Regulation of alternative splicing

Question 1

Explain how Sxl results in exon skipping.

Answer 1

Only in females, Sxl bind to the 3’ of an intron, blocking the association of U2AF and U2 snRNP to the branch-point A. This silences the splicing of the specific intron Sxl is bound to, thus resulting in exon skipping.

Question 2

What is the cascade of regulated splicing that controls sex determination in Drosophila?

Answer 2

1. Sxl leads to skipping of exon 4
2. Tra leads to alternative 3’ splice site usage and leads to exclusion of exon 2
3. In Dsx gne, Rbp1 and Tra2 are SR proteins that bind to ESE sites in the exon 4 of females. As a result, only exon 4 is included in females while excluded in males
   Dsx is a transcription factor that regulates expression of genes important in male and female development.

Question 3

What are the three classes of mutations affecting RNA splicing?

Answer 3

1. Mutations affecting cis-acting elements of a specific gene such as
   mutations in ESS, U1 binding site, etc.
2. Mutations in core splicing factors such as mutations affecting
   spliceosomal proteins such U4/U6/U5 tri-small nuclear
   ribonucleoprotein particles in retinitis pigmentosa.
3. Mutations in trans-acting factors (RNA binding proteins) that
   regulate splicing

Question 4

What are therapies to modulate RNA mis-splicing?

Answer 4

• Small molecules have been developed that
  target splicing factors to modulate their
  activities.
• Antisense oligos (ASOs) are designed to
  recognize specific RNA splicing regulatory
  elements.

Question 5

Give a concrete example of disease caused by mis-splicing.

Answer 5

Retinitis pigmentosa is a progressive disease that is characterized by initial night blindness and loss of peripheral vision, followed by central retinal degeneration. Affected by mutations in U4/U6/U5 of spliceosome.

Question 6

What is the mechanism of mutually exclusive exons?

Answer 6

When one exon is incorporated in the final product, the neighboring specific exons don’t get incorporated => mechanisms that regulate this
* Steric interference => There is an overlap between U1 and U2 binding sites, thus preventing both to bind at the same time; this determines which exon is sliced
* Spliceosomal incompatibility => Some introns have AU-AC sequences instead of GU-AG sequences, the specific snRNPs (U11/U12) used to splice these introns are not compatible with typical snRNPs
* Nonsense mediated decay => Exons are regulated by combinations of splicing activators and repressors; RNA that contain exons that are not multiple of 3, nonsense mediated decay degrades them