Alternative splicing

Question 1

How many distinct proteins do humans have?

Answer 1

> 100,000

Question 2

How many genes do humans have?

Answer 2

≈ 20,000

Question 3

How do humans produce more proteins than there are genes?

Answer 3

Alternative splicing! >90% of pre-mRNAs are alternatively spliced. Exons can be skipped by blocking spliceosome recruitment to them. Not possible in other organisms with less complex genome architecture. Different proteins with different activities are produced.

Question 4

What are mutually exclusive exons?

Answer 4

Exons that never appear in the same mRNA. They are often tandemly arranged and exhibit high similarity (probably derived by exon duplication).

Question 5

What are constitutive exons?

Answer 5

Exons that are always present in the mRNA transcript.

Question 6

What are cassette exons?

Answer 6

Exons that can either be skipped or included in an mRNA.

Question 7

Can exon length be altered?

Answer 7

Yes - some contain multiple 5’ or 3’ SS so differential splicing means they are shorter or longer.

Question 8

What happens if an intron is retained?

Answer 8

The transcript usually gets degraded (premature stop codon introduced).

Question 9

Can mRNA length be altered?

Answer 9

Yes - multiple exons can contain promoters or polyA sites so transcription starts later or ends earlier in some genes than others. E.g. SXL and Dsx in drosophila sex determination.

Question 10

How can you determine if an exon is alternatively spliced?

Answer 10

• Use RT-PCR; design primers flanking exons of interest. You get different length products (e.g. in different conditions) depending on whether the exon was included or not. Low throughput as you need sequence knowledge to design primers.
• Use next-gen sequencing; high throughput and allows us to look at all mRNA in a sample. Reverse transcribe sample, sequence it and map it back to genes. Can see different splicing patterns.

Question 11

How is the DScam pre-mRNA able to produce ≈ 38,000 isoforms of mRNA?

Answer 11

Mutually exclusive splicing with different exon blocks.

Question 12

Do isoforms show tissue specific expression?

Answer 12

Yes - over half do.

Question 13

What can splicing mistakes result in?

Answer 13

Mutations that cause disease and many cancers.

Question 14

Which exons have strong (perfect base pairing) splice sites?

Answer 14

Constitutive exons.

Question 15

Which exons have weak (imperfect base pairing) splice sites?

Answer 15

Alternatively spliced exons.

Question 16

Which proteins can regulate alternative splicing?

Answer 16

• SR proteins (promote splicing).
• hnRNPs (inhibit splicing).
• Tissue specific RBPs (great variation between families - many different domains).

Question 17

How do various proteins regulate alternative splicing?

Answer 17

Alternative splicing is not just expression dependent.
- Variation in abundance and balance of different splicing regulatory proteins. Enhancers and repressors can work against each other. E.g. different splice sites are favoured.
- Regulatory proteins recognise cis-acting motifs within pre-mRNA (introns and exons) and promote (exon inclusion) or repress (exon skipping) splicing. Again, combination dictates splicing outcome.

Question 18

What is the hnRNP 1 protein called?

Answer 18

Ptbp1.

Question 19

What is positive splicing regulation?

Answer 19

Splicing enhancers promote exon definition which leads to EXON INCLUSION. E.g. Tra in the drosophila sex determination pathway.

Question 20

What is negative splicing regulation?

Answer 20

Splicing repressors inhibit exon definition which leads to EXON SKIPPING. E.g. SXL in the drosophila sex determination pathway.

Question 21

What does CLIP stand for?

Answer 21

Cross-linking and immunoprecipitation.

Question 22

What is CLIP?

Answer 22

RBPs are UV cross-linked to their RNA targets, then pulled down with Abs. The RNA is sequenced to work out binding site.
Like ChIP but uses UV to cross-link instead of formaldehyde and looks at RNA instead of DNA.

Question 23

Can position of RBP in relation to exon determine whether exon is included or skipped?

Answer 23

Yes - RBP downstream = included, RBP upstream = skipped.

Question 24

What are hypothesised molecular mechanisms for downstream intron binding leading to exon inclusion?

Answer 24

• Stabilises the U1 snRNP.
• Alters RNA structure to increase U1 interaction.
• Promotes intron definition.

Question 25

What are hypothesised molecular mechanisms for upstream intron / exon binding leading to exon skipping?

Answer 25

• Competition with core spliceosome components.
• Competition with SR proteins.

Question 26

What needs to be considered when trying to predict a splicing pattern (using a computational model)?

Answer 26

• Pre-mRNA sequence (RBP targets, strength of splice sites).
• Regulator protein abundance.
• Regulator protein interactions (synergistic or antagonistic effects).
• RBP binding context (upstream or downstream).

Question 27

What is an example of mutually exclusive splicing in mammals?

Answer 27

Exons 18a and 18b in the FOXP1 TF gene.
18a is included in ES cells and this FOXP1 TF isoform promotes expression of pluripotency associated genes.
18b is included in differentiating cells (change stimulated by increase in expression of MBNL RBP) and this FOXP1 TF isoform promotes expression of differentiation genes.

Question 28

What is an example of mutually exclusive splicing in plants?

Answer 28

Exons 2 and 3 in the FLM TF gene.
2 is included in lower temperatures (beta TF isoform), binds the flowering repressor SVP and together they bind promoters (no flowering).
3 is included in increased temperatures (delta TF isoform), which can’t bind SVP, can’t bind promoters (promotes flowering).

Question 29

What are the proposed molecular mechanisms for mutually exclusive splicing?

Answer 29

• Steric hindrance (exons too close for spliceosome to assemble at both)
• Spliceosome incompatibility (2 exons can’t be defined together because they use different spliceosome machinery (major and minor))
• Nonsense mediated mRNA decay (NMD) (mRNA produced with both exons is degraded)
• Competing secondary structures (only secondary structures formed with 1 exon forms the right structure, and not both)

Question 30

What determines sex in drosophila?

Answer 30

Dosing of X chromosome genes. This drives alternative splicing of the sex lethal (Sxl) gene which drives a cascade of male or female gene expression patterns.
Sxl is only expressed in females (exon 3 skipped) - in males exon 3 is included which leads to premature termination.

Question 31

How does the Sxl protein reinforce sex?

Answer 31

It autoregulates its expression - binds at its promoter to enhance expression, and also binds intronic silencers at exon 3 and promote its skipping.

Question 32

What is the cascade occurring in female drosophila in response to Sxl expression?

Answer 32

• Sxl promotes female specific (alternative) splicing of transformer (Tra).
• Exon 2 containing a premature termination codon is skipped - it is included and Tra is degraded in males.
• Tra forms a complex with SR and SR-like proteins and binds exon 4 of doublesex (Dsx) pre-mRNA.
• This promotes exon 4 inclusion in the mRNA.
• There is polyadenylation after this exon 4 in females (exon 4 skipped and polyA after exon 6 in males).