euk RNA - polyadnelyation and splicing Flashcards
why is regulating gene expression important in organisms?
particularly in complex organisms like eukaryotes, they can regulate when where and the amount of protein produced
TIME, SPACE AMOUNT
describe ways in which you can post transcriptionally modify RNA?
only EUKARYOTES undergo RNA processing/post transcriptional modifications
- splicing of introns- dependent or independent
- 5’ capping, 3’ polyadenlyation
- RNA editing - apop48/100, amylotrophic lateral schlerosis
and then back splicing, adenosine methylation and RNA editing
what does RNA processing mean?
any modification to the mRNA molecule following transcription (only eukaryotes)
> generally refers to 5’capping, 3’polyAdenylation, splicing, rna editing
what is the importance of alternative polyadenlyation?
> most APA sites found in the 3’ UTR
so mRNA is same length but the stability or translation efficiency can be regulated this way
> contribute to genetic diversity by increasing size of the proteome/creating different mRNA transcripts if APA signals are found throughout the mRNA
what is the biological importance of polyadenlylation?
> protects pre-mRNA from
being degraded by exonuclease activity
> can help efficiency of translation by using the PABP which creates a closed loop mRNA and tethers eIF4F to the start of mRNA
distinguish between mutations and RNA editing
mutation - change in the DNA base sequence
RNA editing- change to the open reading frame of the mRNA molecule
so RNA editing is a form of epigentic modification
describe mechanisms in which RNA editing can occur
via base deamination where the amino group is replaced by an oxygen
> uses specific deaminases A-i C-U
or INDELS which then create a new open reading frame for that RNA transcript
why is 5’ capping important in premRNA?
can help to stabilise mRNA and protect it from exonucelase degradation
aids with splicing and polyadenylation
also crucial for translation initiation as it acts as a docking station for ribosome. enables the eIF4E to bind to the 5’ end of pre mRNA transcript
which of the following uses a spliceosome
A alternative polyadenylation
B spliceosome dependent splicing
C rna editing
D spliceosome independent splicing
OPTION B
spliceosome is a protein:rna complex
> made up of 5 rna which then combine with proteins to create 5 different snRNPs
what does snRNA mean?
small nuclear RNAS - u1,2,4,5,6
small nuclear ribonucleoproteins = spliceosome components for spliceosome dependent splicing
which of the 6 RNA PTM processes occur cotranscriptionally?
5’ cap - quite quick, after 40nt have been transcribed
Splicing - of the first intron only
RNA editing - quite a quick process
which of the 6 RNA PTM processes occur post-transcriptionally?
Splicing - the whole gene body
Adenine meth
Backsplicing - exon spliced to itself in 3-5 direction
polyadenylation (cleavage)
how does the POLYAA help with efficient translation
PABP helps with nuclear export
once in cytoplasm exchanges with other cytoplasmic proteins allowing 3’ and 5’ to interact
allows cirularisation of mRNA
what is the difference between UTR-APA and CR-APA
APA sites in different parts of the gene body, either coding or nc regions
UTR-APA will change length of the final UTR which affects stability and localisation
CR-APA is upstream of the stop codon so the final protein is trunchated
outline an example of CR-APA
For IgM if distal PAS used then longer membrane bound protein made
proximal PAS used then protein lacks transmembrane domain and IgM is secreted instead
outline an example of UTR-APA
Brain derived neurotrophic factor has 2 APA which determines ^mRNA localisation^
short UTR = mRNA cell body
long UTR = mRNA in dendrites
how do we identify APA? (experimental methods)
use northern blot to spot different sized mRNA molecules
use FISH to see where the mRNA localise/ if this changes
use NextGen/high throughput of cDNA with primers for polyAAA
how is APA regulated? (how to choose if proximal or distal APA site is used)
generally distal PAS have a stronger concensus sequence
rate of transcription can regulate which site is used
slow = proximal
fast = distal
regulatory RNA binding proteins can also alter ^accessibility^ of the PAS
what is the role of spliceosome proteins?
recognise the 3’ splice site and stabilise base pairings between RNA-snRNA
able to remodel spliceosome
where are the splice site consensus sequences found?
