SOUTHALL - Alternative splicing Flashcards
Key theme:
Diversifying the proteome: with a certain set number of genes, you can create many different types of proteins
Splicing (review)
- Hydroxyl group of branch site A attacks the phosphate link between the last base of the exon and the first base of the intron
- Last base of exon performs hydrophilic attack on the phosphate bond between the last base of intron and the first base of the next exon
- Product: 2 exons ligated together and an exon in a lariat structure
- Does not require ATP
- Can splice over large distances (10’s of kb)
- Requires spliceosome and spliceosome cofactors to coordinate:
o Bringing splice sites into proximity
o Utilizing the correct splice site
o Selecting the correct exons
o Avoiding cryptic splice site: an mRNA sequence that has the potential for interacting with the spliceosome. Mutations, including splice site mutations, in the underlying DNA, or errors during transcription, can activate the cryptic splice site in a part of the transcript that usually is not spliced, causing a frameshift to produce a non-functional protein
Spliceosome
- Contains core of 4 small nuclear ribonucleic particles (snRNPs) + ~50 accessory proteins
- snRNPs are small nuclear RNAs (snRNAs) that associate with proteins
o the 5 snRNAs are: U1, U2, U4-U6, U5 - Attach to pre-mRNA transcript and form complexes in a sequential manner
Spliceosome Steps
- U1 binds 5’ end of intron (donor site) and U2 binds 3’ end of intron (acceptor site)
* U2AF binds to the polypyrimidine tract upstream of 3′ splice site to facilitates spliceosome assembly by recruiting U2 - U1 & U2 interact together to bring the 2 exons together, looping out the intron, forming the pre-spliceosome (complex A)
- The preassembled tri-snRNP (U4–U6+U5) is recruited to form complex B – a pre-catalytic spliceosome that consists of all core components
- complex B undergoes a series of rearrangements, ejecting U1&U4, to form a catalytically active complex B (complex B*)
- Complex B* then carries out the first catalytic step of splicing, generating complex C, which contains the free exon 1 and the intron–exon 2 lariat intermediate
o Hydroxyl group of branch site A performs hydrophilic attacks the phosphate link between the last base of the exon 1 and the first base of the intron - Complex C undergoes additional rearrangements and then carries out the second catalytic step, ligating 5’ exon and 3’ exon, resulting in a post-spliceosomal complex that contains the lariat intron and spliced exons
o Last base of exon 1 performs hydrophilic attack on the phosphate bond between the last base of intron and the first base of exon 2 - Release of spliced mRNA and lariat. Helicase will utilize ATP to act on the lariat to release the snRNPs so they can be re-used
what is alternative Splicing
Pre-mRNA primary transcript can have 2 or more splicing pathways (due to alternative splice sites) to create related but different mRNA’s
what is AS dictated by
- RNA sequences
- Splice site strength:
o Ability to bind general factors (ex. U1, U2)
o Presence of absence of ESE - Splicing factors
genes that undergo splicing:
A lot of eukaryotic organisms undergo splicing, but the % of genes that undergo splicing also depends on the complexity of the organism
Ex. Yeast - ~0.1% of genes undergo splicing while for humans, 95% or genes undergo splicing
The number of genes in an organism’s genome is not a good assessment of protein diversity:
C. elegans have the same number of genes as humans but do not produce a diversity of proteins as in humans
Result of AS
Alternative splicing results in huge variation in the final mRNA and protein translated from them. This is due to many possibilities of:
* Exons retained or skipped
* Introns excised or retained
* 5’ & 3’ splice site positions moved: exons longer or shorter
Ex: DSCAM (Down Syndrome Cell Adhesion Molecule) Gene in Drosophila
- Membrane-anchored cell surface protein
- A role in neuronal development for axon and dendrite self-avoidance (in Drosophila)
- 24 exons permits 38,016 protein variants
o Has multiple exons – colored ones = exons that have alternative splice sites, resulting in different lengths. Those exons have as many as 49 alternatives, resulting in many different mRNA transcripts which will produce a variety in the specific immunoglobin regions of the translated protein
supplements transcriptional control
Alternative splicing can serve as a method of regulation
- Can insert or delete specific domains in specific cell types or in different conditions to control the expression of the gene or the function of the resulting protein
