Gene Structure Flashcards
What is alternative splicing?
Alternative splicing generates different proteins from one gene
Genes contain multiple exons
Changing which exons are found in the final RNA changes the function of the protein
What is the mechanism of splicing?
Doesn’t require ATP
Can splice over large distances (10s of kilobases)
Pre-mRNA contains introns and exons
OH group from branch point adenine attacks phosphyl bridge between last base of exon 1 and first base of intron
Lariat structure formation
OH from last base of exon 1 attacks bridge between exon 2 and lariat
Formation of spliced mRNA and lariat structure
Look at image
What are the spliceosome and spliceosome co-factors responsible for?
Bringing splice sites into close proximity
Using the correct splice site
Exon skipping
Avoiding cryptic splice sites
What is a cryptic splice site?
Selection of alternative splice sites: tissue and developmental-stage specific
Splice site selection must be tightly regulated
Many genetic diseases can be caused by point mutations that activate cryptic splice sites or delete splice sites
Cryptic splice sites can be useful when pre-existing and damaging when introduced by a mutation
Changes frame of protein so rest of protein sequence is missense
Missing important part of coding sequence so protein looses its function
What is the structure of the spliceosome?
Made of 5 small nuclear RNAs (snRNAs)
snRNAs associate with proteins (at least 50) to form 4 small nuclear ribonucleic particles snRNPs
snRNPs: U1, U2, U4, U5, U6 (4 and 6 combine)
Spliceosome is made of snRNPs + accessory proteins
These attach to pre-mRNA transcript and form complexes
Specific complexes are formed in a sequential manner
What are the steps of assembly and action of the spliceosome?
1) U1 and U2 assemble onto pre-mRNA in a co-transcriptional manner
U1 binds to splice donor site
U2 binds to start of 3’ exon (acceptor site)
2) The U1 and U2 snRNPs interact with each other to form the pre-spliceosome (complex A)
Brings ends of introns together
3) The preassembled tri-snRNP U4-U6-U5 is recruited to form complex B (tri-catalytic complex)
4) Complex B undergoes a series of rearrangements to form a catalytically active complex B
U1 and U4 are eliminated from the complex –> now made of U5, U6, U2
5) Complex B carries out first catalytic step of splicing, generating complex C which contains free exon 1 and the intron-exon2 intermediate
6) Complex C undergoes rearrangements and then carries out second catalytic step
Results in post-spliceosome complex that contains lariat intron and spliced exons
7) Release of spliced mRNA and lariat
RNA helicase unwinds RNA of lariat so RNA and U2, U5, U6 can be recycled
DSCAM gene as an example of transcript variation
Example DSCAM (down syndrome cell adhesion molecule) gene
Expressed in developing neurons
Protects neural projections in dendrites from forming connections with themselves
If protein on a dendrite is the same then it will avoid it
Some exons are always included and other exons are randomly selected
24 exons permits 38,016 protein variants
How is alternative splicing a method of regulation? Transcriptional control
Insertion/deletion of specific domains
Ex. It can regulate antibody and neuropeptide production
How is alternative splicing a method of regulation? mRNA production
Splicing can affect amount of mRNA created
A premature stop codon in an exon that is not the final exon
If this exon is included it leads to nonsense-mediated RNA degradation
Unspliced intron leads to unprocessed RNA which is not transported into the cytoplasm or if it is transported then truncated due to premature stop codon in the intron
Exons are retained or skipped
Introns are excised or retained
5’ and 3’ splice site positions are moved (cryptic splice sites) to make exons longer or shorter
What are the effects of alternative splicing?
Rate of translation of mRNA
mRNA degradation and susceptibility
Insertion/deletion of amino acids
Insertion/deletion of functional domains
Polypeptide truncation due to premature stop codon
Protein properties and functions can change due to splicing:
Make a smaller/larger protein
Soluble or membrane bound
Subcellular location changes
Affinity changes for substrate
What are the 3 groups of alternatively spliced transcripts
5’ transcript ends differ from one another
3’ ends differ from one another
Middle portions differ
5’ ends differ
5’ ends differ due to different transcription start sites
Example skipping the starting exon
Example in mouse alpha amylase gene:
In the liver transcription is initiated further down so the starting exons are skipped
Leads to salivary gland and liver having different affinities in tissues
3’ ends differ
5’ ends the same, 3’ ends differ
When different poly A sites are used for transcription termination
Example in immunoglobin chains of antibodies:
Alternative 1: polyA site is after exon 4
Alternative 2: polyA site can after exon 6 so have 2 extra exons which code for a transmembrane anchor
Centre differs
5’ ends the same, 3’ ends the same but middle differs
Can’t be explained by differential promotor use or cleavage
Example: troponin T gene in skeletal muscle
64 different ways found in different muscle types
Tissue specific splicing factors act on the pre-mRNA to decide which exons are included
Used to screen for people thought to have heart attacks as it expresses a specific muscle type of troponin T gene
Alternative splicing vs exon shuffling
Alternative splicing and exon shuffling are different mechanisms for creating diversity
Alternative splicing acts on RNA to create diversity during lifespan of an organism
Exon shuffling created diversity on evolutionary scale over many generations
What is Exon shuffling? How does it occur?
