ISALAN - exon shuffling Flashcards
Eukaryotic genes are split into introns and exons, allowing for:
- Alternative splicing: RNA gets matured in different ways by having different exon
combinations, so the same gene can make different proteins within the cell - Exon shuffling: a molecular mechanism for the formation of new genes. It is a process
through which two or more exons from different genes can be brought together, or the same exon can be duplicated, to create a new exon-intron structure.
Eukaryotic proteins are mosaics of motifs/ domains
– contains alpha helices, beta sheet, zinc
finger domains, etc. combined together
* Each domain = 40-100 amino acids
* Each domain have different functions: to stabilize proteins, allow DNA or protein
binding, enable catalysis, etc.
Exon (general)
Each exon corresponds to each domain
* Any duplication, permutation, rearrangement of the exons in new genome positions, thus manipulation of these building block domains, can generate novel gene & proteins with diverse structures & functions.
Manipulation of exons can be done through domain duplication or shuffling
Mechanisms of domain duplication:
- Unequal crossing over between repeat sequences in the motifs
- Replication slippage of very short motifs
Mechanisms of domain shuffling
– structural domains from different genes are joined together
- Illegitimate (non-homologous) recombination
- LINES
- Transposons
Illegitimate (non-homologous) recombination
- Microhomologies between similar domains in unrelated genes can cause nonhomologous recombination.
- The microhomologies are nicked by TOPO1 and the non-homologous ends are ligated,
resulting in duplication of certain domains and shuffling of downstream genes as well - Ex. ⍺A-crystallin gene in hamster transfected into mouse
LINES
(more common retrotransposition process)
* During transcription of LINES, if a LINE is between exons, its transcript can include the adjacent exon as well (produce a read-through transcript)
* After retro transposition to cDNA, when the LINE is inserted back into another region of DNA, the downstream exon will also be included, which will introduce a new domain into a different gene/ genome region.
* Overtime, some elements of the original gene can be degraded or the new domain might be manipulated, etc. allowing for diversification and evolution
* LINES can also induce ds breaks which can induce domain shuffling through the mechanisms of ds break repair (homologous recombination)
Transposons
Specific transposons: Mutator-like transposable elements (MULEs/Pack-MULES) contain introns and exons within them
* When these transposons are migrate to different regions, they carry the exons and introns with them as well and they can also collect/accumulate sequences, forming internal hybrid genes
* These exons do not contain STOP codon/ translation initiation signals, so they are not folding by themselves, but are coding sequences (not translated when they are on their own) However, when the introns and exons shuffled into a new region with translation initiation signals, they will be expressed along with the new gene region
Domain shuffling effects:
can disrupt the reading frame of all exons downstream if the added exons do not have sequences that are an exact multiple of 3
Exons are flanked by different intron classes:
- Class 0: 0 base between exon phases – complete sets of codons on both exons flanking the intron (intron lies between 2 complete codons)
- Class 1: 1 extra base added on 1 exon, so the following exon must begin with 2 bases to have a complete codon (intron located after 1st base)
- Class 2: 2 extra bases added on 1 exon, so the following exon must begin with 1 base to have a complete codon (intron located after 2nd base)
Therefore, shuffled exon must be inserted into a region with the same flanking intron classes, otherwise it will produce a frameshift in the resulting coding sequence
Shift in reading frame will cause negative selection on the inserted exon:
* It will be a target for purification of the frame shift/ gene restoration and the inserted exon might not be preserved.
Benefits of Exon shuffling:
- Exon shuffling is a driving force for eukaryotic evolution because it is important for cell-cell communication (ex. in ECM, cell adhesion, cellular receptors) which led to the development of complex body plans & multicellular structures.
* Ex. 6.4 % of human genes showed evidence of exon shuffling vs 1 % of plant Arabidopsis genes (less complicated body parts)
* Excess of symmetric exons compared to asymmetric exons indicate many effective exon shuffling events - Allow for formation of domains that are permissive of complex structures (eg. 1-1 exon shuffling)
- Allow for Protein-Protein Interactions (PPI)
1-1 exon shuffling
- 1-1 exon shuffling is more common in humans/ animals over evolutionary time which could provide evolutionary advantage to more complicated organisms (vs. plants = mostly 0-0)
o Monsiga brevicollis (a marine choanoflagellate) & Arabidopsis thaliana
-1-1 shuffling present was explained by chance
-If shuffling occurred then it was 0-0
o Vs. Trichoplax adhaerens
-Distinct presence of 1-1 shuffling - Phase 1 introns frequently interrupt glycine codons (GGT,GGC,GGA,GGG) which are useful to form glycine linkage between domains.
This allows for formation of flexible linkers between domains to overcome structural limitations in metazoans
Ex: α2 Type 1 collagen gene
* Code for highly repetitive sequences with glycine repeats
o Repeated tripeptide: gly-X-Y, where X: often proline & Y: often hydroxyproline
* Occurs from nature favouring 1-1 exon shuffling, resulting in duplication of exons with glycine linkages
* These glycine repeats allow 3 collagen chains to be linked together in a tight, complex triple helix structure, so a long, complicated repetitive protein can be formed
Protein-Protein Interactions (PPI)
- Members of a signaling pathway can have similar domains with different quantities and orders allowing for complicated protein-protein interactions via partial dimerization (self-interactions) between domains
Ex. Blood clotting cascade: Tissue plasminogen activator (TPA)
o Contains 4 exons which are shuffled from domains of other members of the blood clotting cascade (fibronectin, plasminogen, epidermal growth factor)
o Therefore, TPA allows all these proteins to interact via self-interaction (dimerization) with the corresponding domains in TPA
- Shuffling of domains that already promote interactions with other proteins &/or promiscuous domain types [ex. polyglutamine domain that allows TATA binding protein to bind to many different transcription factors] will allow for even more diverse interactions of proteins (form a hub with several protein-protein interactions)
Exon shuffling, PPI, and diseases:
Ex. Amyloid Precursor Protein (APP)
APP undergoes protease processing by either:
* α-secretase – results in soluble APP or
* β-secretase then λ-secretase – which apart from soluble APP, results in a stable β-amyloid protein that can become amyloid plaques and cause Alzheimer disease
How APP is processed depends on the presence of a Kunitz-Type Protease Inhibitor (KPI) domain sequence which inhibits α-secretase by binding to its trypsin domain
Therefore, shuffling of KPI domain onto APP can result in interaction with α-secretase, inhibiting it and causing Alzheimer’s disease