- e.g. it can regulate antibody and neuropeptide production
- Methods:
o Include exon that has premature STOP codon – triggers non-sense mediate decay and results in RNA degradation and no protein produced
o Introns left in – efficiency of export from nucleus = drastically reduced. This means active splicing = coupled with export from nucleus – used in genetic engineering: place intron in transgene to express desired gene at high level
Alternative splicing effects in mRNA and proteins:
- Rate of translation of mRNA
- mRNA degradation susceptibility (non-sense mediated decay)
- Insertion/deletion of amino acids
- Insertion/deletion of functional domains
- Polypeptide truncation due to stop codon
- Alters protein’s properties and functions:
o Whether soluble or membrane bound (depends on domain added/deleted)
o Subcellular location changes (change in localization)
Ex: Secretory pathway calcium ATPase
3 different isoforms with 3 different N-terminal domains
Results in 3 different localization within the cell: in ER, Golgi, Peroxisome
o Affinity changes with substrates (if the protein is an enzyme)
specificity of AS
- Certain variants alternative splicing can be tissue and developmental-stage specific (to produce proteins specific to/ only necessary in certain tissues or developmental stages
- Can be regulated by specific conditions/ environments the cell is in
o Cell stressed, received certain signals impact alternative splicing patterns
splice site selection
splice site selection must be tightly controlled throughout development & in adults
BECAUSE if it goes wrong, can cause many types of genetic diseases – mutations can:
generate cryptic splice sites or delete splice sites, resulting in deletion of exons, truncated exons, inclusion of exons that are not normally included
* If in frame = not as severe effects but if also change reading frame downstream = cause non-sense proteins
* Splice Disease Database shows 2337 splicing mutations associated with 303 genes and 370 diseases
3 Groups of alternatively spliced transcripts:
1) 5’ transcript ends differ from one another
2) 3’ ends differ from one another
3) Middle portions differ
5’ Ends Differ
- Resulting from different transcriptional start sites
- Then pre-mRNA processed differently due to recruitment of different splicing complexes
o Donor site on exon 1 is stronger than donor site on exon 2 so in the pre-mRNA transcript resulting from TSS where exon 1 is included, exon 2 is spliced out.
Ex: Mouse α-amylase gene
* Hydrolyses alpha bonds of alpha linked polysaccharides
* In salivary gland and liver, use different transcription start sites, resulting in inclusion/ deletion of different exons, giving rise to different activities in different tissues
3’ Ends Differ
- Due to different poly(A) sites utilised for transcriptional termination
Ex. Immunoglobulin chains of antibodies - 2 poly(A) sites: after exon 4 or exon 6 – resulting in 2 different pre-mRNA transcripts
- Inclusion of exon 5 & 6 (terminating at exon 6 poly(A) site) that code for transmembrane anchor allows for the version of IgM to be membrane-bound, while utilization of exon 4 6 poly(A) site results in secreted form of IgM
Centre Differs
- Can’t be explained by different TSS (different promoter use) or Termination sites (different cleavage)
- About machinery for including/ excluding different exons (alternative splicing)
Ex: troponin T gene (skeletal muscle)
- 64 different ways of exon combination (found in different muscle types)
- Resulting from different tissue-specific splicing factors acting on the pre-mRNA (to include/ exclude different exons in each splicing version)
- Can be used to identify heart attack: perform PCR on blood sample to see if the heart version of troponin T is present. If present = heart cells have ruptured, and the heart troponin T has been released into the bloodstream
Regulation of alternative splicing
By: tissue specific splicing factors
- Proteins that recognize cis-acting sequences within the RNA transcript
- Can promote or inhibit its nearby splice sites
Ex:
a) presence of splicing factor promotes inclusion of exon 2 by making splice acceptor site on 5’ end of exon 2 a strong one
b) presence of splicing factor prevents inclusion of exon 2 by silencing the splice acceptor site on 5’ end of exon 2
Therefore, presence of splicing factors in different cell types can regulate the alternative splicing of specific genes
Example 1 of regulation of alternative splicing:
Drosophila sex determination
- Controlled by an alternative splicing cascade where each gene product controls the splicing of the next gene in the hierarchy.
