can change behaviour of mRNA by (3)
block protein binding
disrupt RNA structure
target RNA for degradation
Conserved sequences
5’ and 3’ splice sites
branch site,
these define the limits of introns
—>recruit the splicing machinery required to remove intron and join exons
Step 1 of splicing
cut at 5’ splice –> bonds branch site
Step 2 of splicing
Cut at 3’ splice site to form ‘lariat’ (loop)
ligation of 2 exons
Translation mechanism
complex around 5’ prime end of mRNA and ribosome sub unit
eiF4E and G bind cap
eiF4G bind polyA binding protein at 3’ end - circularise mRNA
Circularised mRNA leads to
Inintiation linked to termination
Natural anti-sense RNA is due to
bi-transcription of genes
e.g. Msx1 - murine tooth development
Frq - cicardian rhythm
Therapeutic use of antisense
correct defects in expression from mutations
e.g. thalassaemia
Thalassaemia
splicing in wrong place
incorporation of introns
Duchenne Muscular Dystrophy
mutation in 23 stop codon
antisense used to create shortened dystrophin instead of truncated
Spinal Muscular Atrophy
Mutation in exon 7
exclusion of exon - enhance sequence element mutation
unstable, shorter form
mRNA
antisense brings in SF2/ASF protein blocked by mutation by binding to it
Most mutations
stop mRNA and protein being produced
unstable
Antisense problem
unstable
difficult to transfer to cell
natural mechanisms in cell block function and degrade
Solutions to antisense problems
modify ribose/backbone
change phosphate
change hydrogen
Morpholino
ring structure instead of ribose
Benefits of modified nucleic acids (3)
stability
easier delivery
not recognised as nucleic acids - not sequestered
Produce antisense in cell
by introducing gene in cell that does so
e.g. U7 sRNA
Ribozyme action (5)
RNA ligation and cleavage RNA virus genomic replication Intron splicing cleave tRNA peptide bond formation
Hammerhead ribozyme (4) example function
plant RNA viruses
cleave individual genomic RNAs during rolling cell replication
produce long linear genome
usually cis cleavage
Hammerhead structure
T junction
cleavage site at H
Adapt hammerhead (2) to
by…
possible uses
to trans
sequence stems I and II to target any sequence
possibly use in cancer and HIV
Aptamers/decoys
not antisense
short RNA sequence - bind specific substrate
block activity
engineered
SELEX used to select interacting molecule
SELEX definition
Systematic evolution of ligands by exponential enrichment
SELEX procedure (5) evolution
random RNAs bind to target enrich RT into DNA template select RNA bind tightly
Inhibit HIV replication by aptamer
specific protein
TAR decoy aptamer
bind TAT protein
Aptamer use (4) examples
target validation
diagnostic
therapeutics
screening
RISC definition
RNA induced silencing complex
protein and miRNA/siRNA
30nt pair dsRNA does not
not induce interferon response
siRNA (4)
features
21-23 nucleotide
perfect complimentary to RNA
viral defence mech
degradation RNA - endonucleolytic cleavage
miRNA (4)
features
21-23 nucleotides
imperfect complimentary
gene regulatory mechanism 33- 80%
block in translation - deadenylation, decapping and degradation
Pri-miRNAs —> miRNA/siRNA (4)
longer precursor cut by Drosha
recognised and cut by Dicer
recruit Ago with other factors –>RISC
Ago drives repression of translation
miRNA translation
RNA pol II and III
independent genes or within pre-mRNA introns
Multi-miRNA precursor cleaved to individual
dsRNAs (3)
part immune defence against exogenous genetics
e.g. transposons and viruses
both produce dsRNA
viruses can suppress RNAi
DICER domains
dsRNA binding domain
Paz domain
2 RNAse III domain - catalytic
Paz domain of dicer
recognise 3’ end
preferentially 3’ overhang
used to measure where to cleave - molecular ruler principle
position RNAse III domains
RNAse III domain dicer
2 active sites
cut RNA each side
RISC function
directs either mRNA cleavage or translational repression
catalytic component - Argonaute
Piwi domain - cleaves mRNA
Piwi domain of argonaute
resembles RNase H
PAZ domain positions it
Dicer complex hands to
Argonaute complex
Cap - dependent regulation translation (4)
micro RNA bind 3’ end untranslated between stop codon and polyA tail
- displace eiF4E and G
argonaute interacts with miRNA and cap
recruit enzyme for degrad
Direct regulation translation
inhibition
Indirect regulation translation
de-adenylation
after miRNA regulation action
move to P bodies
mRNA degradation
miRNA production linked when cell is
stress –>
oxidative stess
AA starvation
siRNA and miRNA delivery
transfection
transcription of two complimentary RNA/loop structure
cojugation with cholesterol/liposomes/peptide, polymers/antibodies
nano particles
viral vectors
stem
aptamer conjugation - bind extracell receptor
siRNA design
modified 2’ base overhangs
shRNA can be
taken up by cells and used for function
Viral delivery siRNA/miRNA
lentiviral delivery
or adenoviral
Multiple treatment for HIV (3)
ribozyme target cell mRNA - resistant/slow progression
TAR decoy target viral protein - bind and inactivate
shRNA target viral RNAs - inhibit
