L15 - Control of Gene Expression - RNA Flashcards
What 3 things can achieve different isoforms?
Alternative splice sites
Start sites
Poly-adenylation sites
What is subcellular localisation used for?
Used to target translation to the part of the cell its needed
What 2 things can translation be regulated by?
Directly by sequences in untranslated regions
Globally by regulation of eIFs
Some mRNAs have a second open reading frame that can be?
Regulated independently
What % of Drosophilla and human genes are alternatively spliced?
40% Drosophila
75% human
What are the 4 types of alternative splicing?
Optional exon
Optional intron
Mutually exclusive exon
Internal splice site
Why are splice donor and acceptor sequences found so frequently?
They are only two bases
What is a cryptic splice site?
A site that is favoured over neighbouring splice sites
What else effects the choice of splice site in RNA?
Other sequences
Regulation of alternative splicing - sex determination in Drosophila genes
Sxl – sex lethal
Tra – transformer
Dsx – double sex
Sex determination in Drosophila males method
Transcripts for sxl and tra are spliced to produce inactive isoforms
Transcripts for dsx are active and spliced to produce a male specific repressor protein
- Represses transcription of genes required for female development
Sex determination in Drosophila females method
Two X chromosomes allow a small amount of active Sxl protein - alternative promoter
Sxl represses splicing by blocking binding by U2AF
This feeds back on the sxl transcript to make more of itself and also binds to tra transcripts
Results in a female specific isoform of Dsx
Site of polyadenylation on mRNA - B lymphocytes
They produce two antibody isoforms
Antibody gene has two positions for cleavage and polyadenylation of RNA transcript
Site of polyadenylation - when the cell produces the long transcript
First stop codon is spliced out
Results in the translation of a transmembrane domain
Site of polyadenylation - when the cell produces the short transcript
Splice acceptor is lost and first stop codon isn’t lost
Results in the antibody being secreted
Alternative start sites - optimal sequence
Kozak sequence - ACCAUGG
What happens if the start sequence is not perfect - leaking scanning
Small ribosome can scan past the first AUG
- Stops at a second or third AUG
All in the same reading frame, so isoforms differ only by the sequence of there N-terminus
What favours the first AUG?
High levels of eIF-4F in a cell
Regulated nuclear transport - regulating mRNA - HIV small genome integration
HIV has a small genome that is integrated into the host genome
After integration the entire genome is transcribed in one piece
- Alternative splicing allows for many different protein products to be made
Regulated nuclear transport- why is full length RNA needed to make new virons?
Un-spliced RNAs cannot leave the nucleus
Rev protein binds to HIV introns and interacts with nuclear pore to allow unspliced RNA to leave
Rev levels distinguish two phases of infection
What is Rev protein involved in?
Involved in binding to HIV introns to allow unspliced RNA to leave
Signals in the untranslated region of mRNA can target it to part of the cell – regulating mRNA
Intermolecular base pairing within the 3’ UTR forms stem loops
These are recognized by cellular proteins
Gives rise to localised translation
Translated control elements in mRNA - regulating mRNA - ferritin
Stores iron in the cell - reducing the available Fe
Translated control elements in mRNA - regulating mRNA - transferrin
Receptor imports iron into the cell - increasing the available Fe
Aconitase if low Fe in cytoplasm method
Binds to stem loops in the 5’ UTR of ferritin mRNA and blocks translation
Binds to stem loops in the 3’ UTR of transferrin mRNA and blocks its degradation
Aconitase if high Fe in cytoplasm method
Binds to iron in the cytoplasm and goes through a conformational change Releases the mRNAs - Ferritin mRNA translated - Transferrin mRNA degraded Rapid and strong regulation
Global regulation by eIF2 and eIF2B – regulating translation
If the cell is…. it turns down global translation by phosphorylating eIF-2
o Entering G0
o Infected by a virus
o Lacking nutrition
This causes eIF-2b to bind to eIF-2 very tightly, blocking its recycling
eIF-2 and GTP
Active - binds to Met tRNA to start ribosome scanning
eIF-2 and GDP
Inactive
What is eIF-2b required for?
The dissociation of GDP from eIF-2 to activate it
What are IRES?
Internal ribosome entry sites are stem loops in RNA
IRES allow more than one gene to be present on mRNA – regulating translation
They can initiate formation of the ribosome independent of the cap/polyA initiation complex
eIF-4G
Is required for IRES based initiation
Binds to the IRES stem loop
Where are IRES often found?
In viral transcripts
Viruses favour translation of their transcripts by cleaving eIF-4G into a form that cannot bind eIF-4E but still binds IRES
What happens to eIG-4G during apoptosis?
Cleaved into a form that cannot bind eIF-4E but still binds IRES
Certain genes required during cell death utilize IRES and they continue to be translated
RNA stability - regulating transcription - RNA degradation
The half-life of different mRNAs varies greatly
Poly A tails start at 200 in length, but an exonuclease cuts them down to 30nt
- At this point they are decapped and degraded
What is re-adenylation?
Some mRNAs are re-adenylated in the cytoplasm to activate them or to extend half-life
Which factors promote translation but block degradation?
DAN competes with eIF-4E for binding to the cap
