Alternative splicing Flashcards
How many distinct proteins do humans have?
> 100,000
How many genes do humans have?
≈ 20,000
How do humans produce more proteins than there are genes?
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.
What are mutually exclusive exons?
Exons that never appear in the same mRNA. They are often tandemly arranged and exhibit high similarity (probably derived by exon duplication).
What are constitutive exons?
Exons that are always present in the mRNA transcript.
What are cassette exons?
Exons that can either be skipped or included in an mRNA.
Can exon length be altered?
Yes - some contain multiple 5’ or 3’ SS so differential splicing means they are shorter or longer.
What happens if an intron is retained?
The transcript usually gets degraded (premature stop codon introduced).
Can mRNA length be altered?
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.
How can you determine if an exon is alternatively spliced?
- 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.
How is the DScam pre-mRNA able to produce ≈ 38,000 isoforms of mRNA?
Mutually exclusive splicing with different exon blocks.
Do isoforms show tissue specific expression?
Yes - over half do.
What can splicing mistakes result in?
Mutations that cause disease and many cancers.
Which exons have strong (perfect base pairing) splice sites?
Constitutive exons.
Which exons have weak (imperfect base pairing) splice sites?
Alternatively spliced exons.
Which proteins can regulate alternative splicing?
- SR proteins (promote splicing).
- hnRNPs (inhibit splicing).
- Tissue specific RBPs (great variation between families - many different domains).
How do various proteins regulate alternative splicing?
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.
What is the hnRNP 1 protein called?
Ptbp1.
What is positive splicing regulation?
Splicing enhancers promote exon definition which leads to EXON INCLUSION. E.g. Tra in the drosophila sex determination pathway.
What is negative splicing regulation?
Splicing repressors inhibit exon definition which leads to EXON SKIPPING. E.g. SXL in the drosophila sex determination pathway.
What does CLIP stand for?
Cross-linking and immunoprecipitation.
What is CLIP?
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.
Can position of RBP in relation to exon determine whether exon is included or skipped?
Yes - RBP downstream = included, RBP upstream = skipped.
What are hypothesised molecular mechanisms for downstream intron binding leading to exon inclusion?
- Stabilises the U1 snRNP.
- Alters RNA structure to increase U1 interaction.
- Promotes intron definition.
