6th March - What effects splicing patterns?

Question 1

In a simple gene which has two alternative 5’ss, not subject to complex regulation, what determines which ss is used?

Answer 1

The position of the splice site
The strength of the splice site

If both splice sites are weak then splice site choice is dependent upon U1 binding
If both splice sites are strong then U1 snRNPs will be bound at both sites, thus in this case the 5’ss closest to the 3’ss is used

Question 2

What is the common function of SR proteins?

Answer 2

promote splicing

Question 3

What do SR proteins bind to in DNA?

Answer 3

The exonic splicing enhancer (ESE)

Question 4

How might SR protein binding to the ESE affect alternative splicing?

Answer 4

It might promote exon inclusion

Question 5

What is the function of SRSF1?

Answer 5

Recruits the U1snRNP and stabilises its binding and similarly stabilisies U2AF65:U2AF35s interaction

Question 6

Which splice site does SRSF1 favour?

Answer 6

The downstream splice site

Question 7

What do HnRNPs bind to in the DNA?

Answer 7

Exonic splicing silencers

Question 8

What do HnRNPs do in alternative splicing?

Answer 8

They promote exon skipping and use of the upstream splice site

Question 9

What are the 4 models of action for HnRNPs?

Answer 9

Smothering - hnRNPA1 propagates along the exon therefore covering it

Direct competition for sites - hnRNPA1 can also bind to an intronic splicing silencer (ISS) before the 5’ss leading to inhibition of U2 binding

Looping - hnRNPA1 molecules bond to 21SSs form a loop by associating with one another, this loop is then missed out of splicing

Hyperstabilisation - binds U1 so strongly that it prevents the next step of splicing, i.e. U6 can’t displace U1

Question 10

What is the main function of polypyrimidine tract binding protein (PTB)?

Answer 10

Involved in tissue specific regulation, especialluy in the brain and muscle

Question 11

What does the PTB bind to?

Answer 11

CU rich regions

Question 12

Why can’t the binding sites of PTB be predicted?

Answer 12

They have an unknown RRM order with flexible linkers inbetween

Question 13

How is exon 3 repressed in smooth muscle?

Answer 13

Exon 3 is normally dominant but in smooth muscle it is repressed by PTB and other proteins.
Believed PTB makes a loop at each end of the exon to promote exclusion

Question 14

Outline the experiment that demonstrated exon 3 was repressed in smooth muscle by the formation of hairpin loops at the beginning and end of exon 3

Answer 14

Used SMI to count the number of molecules of PTB bound to each RNA
Found that 5-6 PTBs were bound to each RNA
They used a mutant substrate which showed that 3 bound to the upstream tract and 2-3 to the downstream tract therefore they ruled out smothering
Upstream PTBs interfered with U2AF binding

Question 15

How is the neuronal isoform of src repressed in all cells other than neurones?

Answer 15

PTB binds to the CU-rich elements flanking the N1 exon allowing its interaction with SL4 of U1 snRNA therefore preventing U1 from interacting with the complex of the downstream splice site i.e. U1 is hyperstabilised

Question 16

What did CLIP Seq and RNAi/RNA seq demonstrate about HnRNP binding and function?

Answer 16

The function of many HnRNPs depends upon where they bind:

• Upstream of an exon, in an exon or flanking it, they promote exclusion
• Downstream of an exon they may stimulate exon inclusion

Question 17

What percentage of SNP mutations cause disease by affecting splicing?

Answer 17

30%

Question 18

Can a synonymous mutation (e.g. Val (GUU) –> Val (GUC)) affect splicing?

Answer 18

Yes

Question 19

Do both splice sites work synonymously?

Answer 19

Yes, if U1 doesn’t bind to the 5’ss then 3’ss won’t attract U2AF to attract U2 –> exon skipping

Question 20

What are intronic splicing enhancers?

Answer 20

Sequences deep inside introns that might come together to bring exons close together by stabilising their secondary structure within the intron, thus promoting splicing