- Therefore, different gene products are made in male and female fruit flies – goes on to regulate the development and behavior of the different sexes
Top of hierarchy: Sxl (sex lethal gene)
o Master sex determination gene in somatic cells
o Its Exon 3 has a STOP codon
o Sxl autoregulates its own splicing to not include exon 3
- Initial production is stimulated by the ratio of sex chromosomes to autosomes (X:A) of 1 in female
o stimulate differential expression of certain TF during early stage of development (blastoderm stage)
o the TF stimulates expression of sxl from a different promotor so exon 3 is not included, producing a full functioning protein - After early stage of development, conventional promotor is used & exon 3 is included
- In females, the presence of sxl silences the splice site of exon 3 (inhibits splicing in of exon 3) by preventing binding of U2AF at 3’ ss, resulting in exon 2 splicing with exon 4 instead and exon 3 is excluded, producing a full functioning protein, more sxl production + autoregulation.
- Lack of sxl in males (due to X:A ratio of 0.5) results in exon 2 splicing with exon 3 which includes a STOP codon, producing a truncated protein product.
Sxl continues to regulate splicing of transformer (tra) gene downstream of the cascade
- Contains STOP codon in exon 2
- Lack of sxl in males results in production of non-functioning truncated Tra protein
- In females, presence of SXL binding at the proximal splice site of exon 2 (in intron 1) prevents U2AF binding at the conventional 3’ SS
- So U2AF binds cryptic site in exon 2 (known as the ‘distal splice site’) instead, allowing for the splicing out of the stop codon, producing a full functioning Tra protein in females
1St Tra target: doublesex (dsx) gene
- Encodes a transcriptional repressor that determines development
- Tra is rcruited by Tra2 to an Exon splicing enhancer (ESE) on exon 4
- Tra recruits members of the U2AF family to promote splicing to exon 4 (inclusion of exon 4) which has a sequence that promotes transcription termination
- Leads to female dsx mRNA producing a shorter protein isoform with different properties
- Lack of Tra in males results in only Tra2 at the ESE on exon 4 and exon 4 is not included in the dsx mRNA, producing a longer dsx protein
- The different proteins which are TF will bind to different targets, recruit different complexes to regulate gene expression
2nd Tra target: fruitless (fru) gene
- Encodes a transcriptional regulator that determines development
- Has STOP codon in a region of exon 2
- Presence of Tra promotes splicing from the end of exon 2 so the STOP codon is included in the female mRNA transcript
- Lack of Tra in males results in splicing from a region in exon 2 before the STOP codon, so the STOP codon is not included in the male mRNA transcript
- Results in the inclusion of a male specific region at the N-terminus of fru in males, but not present in females, so the male isoform will go on to regulate male-specific phenotype and behaviors
Summary of the Drosophila sex determination cascade (females)
- Sxl is initially expressed in response to X:A ratio of 1 in early stage of development
- Sxl autoregulates its own splicing to NOT include exon 3 (act negatively)
- Sxl regulates Tra splicing to exclude STOP codon in exon 2 (act negatively)
- Presence of Tra forming complex with Tra2 promotes splicing in of dsx exon 4 (act positively) to include transcription termination site to produce shorter dsx isoformPresence of Tra promotes splicing at the end of exon 2 in fru which includes a STOP codon to exclude the male-specific region of fru
Leads to female phenotype and behavior by informing somatic tissues to become female (ex. brain to give rise to female specific behaviors)
Summary of the Drosophila sex determination cascade (males)
Due to X:A ratio of 0.5, resulting in lack of sxl and thus lack of Tra, promotes production of male isoform of dsx (longer) and fru (include male-specific region) leading to male phenotype and behavior. Male fru can go on to regulate targets in the CNS.
Male isoform of fru controls many aspects of male courtship behavior
* Orientation during beginning of mating
* Tapping female’s abdomen
* Specific singing by beating of wings at a certain frequency
* Licking of female genitals
* Attempting copulation
Alternative splicing of fru (diverge from male isoform) has a profound effect on the sexual behaviors of flies males become interested in other males due to lack of male fru isoform to instruct its behaviors
Sxl can also inhibit translation MSL-2 which is involved in dosage compensation
MSL-2 complex increases gene expression across the X chromosome
Males only have 1 X chromosomes, so its genes on x chromosome will only be expressed at half the amount
In males, lack of Sxl permits MSL-2 complex formation, so males can express the same amount of genes as in females
