SOUTHALL - Alternative splicing

Question 1

Key theme:

Answer 1

Diversifying the proteome: with a certain set number of genes, you can create many different types of proteins

Question 2

Splicing (review)

Answer 2

1. Hydroxyl group of branch site A attacks the phosphate link between the last base of the exon and the first base of the intron
2. 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
3. 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

Question 3

Spliceosome

Answer 3

• 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

Question 4

Spliceosome Steps

Answer 4

1. 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
2. U1 & U2 interact together to bring the 2 exons together, looping out the intron, forming the pre-spliceosome (complex A)
3. The preassembled tri-snRNP (U4–U6+U5) is recruited to form complex B – a pre-catalytic spliceosome that consists of all core components
4. complex B undergoes a series of rearrangements, ejecting U1&U4, to form a catalytically active complex B (complex B*)
5. 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
6. 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
7. 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

Question 5

what is alternative Splicing

Answer 5

Pre-mRNA primary transcript can have 2 or more splicing pathways (due to alternative splice sites) to create related but different mRNA’s

Question 6

what is AS dictated by

Answer 6

• RNA sequences
• Splice site strength:
  o Ability to bind general factors (ex. U1, U2)
  o Presence of absence of ESE
• Splicing factors

Question 7

genes that undergo splicing:

Answer 7

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

Question 8

Result of AS

Answer 8

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

Question 9

Ex: DSCAM (Down Syndrome Cell Adhesion Molecule) Gene in Drosophila

Answer 9

• 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

Question 10

supplements transcriptional control

Answer 10

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

Question 11

Alternative splicing effects in mRNA and proteins:

Answer 11

• 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)

Question 12

specificity of AS

Answer 12

• 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

Question 13

splice site selection

Answer 13

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

Question 14

3 Groups of alternatively spliced transcripts:

Answer 14

1) 5’ transcript ends differ from one another
2) 3’ ends differ from one another
3) Middle portions differ

Question 15

5’ Ends Differ

Answer 15

• 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

Question 16

3’ Ends Differ

Answer 16

• 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

Question 17

Centre Differs

Answer 17

• Can’t be explained by different TSS (different promoter use) or Termination sites (different cleavage)
• About machinery for including/ excluding different exons (alternative splicing)

Question 18

Ex: troponin T gene (skeletal muscle)

Answer 18

• 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

Question 19

Regulation of alternative splicing
By: tissue specific splicing factors

Answer 19

• 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

Question 20

Example 1 of regulation of alternative splicing:
Drosophila sex determination

Answer 20

• 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

Question 21

1St Tra target: doublesex (dsx) gene

Answer 21

• 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

Question 22

2nd Tra target: fruitless (fru) gene

Answer 22

• 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

Question 23

Summary of the Drosophila sex determination cascade (females)

Answer 23

1. Sxl is initially expressed in response to X:A ratio of 1 in early stage of development
2. Sxl autoregulates its own splicing to NOT include exon 3 (act negatively)
3. Sxl regulates Tra splicing to exclude STOP codon in exon 2 (act negatively)
4. 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)

Question 24

Summary of the Drosophila sex determination cascade (males)

Answer 24

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

Question 25

Example 2 of regulation of alternative splicing:
K+ channel gene SLO

Answer 25

• Contains STREX domain which increases sensitivity to Ca2+
  1. Activation of neurons (neuron depolarization), results in high level of intracellular Ca2+
  2. high level of Ca2+ phosphorylates a specific factor which binds to CARRE site within the SLO pre-mRNA
  3. This Promotes splicing out of the STREX domain

Therefore, when Ca2+ is high, the SLO K+ channel gene product is less sensitive to Ca2+
which is a type of Homeostatic mechanism

Question 26

2 types of tissue specific splicing factors

Answer 26

1. Serine-arginine-rich proteins (SR)  promote splicing in
   * Has RNA recognition motif @ N-terminus & SR domain @ C-terminus
   * Influence splice site selection in 2 ways:
   o Bind 5’ SS & promote U1 binding (promote splicing from the donor site)
   o Bind ESE within downstream exon and stimulate/ stabilize U2AF binding (promote use of the acceptor site to splice the downstream exon in)
2. Heterogenous nuclear Ribonucleoproteins (hnRNPs)  inhibit splicing overall
   * Prevents binding of U1 or U2AF factors

Question 27

Close link between splicing and transcription

Answer 27

• Splicing occurs as transcription is still going on
• Therefore, rate of elongation (RNA pol speed) can affect the splicing pattern
  o Slow: more likely to INCLUDE exons with weak acceptor sites
  o Faster: more likely to SKIP exons with a weak acceptor site

Been shown in vivo in Drosophila using a mutant line with a lower RNA polymerase II elongation rate

Question 28

Identification of occurrence of alternative splicing
By Genome Wide Analysis using:

Answer 28

1. Microarrays
   * Slides with different oligonucleotides printed on them, corresponding to different regions of the genome
   * Hybridization pattern of extracted DNA from tissues will correspond to specific patterns
2. Nowadays = Next-Generation Sequencing
   * Sequence all mRNA produced in a cell and perform analysis to see which exon is included/ excluded and compare across tissues to see where alternative splicing has occurred

Question 29

Further identification methods:

Answer 29

• Alternative Splicing in complex tissues – how do you know which cells have a specific splice pattern?
• How can you rapidly identify the target genes of certain splice factors in specific cell types?

As the pre-mRNA transcript exit RNA pol, factors bind to the transcript to regulate splicing patterns ex. U2AF

• Can tag U2AF factor with Dam from E. coli which methylates adenine in specific GATC sequences within DNA
• Dam tagging allows us to profile protein-DNA interactions in a cell-specific manner in Drosophila
• Therefore, we can see where splicing factors are interacting with DNA as genes are being transcribed and spliced

Question 30

Sxl further research

Answer 30

Sxl shown in strongly associate with a set of transcriptional start sites

Provide basis for ongoing research and development:
* Can Sxl bring about sexual dimorphism in the nervous system through transcriptional regulation, as well as alternative splicing?
* How is Sxl recruited to the DNA/chromatin?
* Does Sxl require its RNA binding function to be recruited to the DNA/chromatin?