Exon shuffling: exons jump around in the genome so exons from different genes are rearranged or combined to give new gene structure
Many eukaryotic proteins are mosaics of motifs
How it occurs:
Illegitimate non-homologous recombination during meiosis - slightly misaligned
LINES (long interspersed nuclear elements): An exon nearby the LINE element can be transcribed and included in the RNA, then moved to a different location
DNA transposons (similar to LINES): can collect exons and move them into genes
What are tissue specific splicing factors?
Tissue specific splicing factors: proteins that recognise cis-acting factors within the RNA transcript
Bind to pre-mRNA before it is spliced and it decides which exons are included
Promote or inhibit splice sites in different cases
Factor binds 1st intron and promotes splicing of exon 1 to exon 2
Factor binds 1st intron and inhibits splicing of exon 1 to exon 2, allowing splicing of exons 1 and 3
Sex lethal (Sxl) gene
Autoregulates its own splicing
If sxl is present: inhibits inclusion of exon 3 which contains a stop codon = production of sxl protein
In males: No expression of early promotor so never get sxl protein
In females: Early promotor causes burst of sxl so stop codon is never included so get sxl protein
Sxl determines sex in somatic cells
Transformer (Tra) gene
Tra splicing is regulated by sxl
In males: no sxl –> tra with early stop codon –> short protein with no function
In females: sxl binds at proximal splice site in intron 1 –> prevents U2AF binding –> binds cryptic splice site in exon 2 (distal splice site) –> allows splicing out of stop codon –> tra protein is expressed
Sxl promotes exclusion of an exon
Doublesex (Dsx) gene
Dsx is responsible for sex determination
Tra is a splicing factor and regulates dsx (doublesex) gene
Dsx encodes a transcriptional repressor that determines development
Tra promotes inclusion of an exon
Females: Tra is recruited to exon splicing enhancer (ESE) by tra 2 –> tra binds to exon 4 –> recruits U2AF –> inclusion of exon 4 –> exon 4 contains stop site –> transcription termination
Dsx mRNA ends with exon 4 –> shorter protein isoform with different properties
Males: exon 4 is not included
2 isoforms of dsx protein –> regulates genes in different ways to get more female or male characteristics
Fruitless (fru) gene
Fru is responsible for sex determination
Tra also regulates splicing of fru (fruitless) gene
Encodes a transcriptional regulator that determines development
In females: tra promotes splicing from the end of exon 2 –> inclusion of stop codon –> transcription termination no functional protein (truncated protein) and no male isoform is produced
In males: tra is absent –> get splicing from start of exon 2 (before stop codon) –> excludes stop codon –> functional protein
Tra forms a male specific isoform of fruitless which is important for male specific behaviour
Explain the summary of the sex determination hierarchy in drosophila
Splicing factors can act positively (ex. Tra) to promote the use of a splice site
Or act negatively (ex. Sxl) to inhibit the use of a splice site
In females: 2 copies of X –> Sxl expression –> regulates it’s own splicing –> regulates Tra –> functional tra –> splices dsx and fru differently to get female characteristics
Sxl also regulates MSL-2 which is important for dosage compensation
In males: no Sxl expression –> no Tra –> male versions of dsx and fru to give male characteristics
How does fruitless control mating behaviour?
Fruitless controls male mating behaviour
Male orientates itself at 45 degree to female, taps abdomen of female, sings, licks and attempts copulation
How is alternative splicing used for response to signals with SLO gene?
SLO gene encodes potassium channel which is important for action potential in neurons
STREX domain –> SLO is reacts quicker to allow K+ to pass through channel
During action potential calcium is high –> binds to camkinase proteins –> regulates factor that binds to CAR region in pre-mRNA –> exclusion of STREX domain –> potassium channel is less sensitive and slows down action potential
What are the regulating factors?
ESE - exonic splicing enhancer
ISE - intronic splicing enhancer
ESS - exonic splicing silencer
ISS - intronic splicing silencer
SR proteins - Stimulate splicing
hnRNPs - hinder splicing, bind to exon silencing elements
What are SR proteins?
Serine arginine repeat regions within splicing factors
Bind 5’ splice site and promote binding of U1 snRNP to promote splicing
Can bind within exonic splice enhancers (ESEs) within downstream exon to promote U2AF binding and promote splicing
What happens if RNA polymerase is elongating at a slower or faster rate?
Splicing occurs as transcription is still going on
Rate of elongation can affect splicing pattern
Slow: more likely to include exons with weak acceptor sites
Fast: more likely to skip exons with weak acceptor site (as protein won’t bind tightly)
What controls regulation of splicing?
