RNA biology lecture 5 Flashcards
1
Q
Expanding the size of the proteome
A
- ↑ no. of available components = key for evolution of more complex organisms
- ↑ proteins by ↑ no. of genes or alternative splicing
- Alternative processing reactions inc/ alternative pre-mRNA splicing, alternative polyadenylation + pre-mRNA editing
2
Q
Expansion of protein by alternative splicing
A
- Allows permutation of exons allowing cells to make new proteins from a single gene
- Normally exons = next to each other
- Certain exons can be present or absent in some isoforms
- ↑ diversity w/o uparrow DNA contect
3
Q
Common alternative splicing mechanism
A
- Exon skipping (certain exons skipped or included)
- Intron retention (mRNA differ depending on whether or not they have introns in mature mRNA, creates mRNA w/ diff coding capacity)
- Alternative 5’ donor sites of 3’ acceptor site (additional sites activated in certain circumstances → cystic exons, creates variety of transcripts)
- Mutually exclusive exons (inclusion/ exclusion of exons → mRNA that differ in coding capacity)
- Alternative promoters (makes greater variety of possible transcripts)
4
Q
Alternative splicing leads to diversity
A
- e.g. Dsipshilia Dscam gene → more than 38,000 isoforms
- Creates plasticity needed to govern complex exon connections
- Relies on arrangement of certain exon clusters that define certain exons
- 12 alternative variants of exon 4, 48 of exon 6
5
Q
Trans factor
A
- In addition to spliceosome, additional sequence elements are present in pre-mRNA + additional trans-factors
- 2 groups of trans-factor: +ve splicing factors (SR proteins) -ve factors = hnRNPs
6
Q
Recognition of splice sites in introns alone x explain alternative splicing
A
- If was only dependent on intronic sequences, alternative splicing = hard to explain
- Some introns = huge
- Exon definition model: recognition of splice sites = initiated from exon-centric view
- Exon bridge linking 3’ + 5’ splice sites of different exons so pan-exon association
7
Q
Regulating splice site recognition w/ SR proteins
A
- Within exon sequences, have sequences that modulate recognition of a splice site
- Could be 3’/5’ weak splice site
- Exonic splicing enhancer = recognised by SR proteins, +ve effect by facilitating interaction w/ U2AF + U1snRNA
- This is converted to a cross intron assembly
8
Q
Initiation of exon regulation via silencer sequences
A
- Exons also have exon splicer silencer (ESS)
- -ve factors bind these sequences, prevent recognition of 5’/3’SS → exon exclusion
- 3’/5’ SS x defined but exon sites for other exons either side may be defined → cross exon boundary
9
Q
Splicing of individual exons
A
- Competition btw +ve and -ve factors at overlapping enhancer/silencer sequences → skipping/exclusion
- Binding affinity + conc. of factors = key
- Silencing factors can bind intronic splicing silencers
- Regulation = through exon definition, presence/absence of exon enhancer seq + assoc of proteins like SR + hnRNP
- IF [hnRNP] ↑, can nucleate from ESS + create steric hindrance over 3’SS, if [SR] ↑, prevents hnRNP blocking 3’SS
- ↑ complicated
10
Q
Example - sex determination in Drosphilia
A
- Depends on ratio btw X chromosome + autosome
- Regulator promoter only active in female early embryos → Sx1 gene transcription → functional protein that regulates expression from a diff promoter
- In males, lack of Sx1 → activation from 2nd promoter → pre-mRNA that x spliced, exon included that has stop codon → non-functional protein
- Sx1 regulates other pre-mRNA that regulate other splice factors e.g. Tra
- Tra pre-mRNA excluding exon → functional Tra by Sx1 preventing recognition of 3’SS → stop spliced out
11
Q
DNA methylation
A
- Regulation of alternative splicing = linked to transcription + pol speed
- Achieved via coupling DNA meth. to Pol speed
- Alternative splicing of exon 4-6 = lymphocyte development, weak 3’SS
- If pol transcribes slowly btw 5+6, weak 3’SS engages w/ spliceosome + assembles spliceosome, if fast x
- CTCF btw exon 5+6 forms obstruction on DNA, if DNA x methylated CTCF forms block, slows elongation
- Splicing patterns can be inherited by epigenetic modification
- Chromatin modifications can direct inclusion/exclusion of exons e.g. =ve regulator PTB
- Chromatin adaptors assoc. w/ certain chromatin modifications
12
Q
Alternative polyadenylation
A
- ↑ polyA sites in pre-mRNA, usage can be regulated
- polyA site in pre-mRNA us of final stop, alternative usage = coding region APA
- If polyA site = ds of stop codon in UTRs, UTRAPA used
- Coding APA → production of mRNA w/ different coding potential, UTRAPA → transcript diversity w/ mRNA isoforms
- E.g. Hlgm heavy chain : alternative polyA + splicing, dependent on 2 polyA sites that create alternately cleaved poly-A, recognition of polyA site, in immature B cells, ↓ levels of cleavage, us polyA site = suppressed
13
Q
Alternative cleavage + polyadenylation
A
- 70% of human genes undergo alternatively
- Alternative 3’ end processing → mRNA w/ different 3’ UTRs
- This x Δ protein coding info but effects expression + mRNA localisation
- e.g. cancer mRNA has shorter UTRs, avoiding potential miRNA targets, achieved w/ different polyA sites ds of coding
- UTR has sites e.g. mRNA target sites, dstab elements
14
Q
RNA editing
A
- substituting base within mRNA
- E.g. ADAR Δ A→I
- During translation I is read as G → sub of aa in final product
- Seratonin receptor e.g. 5HT2c receptor Δ 5 codons by A→I
- E.g. C→U by APOBEC1
- C-U in CAA → UAA (stop codon), happens in apolipoproteoin, makes shortened 2153 aa
- In liver, CAA x recognised by comp factors → whole protein 5463 aa - Epitranscriptome
- All 4 nucleotides can be modified by methylation, ↑ variety, both coding + nc transcripts
- Most common = MGA methylation of A w/ METTL4 + WTAP methyltransferase + erasers like FTO
- Read by readers like hnRNPC, stimulate transcription/alternative splicing
- Pseudouridyl of nucleotides through snoRNA guided mechanism or through PUS
- Pseudouridyl at UGA stop → ‘read through’ by ribosome
