Lecture 6 - Regulatory Mechanisms For Quality Control And Turnover Of Pre-mRNA Flashcards
Why do RNA’s need to be degraded - homeostatis and quality control?
Some are bi-products of transcription and need to be removed and resources recycled
They weren’t made correctly and could damage cell if made into a protein
Why do RNA’s need to be degraded? - gene regulation
If they never went away how could you have gene regulation
Highly regulated mRNA’s tend to have short half lives
How do exonucleases degrade RNA?
From the end 3’ - 5’ or 5’ - 3’
How do endonuclease degrade RNA?
Internally
What is the major human 5’ - 3’ exonuclease present in the nucleus?
Xrn2
What does xrn2 degrade?
Transcriptional termination of Pol II and pre-mRNA degradation (when splicing is disturbed or decapping has occurred).
Where does xrn2 degrade?
Cytoplasm
What needs to happen before xrn2 can degrade?
Decapping
What is the major 3’ - 5’ exonuclease activity in the nucleus an cytoplasm?
The exosome
What is the exosome involved in?
Processing and degradation
Where is most RNA degraded in?
The nucleus
When can RNA be degraded?
During or after its synthesis
Do introns need to be degraded?
Yes
What happens in the quality control pathway after the start of RNA transcription?
Possible attenuation, capping, splicing and 3’ end cleavage, possible RNA editing and nuclear export
What happens when RNA transcriptions goes wrong after the start of transcript?
RNA transcript aborts, nonfunctional mRNA sequences, retention and degradation in the nucleus.
See other cue cards
Degradation of proper mRNA’s - what do you need to do to do this?
Get rid of polyA tail (deadenylase) and 5’ cap
What happens first poly A tail degradation or the cap?
PolyA degradation
Is the polyA tail in the proximity of the 5’ cap?
Yes
Regulated turnover - what is this?
mRNA was made properly but it is no longer needed
What is a half life?
The time it takes for half of a message to go away
The untranslated region - Why is the 3’ UTR so important?
Encodes regulatory features, RNA binging proteins can bind here as can microRNA binding sites
What can affect deadenylation if the 3’ UTR?
Specific proteins and specific RNPs
How are RNA’s recognised by specific proteins?
Through contact or through proteins recognising sequences
Where are ARE-mediated mRNA found?
In the 3’UTR
What recognises the ARE?
Proteins which stabilise (HUR) and destabilise (TTP) the mRNA
Non-ARE mediated mRNA can also be involved with stabilising and destabilising of mRNA in the 3’UTR what are these and do they stabilise or destabilise?
UCR elements are recognised by PTB which stabilises
Stabilisation/destabilisation of mRNA via 3’UTR = how does miRNA- mediated mRNA happen?
MiRNA regulators work in a risc complex. MiRNA binds to argonot which takes it to the mRNA promoting deadenylation.
What are the ARE’s which destabilise mRNA?
TTP, KSRP and AUF
What are the ARE’s which stabilises mRNA?
HUR and sometimes AUF
The factors which stabilise or destabilise mRNA aren’t regulated by signaling, true or false?
False - they are
How do the stabilising or destabilising factors work?
By recruiting exonucleases
microRNA regulation of mRNA stability - how are these generally transducer?
As pol II transcripts found in introns
microRNA regulation of mRNA stability - what do these bind to?
Complementary sequences
Reporter assays - how are these done - step 1: promoter insertion and reporter
You add a promoter you are interested from your mRNA to a reporter gene (this is not your mrna). You would put it either upstream or downstream of the reporter depending on where the promoter is
Reporter assays - how are these done - step 2: transfect
You transfect it into cells and incubate it until protein is formed. You then measure the protein or the mRNA over different timeframes
Why do we use reporter assays?
To test the importance/ regulation of mRNA in different contexts e.g. we can see if a gene is getting down regulated.
What are the controls of the reporter assay?
Transfection of another reporter to make sure you know how much reporter you are getting in.
What are the requirements of a reporter assay in terms of something that can be quantified?
Luciferase, GFP and lacZ
What is a caveat of reporter assays?
Sometimes the reporter does not integrate into the native location of the gene in the genome and therefore you can’t see any interesting effects.
Cell lines should also be mimicked in its natural environments
How do you measure promoter activity?
Reporter assay by making numerous deletions upstream of the start site and put each into the reporter plasmid turning it into DNA. You then transfect it into a eukaryotic cell. You can then see what important for transcription
When infected the promoter of luciferase drops and when you stop transcription the promoter of luciferase also drops - what does this mean?
When transcription stops luciferase promoter drops meaning that infection stops transcription.
What would you expect to see if the RNA had a long half life after turning off transcription?
Rna still being present
How do you measure the half life of a mRNA?
Stop transcription and measure how long the mRNA lasts
How do you stops transcription?
Inhibitors to block
What inhibitors block transcription?
Actinomycin D
Alpha-amanitin
How does actinimycin D strop transcription?
intercalated into DNA and blocks transcription and elongation
How does alpha-amanitin stop transcription?
Binds to subunit of RNA pol II
The steps of measuring half life of mRNA?
Add a inhibitor to stop transcription at time 0. Then harvest rna at different time points and measure it. Make a graph and determine the point at which you have half the amount of mRNA than you started with
What is the caveat to using inhibitors to block transcription?
They are toxic to a cell
What effects the half life of mRNA?
Elements in the UTR