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GENES TO GENOME > euk RNA - polyadnelyation and splicing > Flashcards

euk RNA - polyadnelyation and splicing Flashcards

1
Q

why is regulating gene expression important in organisms?

A

particularly in complex organisms like eukaryotes, they can regulate when where and the amount of protein produced
TIME, SPACE AMOUNT

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2
Q

describe ways in which you can post transcriptionally modify RNA?

A

only EUKARYOTES undergo RNA processing/post transcriptional modifications

  1. splicing of introns- dependent or independent
  2. 5’ capping, 3’ polyadenlyation
  3. RNA editing - apop48/100, amylotrophic lateral schlerosis

and then back splicing, adenosine methylation and RNA editing

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3
Q

what does RNA processing mean?

A

any modification to the mRNA molecule following transcription (only eukaryotes)
> generally refers to 5’capping, 3’polyAdenylation, splicing, rna editing

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4
Q

what is the importance of alternative polyadenlyation?

A

> 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

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5
Q

what is the biological importance of polyadenlylation?

A

> 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

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6
Q

distinguish between mutations and RNA editing

A

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

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7
Q

describe mechanisms in which RNA editing can occur

A

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

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8
Q

why is 5’ capping important in premRNA?

A

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

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9
Q

which of the following uses a spliceosome
A alternative polyadenylation
B spliceosome dependent splicing
C rna editing
D spliceosome independent splicing

A

OPTION B

spliceosome is a protein:rna complex
> made up of 5 rna which then combine with proteins to create 5 different snRNPs

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10
Q

what does snRNA mean?

A

small nuclear RNAS - u1,2,4,5,6

small nuclear ribonucleoproteins = spliceosome components for spliceosome dependent splicing

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11
Q

which of the 6 RNA PTM processes occur cotranscriptionally?

A

5’ cap - quite quick, after 40nt have been transcribed

Splicing - of the first intron only

RNA editing - quite a quick process

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12
Q

which of the 6 RNA PTM processes occur post-transcriptionally?

A

Splicing - the whole gene body

Adenine meth

Backsplicing - exon spliced to itself in 3-5 direction

polyadenylation (cleavage)

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13
Q

how does the POLYAA help with efficient translation

A

PABP helps with nuclear export

once in cytoplasm exchanges with other cytoplasmic proteins allowing 3’ and 5’ to interact

allows cirularisation of mRNA

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14
Q

what is the difference between UTR-APA and CR-APA

A

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

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15
Q

outline an example of CR-APA

A

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

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16
Q

outline an example of UTR-APA

A

Brain derived neurotrophic factor has 2 APA which determines ^mRNA localisation^

short UTR = mRNA cell body
long UTR = mRNA in dendrites

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17
Q

how do we identify APA? (experimental methods)

A

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

18
Q

how is APA regulated? (how to choose if proximal or distal APA site is used)

A

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

19
Q

what is the role of spliceosome proteins?

A

recognise the 3’ splice site and stabilise base pairings between RNA-snRNA

able to remodel spliceosome

20
Q

where are the splice site consensus sequences found?

A

5’ site GU
3’ AG
branchpoint adenosine 30nt upstream of the 3’ intron

21
Q

how is splicing regulated?

A

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

22
Q

how can ESE and ISS cis-acting sequences be implicated in disease?

A

if they are mutated, this can lead to alterations in splicing and overall protein function may be trunchated/non functional

23
Q

why is splicing important biologcally?

A

allows diversity of proteins to be generated (via Alt splice)

allows for normal gene expression

24
Q

what is the spliceosome?

A

large highly dynamic RNA-protein complex

spliceosome allows the 2 transesterficiation reactions to happento remove intron and ligate 2 exons together

25
Q

why do the snRNAs have an extensive secondary structures?

A

they allow the active site of the spliceosome to form ready for the transesterifaction reactions

26
Q

name some examples of spliceosome proteins - what are their functions?

A

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

27
Q

how do self splicing introns differ from spliceosomal introns

A

self splicing introns DONT use the spliceosome/proteins/RNA to catalyse

instead the intron is autocatalysed using ^ribozymes^

28
Q

how could stemloop/2nd structure affect splicing?

A

could block the splice site so exon is not included

could affect spliceosome helicase activity and make it harder to unwind

29
Q

how do group 2 self splicing introns splice?

A

they contain a gene encoding for ‘‘intron-encoded-protein’’

which has helicase, r.transcriptase and endonuclease activity so can self-splice the intron easily

30
Q

how does prp8 and IEP (intron encoded protein) relate?

Evidence of evolution relationship

A

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

31
Q

what is the advantage of AS?

A

A.S is implicated in many human diseases

A.S can give rise to protein diverity, important in embryonic development

32
Q

which of the A.S mechanisms is most common?
A intron retention
B alt. splice sites
C exon skipping
D mutually exclusive exons

A

C exon skipping

as seen in the insulin receptor alpha subunit which alters its sensitivtiy

switches in foetus and adult

33
Q

which of the A.S mechanisms is least common?
A intron retention
B alt. splice sites
C exon skipping
D mutually exclusive exons

A

A intron retention

intron is not spliced out and retained in coding sequences
this is likely to induce degradation or trunchation of mRNA molecule

34
Q

how can splicing be regulated by enhancers/silencers?

A

ESE/ISE can create stronger splice sites so more likely to be recognised by spliceosome

35
Q

how can splicing be regulated by rate of transcription?

A

Could lead to exon skipping

rapid transcription then less time for spliceosome to assemble
this could encourage exon skipping if the concensus is weak

36
Q

how could secondary structure of emerging mRNA transcript affect splicing?

A

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

37
Q

what are the 2 major types of splicing associated diseases?

A

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

38
Q

what causes splice site inactivation? What are the consequences?

A

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

39
Q

what causes splicing enhancer inactivation? What are the consequences?

A

synonomous mutations could inactivate splicing enhancers

this could lead to exon skipping

40
Q

what is a synonomous mutation?

A

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)

41
Q

how can trans-acting mutations affect splicing ? What are the consequences?

A

The different proteins/RNA molecules could me mutated and negatively affect spliceosome assembly

So many genes affected

42
Q

how could we treat splicing diseases?

A

could use anti-sense oligonucleotides and target denovo or cryptic splice sites

to mask or activate their effects

GENES TO GENOME (15 decks)

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