Gene Structure 4 Flashcards
Who came up with the intron early theory?
Walter Gilbert
What is the intron early theory?
That introns originated in prokaryotes but were lost due to genome streamlining - suggesting that eukaryotes inherited all introns from prokaryotic ancestors.
Believed introns promoted gene evolution as they permitted the shuffling of genes - produced more complex genes and larger protein collection.
What is the intron late theory?
Stated that introns were unique to eukaryotes and that new introns have been emerging throughout eukaryotic evolution - prokaryotes have never contained introns or the spliceosome.
What is the current intron theory?
Somewhere in between the early and late theory -
Group II introns originated in the mitochondrial ancestor and these invaded the host genome upon mitochondrial endosymbiosis.
(still debated).
Why can introns be a burden to a host?
3
Cell must contain spliceosome and this is a huge complex.
Also increased energy cost as RNAPII is transcribing larger sequences.
Create vulnerability - as need recognition by cis regulatory sequences to be spliced.
What are the 5 “life phases” of introns?
Genomic intron. Transcribed intron. Intron being spliced. Excised intron. Exon junction complex (EJC)-harbouring transcript.
What is the genomic intron?
Still in the DNA and is the location of the gene’s cis regulatory elements.
How are genomic introns involved in transcription initiation?
They contain enhancers, silencers and TF binding sites - mostly found in 5’ introns.
How are genomic introns involved in alternative splicing?
They contain alternative transcriptional initiation sites (promoters).
e.g. alpha-fetoprotein - cell specific expression.
How are genomic introns involved in transcriptional termination?
Intron sequences can regulate polyadenylation and cleavage.
Give an example of how genomic introns effect transcriptional termination.
Human Beta globin - protein can be soluble or membrane bound depending on where transcription is terminated and poly A tail is added.
Soluble - polyA on exon 14.
Membrane bound on exon 13.
What can genomic introns also be host to?
Nested genes - protein coding or non-coding RNA
What is the genomic design hypothesis?
Genes that need complex regulation contain more introns to accommodate their regulatory elements.
Highly expressed genes, which need low regulation have shorter introns.
What is a transcribed intron?
An intron that is present in the pre-mRNA
What is the RNAPII elongation rate and what does this mean for intron transcription?
50kb min^-1.
This means that intron may take hours to be transcribed - this explains the time delay between gene activation and translation of protein.
Give an example of how the gene activation to translation of protein time delay involved in negative feedback loops
Example - HES7 gene (transcription factor) - 19 minute delay between transcriptional initiation and full mature mRNA.
The protein produced inhibits its own transcription.
What is a spliced intron?
An intron present in mRNA - being spliced out by the spliceosome.
Which process is intron splicing linked to and how?
Transcription - linked via C-terminal domain of RNAPII.
How can splicing effect initiation of transcription?
U1-snRNP associates with and recruits TFIIH and TFIID to the 5’ splice site and stimulates phosphodiester bond formation (in mRNA).
How can splicing effect elongation of transcription?
Splicing factors and spliceosomal components can interact with transcriptional elongation factors.
U2 snRNP binding at the 3’ end of the transcript promotes the elongation using RNAP.
How can splicing effect termination of transcription?
U2 snRNP interacts with cleavage/polyA specificity factor (CSPF).
CSPF binds to polyA site and this can promote polyA tail formation and/or splicing via interaction with U2.
What is an excised intron?
An intron that has been removed from pre-mRNA by the spliceosome.
What often happens to excised intron?
They undergo debranching and degradation.
What can be expressed after an intron has been excised?
Embedded/nested genes - often miRNA and snoRNAs.
How long are miRNAs?
~22nt
Which organisms are miRNA found in?
Metazoans, plants and other eukaryotes.
How are miRNAs expressed if embedded in introns?
They are co-expressed with other genes because the lack promoters.
Where do miRNAs bind to and what does this result in?
Bind to 3’UTRs of mRNA and cause degradation/prevent translation.
How long are snoRNAs?
60-150nt
Which organisms are snoRNA found in?
Archaea and eukaryotes.
What do snoRNAs do?
Modify RNAs - rRNAs, snRNAs, tRNAs.
Can also regulate mRNA and are released after splicing.
What are the two classes of snoRNAs?
C/D box snoRNAs - guide 2’O methylation of rRNAs.
H/ACA box snoRNAs - guide pseudoridylation of rRNAs.
What is another feature of snoRNAs?
They are mobile genetic elements via retro-transposition.
Where does the EJC bind?
Binds ~25nt upstream of the exon-exon junction (where an intron has been) on the mRNA transcript.
What are the four core proteins in the EJC?
MAGO, Y14, eF4AIII and MLN51.
What are the four roles of the EJC?
Nuclear transport
Translation activation
mRNA localisation
nonsense mediated decay.
How is the EJC involved in nuclear transport?
EJC (that is bound to the mRNA) interacts with nuclear pore complex and recruits the export factor - ALY/REF.
How much faster is the nuclear transport export rate for spliced transcripts?
10x faster.
How is EJC involved in translation activation?
MLN51 interacts with eIF3, which is a translational initiation factor.
How is EJC involved in cytoplasmic localisation?
Some mRNAs require the EJC to localise them to subcellular locations.
What is nonsense mediated decay?
Degradation of mRNAs that include premature stop codons.
Why do cells have a mechanism in place to degrade mRNAs that have premature stop codons?
To prevent dominant-negative/gain of function.
How is EJC involved in nonsense mediated decay?
mechanism
UPF3 loaded on to EJC during splicing.
UPF2 binds complex in the cytoplasm – ribosome knocks off EJCs unless there is a premature stop codon.
SURF complex forms and binds to ribosome at the premature stop codon.
Because the ribosome has stopped the EJC is left on the mRNA – UPF1 then interacts with UPF2.
UPF1 is phosphorylated, allowing it to bind the RNA and recruit SMG6 and SMG5-SMG7
SMG5-7 promoter deadenylation and decapping.
SM6 cleaves the mRNA.
