Question

Please answer both questions:

A. What are three consensus sequences required for the splicing of major class introns (not minor class introns)?

B. How U1 snRNP recognizes its target in an intron to form the commitment (E) complex to initiate spliceosome formation?

Answer 1

A) Consensus sequences required for the splicing of major class introns:

3' splice sites: CAG
5' splice sites: AAG,CAG

GTAAGT, GTGAGT

B)  The five snRNPs involved in splicing are U1, U2, U5, U4, and U6. Together with some additional proteins, the snRNPs form the spliceosome. Each snRNP contains a single snRNA and several (<20) proteins. The U4 and U6 snRNPs are usually found as a single (U4/U6) particle. A common structural core for each snRNP consists of a group of 8 proteins, all of which are recognized by an autoimmune antiserum called anti-Sm; conserved sequences in the proteins form the target for the antibodies. All the snRNPs except U6 contain a conserved sequence that binds the Sm proteins. The human U1 snRNP contains 8 proteins as well as the RNA.

An SR protein has a variable length of n Arg-Ser-rich region and is involved in splicing.

• U1 snRNP initiates splicing by binding to the 5’ splice site by means of an RNA-RNA pairing reaction.

• The E complex contains U1 snRNP bound at the 5’ splice site, the protein U2AF bound to a pyrimidine tract between the branch site and the 3’ splice site, and SR proteins connecting U1 snRNP to U2AF.

Binding of U1 snRNP to the 5′ splice site is the first step in splicing. The recruitment of U1 snRNP involves an interaction between one of its proteins (U1-70k) and the protein ASF/SF2 (a general splicing factor in the SR class). U1 snRNA base pairs with the 5′ site by means of a single-stranded region at its 5′–terminus which usually includes a stretch of 4-6 bases that is complementary with the splice site.

The wild-type sequence of the splice site of the 12S adenovirus pre-mRNA pairs at 5 out of 6 positions with U1 snRNA. A mutant in the 12S RNA that cannot be spliced has two sequence changes; the GG residues at positions 5-6 in the intron are changed to AU.

E complex =early pre splicing complex,, Splicing can be broadly divided into two stages:

• First the consensus sequences at the 5′ splice site, branch sequence, and adjacent pyrimidine tract are recognized. A complex assembles that contains all of the splicing components.

• Then the cleavage and ligation reactions change the structure of the substrate RNA.

Components of the complex are released or reorganized as it proceeds through the splicing reactions.

The commitment (E) complex forms by the successive addition of U1 snRNP to the 5 splice site, U2AF to the pyrimidine tract/3 splice site, and the bridging protein SF1/BBP (mammalian/yeat). The direct way of forming an E complex is for U1 snRNP to bind at the 5’ splice site and U2AF to bind at a pyrimidine tract between the branch site and the 3’ splice site. Another possibility is for the complex to form between U2AF at the pyrimidine tract and U1 snRNP at a downstream 5’ splice site.