23rd Feb - Components that recognise essential sequences at splice sites

Question 1

Describe the U1 snRNP

Answer 1

Has 4 stem loops

2 stem loops are bound by 70K and U1-A proteins

Question 2

How was it tested that the U1 snRNA would base pair with the 5’ss?

Answer 2

Injected U1 Ab into Xenopus oocytes –> U1 is required for splicing

In nuclear extracts:

• -DNA oligo complementary to the 5’ end of U1 snRNA
• -2’-O-methyl-oligo complementary to the 5’ end of U1 snRNA
• -> The 5’ end of U1snRNA is required for splicing

In cells: Transfection of u1SNRNA genes expressing altered 5’end sequences can affect the choice between alternative 5’ss
–> It recognises potential sites and its base pairing affinity can affect splice site choice

Question 3

How does U1 interact with the 5’ss?

Answer 3

Helix A of U1-C interacts with the minor groove of 5’ss-U1 5’ end duplex

Question 4

What facilitates 5’ss-U1 base pairing?

Answer 4

70K and C proteins, but they don’t recognise specific bases

Question 5

What is the function of U2AF 35 and U2AF65?

Answer 5

They recognise the 3’ss

Question 6

What are UHMs?

Answer 6

RRM derived domains that interact with proteins and specifically with ULM motifs

Question 7

Describe U2AF35

Answer 7

It binds AG at the 3’ss
The alpha helix of the UHM binds the ULM of U2AF65
The beta sheets side is blocked by 2 closely packed CCH Zn fingers preventing DNA binding

Question 8

Describe U2AF65

Answer 8

Recognises the polypyrimidine tract

Question 9

What recognises the branch site?

Answer 9

mBBP/SF1

Question 10

How does SF1 detect the branch site?

Answer 10

Through a number of specific H bonds