5’ site GU
3’ AG
branchpoint adenosine 30nt upstream of the 3’ intron
how is splicing regulated?
there are spliceosome enhancers and silencers
which INDIRECTLY alter spliceosome assembly by binding to RNA-BP
exon = exon-splicing enhancers generally found
intron = intron-splicing silencer
how can ESE and ISS cis-acting sequences be implicated in disease?
if they are mutated, this can lead to alterations in splicing and overall protein function may be trunchated/non functional
why is splicing important biologcally?
allows diversity of proteins to be generated (via Alt splice)
allows for normal gene expression
what is the spliceosome? What is its function?
large highly dynamic RNA-protein complex
spliceosome allows the 2 transesterficiation reactions to happento remove intron and ligate 2 exons together
why do the snRNAs have an extensive secondary structures?
they allow the active site of the spliceosome to form ready for the transesterifaction reactions
name some examples of spliceosome proteins - what are their functions?
prp8 = largest and most conserved. forms a cavity to hold the snRNAs
prp28 = helicase activity and can remodel RNA-RNA/RNA-protein interactions
prp2 = displaces the SF3b protein, freeing the A-branchpoint
how do self splicing introns differ from spliceosomal introns
self splicing introns DONT use the spliceosome/proteins/RNA to catalyse
instead the intron is autocatalysed using ^ribozymes^
how could stemloop/2nd structure affect splicing?
could block the splice site so exon is not included
could affect spliceosome helicase activity and make it harder to unwind
how do group 2 self splicing introns splice?
they contain a gene encoding for ‘‘intron-encoded-protein’’
which has helicase, r.transcriptase and endonuclease activity so can self-splice the intron easily
how does prp8 and IEP (intron encoded protein) relate?
IEP within bacteria resided in archeal host (endosymbiotic)
group 2 introns able to integrate into archeal genome using IEP.
eventually group 2 introns lost their catalytic ability leaving prp8
what is the advantage of AS?
A.S is implicated in many human diseases
A.S can give rise to protein diverity, important in embryonic development
which of the A.S mechanisms is most common?
A intron retention
B alt. splice sites
C exon skipping
D mutually exclusive exons
C exon skipping
as seen in the insulin receptor alpha subunit which alters its sensitivtiy
switches in foetus and adult
which of the A.S mechanisms is least common?
A intron retention
B alt. splice sites
C exon skipping
D mutually exclusive exons
A intron retention
intron is not spliced out and retained in coding sequences
this is likely to induce degradation or trunchation of mRNA molecule
how can splicing be regulated by enhancers/silencers?
ESE/ISE can create stronger splice sites so more likely to be recognised by spliceosome
how can splicing be regulated by rate of transcription?
Could lead to exon skipping
rapid transcription then less time for spliceosome to assemble
this could encourage exon skipping if the concensus is weak
how could secondary structure of emerging mRNA transcript affect splicing?
secondary structure of mRNA can influence
e.g if Alu sequence, both base pairs then can block access of splice site
so exon is skipped
what are the 2 major types of splicing associated diseases?
cis-acting
> single gene is affected (splice site changes, splicing enhancer/silencer changes)
trans-acting
> mutated spliceosome components so multiple genes are affected or abnormal RNA are crreated
what causes splice site inactivation? What are the consequences?
a new stronger cryptic splice site could be created
which could create trunchated protein or intron retention
or complete inactivation of splice site could lead to exon skipping
what causes splicing enhancer inactivation? What are the consequences?
synonomous mutations could inactivate splicing enhancers
this could lead to exon skipping
what is a synonomous mutation?
don’t affect amino acid sequence/ silent mutations BUT can affect gene expression/protein structure
as the interactions between cis and trans acting elements regulatory elements may be altered
(UGC and UGU are still cysteine)
how can trans-acting mutations affect splicing ? What are the consequences?
The different proteins/RNA molecules could me mutated and negatively affect spliceosome assembly
So many genes affected
how could we treat splicing diseases?
could use anti-sense oligonucleotides and target denovo or cryptic splice sites
to mask or activate their effects