RNA sequences - elements within RNA that can recruit proteins
Constitutive or tissue specific trans-acting factors
(example Tra-2 is always present)
Splice site strength: ability to bind to factors (U1 / snRNP) and presence/absence of ESEs
Origins of introns early and late theory
Intron early theory:
Introns originated in prokaryotes and due to evolution lost them to have more compact genomes
Introns were kept in more complex organisms
No evidence for anything that resembles introns in bacteria
Intron late theory:
Introns only evolved in eukaryotes
Prokaryotes and archaea never had introns or spliceosome machinery
Origins of introns theory with LECA
Last eukaryotic common ancestor (LECA)
Prokaryotes invaded archaea like cell, co-opted to generate energy for the cell, became mitochondria of eukaryotes
Bacteria contained retroelements (group II introns / self splicing RNAs) which invaded the archaea genome
Happened after endosymbiosis
Created precursors of introns / origin of spliceosomal introns in pieces
How can introns be a burden to the host?
Spliceosome complex is huge and forms a large part of the genome
So need to transcribe more RNA which requires energy and time (60 nt / s)
Vulnerability as errors in splicing causes mutated proteins ex. Need recognition of cis-regulatory sequences
Roles of introns
Sequence dependent functions ex. intron contains non coding RNA like microRNA
Length dependent functions
ex. Large introns take a long time to transcribe
Splicing dependent functions
ex. interaction between splicing machinery and RNA polymerase
Life phases of an intron
Genomic intron
Transcribed intron
Intron being spliced
Excised intron
EJC-harbouring transcript (marks where exons have been spliced together)
What is a genomic intron?
Still in the DNA
Location of gene’s cis-regulatory elements
Contain transcription initiation sites (modulate main promotor action)
Enhancers, silencers, TF binding sites
Often found in most 5’ introns
40% of TF binding sites are within introns
Alternative transcription initiation due to genomic intron - AFP
Alpha-fetoprotein (AFP)
plasma protein made in the liver and yolk sack in the foetus
regulates osmotic pressure
Tissue specific expression
Alternative transcription initiation due to genomic introns
Use of upstream TSS of exon 1 or TSS in the first intron
Alternative transcription termination due to genomic intron
Intron sequences regulate polyadenylation and cleavage
Different transcription termination depending on which polyA site is used (Intron needs to be harbouring the site)
Example Flt-1 gene
Soluble form is more abundant than the membrane bound form
Membrane bound form has a later polyA site so includes exon 14 (longer protein)
Soluble form has an earlier polyA site so excludes exon 14 (shorter protein)
Nested genes of genomic introns
Introns can encode nested genes
Same orientation or reverse strand
800 in drosophila
May have their own promotor and different expression profile
Non-coding (ex. microRNA) and protein-coding genes
How does length of intron affect timing of when protein is made?
Length of intron affects the timing of when the protein is made
RNA polymerase II has an elongation rate of 50kb / min
Intron transcription may take hours
Time delay between gene activation and translation of protein
Must splice and export from nucleus before translation
HES7 gene transcribed introns
HES7 gene (mus muscula) in mice
Transcription factor
Forms a negative feedback loop
Controls timing of somite segmentation during embryonic development
Somites form vertebrae, bones, cartilage
Made in sequence as organism is growing
Oscillations in HES7 expression
Peak in oscillation forms somites so somites are formed at regular intervals
Timing of expression and feedback loops in transcribed introns
Timing of oscillations affected by transcription
Longer mRNA = transcription and translation takes longer = longer oscillations
Negative feedback loop
Delays due to time for transcription, splicing, translation
HES7 is a transcription factor and represses its own transcription
As protein levels increase, transcription decreases
Once protein levels are low, transcription begins again
Leads to oscillations in protein production
Unstable protein required
Introns are important for producing the correct period of oscillation
mRNA stability, regulatory elements for feedback loops, length of mRNA
Knockout HES7 mouse embryo, then reintroduced gene but without the introns –> oscillations occurring very frequently
How are introns spliced? What do they affect?
Splicing occurs co-transcriptionally
Linked via RNAPII C-terminal domain
Splicing can affect initiation, elongation, termination
How do introns affect initiation of splicing?
U1 of spliceosome binds to 5’ end of intron in the pre-mRNA
U1 promotes binding of pre-initiation complex TFIIH and TFIID
Intron at the start of the gene enhances transcription
How do introns affect elongation of splicing?
RNA polymerase doesn’t always elongate, sometimes falls off
Machinery to make sure RNA polymerase stays on
U1 promotes TAT-SFI which binds to RNApol machinery and enhances elongation
How do introns affect termination of splicing?
Endonucleolytic cleavage and polyA tail addition
If a potential termination (polyA) site close, CPSF protein combines
U2 binds to 3’ end so if U2 is close to CPSF, CPSF is enhanced which increases the probability that transcription will end
U1 binds to 5’ end so if U1 is close, CPSF is inhibited which prevents termination
Prevents early termination in an intron ex. a cryptic termination site
What are excised introns?
When an intron is excised it forms a lariat structure and undergoes debranching and degradation
Embedded RNA genes may be expressed in a removed intron
Can contain non-protein coding RNAs (ncRNAs), such as microRNAs (miRNAs) and small nuclear RNAs (snoRNAs)
Excised introns - mirtrons
Intron is excised (mirtron) by splicing to form pre-miR
Pre-miR is exported from the nucleus and cut up by Dicer to form microRNA duplex
Unwinded and loaded onto RISC which regulates expression of RNA
Either leads to mRNA degradation or affects translation
