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Anyone can expatiate clearly the mechanism of pre-mRNA splicing? - Help (Jun/24/2005 )

Any guys know the mechanism of pre-mRNA splicing well?
Especially,what is the H,E,B,A,C complexes formed in the splicing procedure?


QUOTE (hxygz @ Jun 25 2005, 01:30 AM)
Any guys know the mechanism of pre-mRNA splicing well?
Especially,what is the H,E,B,A,C complexes formed  in the splicing procedure?

Splicing of pre-mRNA occurs within the multicomponent spliceosome complex via two successive chemical steps (reviewed in references 33, 41, 47, and 63). Introns are excised and adjacent exons are spliced together, and this must be achieved with great accuracy, even though the introns may be many thousands of bases in length. Sequence elements at the splice sites themselves, as well as activating enhancer elements, provide the signals within the pre-mRNA that give rise to this specificity. The requirement for accuracy in determination of the precise splice junctions is reflected in a number of steps in which the primary consensus elements are recognized sequentially by separate protein or small nuclear RNA (snRNA) splicing factors. Work with both yeast and HeLa nuclear extracts has led to a model in which the spliceosome is assembled by sequential addition of small nuclear ribonucleoprotein particles (snRNPs) and other splicing factors and by substantial conformational rearrangements before the actual catalytic steps (reviewed in reference 58). A number of the steps leading to the formation of a catalytically competent spliceosome require ATP hydrolysis, either for protein phosphorylation by kinases or for the action of RNA helicases. Initial recognition of splice sites involves binding of U1 snRNP to the 5' splice site, mediated by base pairing between the 5' splice site consensus sequence and the 5' end of U1 snRNA (62, 64, 84). At the 3' end of the intron, the heterodimeric protein U2AF binds to the polypyrimidine tract via the RNA binding domains of the 65-kDa subunit (82). Both of these initial binding events can be assisted by proteins of the SR class (17, 29, 69). In yeast, the branch point sequence is initially recognized by the protein BBP (for branch point binding or bridging protein [known as SF1 or mBBP in mammalian systems]) (2, 3, 5). At this E, or commitment complex, stage, a bridging interaction can already occur across the intron (46), formed either by BBP (2) or by SR proteins (79). Subsequent formation of the A complex, or prespliceosome, involves ATP hydrolysis and the binding of 17S U2 snRNP to the branch point aided by the N-terminal domain of U2AF65 (74) and a DEAD-box helicase protein (21, 60, 75). Recognition of the branch point at this stage involves base pairing between the branch point sequence and a conserved region of U2 snRNA (53, 77, 83), with the branch point adenosine itself remaining bulged out of the intermolecular duplex (56). Formation of the B complex involves the binding of the U4/5/6 tri-snRNP to the A complex (30), a step that is also ATP dependent and that is activated by SR proteins (59). This is followed by conformational rearrangements. In particular, U4-U6 base pairing is disrupted, and U6 becomes base paired with both U2 snRNA (16, 42, 78) and with the intron part of the 5' splice site sequence (26, 36, 68), replacing the U1-5' splice site duplex and forming an RNA bridge, composed of U2 and U6 snRNAs, between the 5' splice site and branch point (41, 51). In addition, U5 snRNA contacts bases just within the 5' exon (49, 68). The spliceosome, poised for catalytic step 1, is now referred to as the C complex and is characterized by the presence of splicing intermediates.