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Splicing - Detailed Scheme

Description of illustration below
The pre-mRNA is shown at the top, having one intron and two flanking exons. Based on sequence comparisons of a large number of introns and functional tests, we know that short sequence motifs in the pre-mRNA are essential for splicing. These are located at the 5' exon-intron border (5' Splice Site), at the 3' exon-intron border (3' Splice Site) and at the so called branch point (BP). Between the branch point and the 3' SS, there is usually a sequence rich in pyrimidines (which are cytidine and uracil) (Py). Apart from these sequences, the remainder of the intron sequence can have any sequence or any length. Introns may be as short as 50 bp or as long as 70-80 thousand bp. It should be remembered that the pre-mRNA is associated with hnRNP proteins and not naked at any time (these proteins are not shown in the figure below).

Based on biochemical experiments, the splicing factors bind to the pre-mRNA in a specific order. Different complexes can be detected (E to C). First, the U1 snRNP binds to the 5' SS, partly by base pairing between the U1 snRNA component and the conserved 5' SS sequence of the pre-mRNA. The U1 snRNP is helped in the binding by special proteins, called SR-proteins. SR-proteins can also help another splicing factor, the U2AF protein complex, to bind to the polypyrimidine region. Next, the U2 snRNP, together with additional splicing factors (such as SF3b and SF3a and SF1) bind. The U2 snRNP binds to the branch point, again by base pairing between the RNA component of the snRNP and the pre-mRNA. PSF is a protein that binds to the pyrimidine rich region. Subsequently, the U4, U6 and U5 snRNPs, already bound to each other in a complex, the U4/U6.U5 tri-snRNP, bind to the pre-mRNA. At this stage the U1 snRNP seems to be removed. Energy, in the form of ATP, is needed at several steps in this scheme. It is not needed for the cleavage and ligation of the pre-mRNA, but presumably for conformational changes in the spliceosome. Additional splicing factors such as SF4 bind, and the first transesterification reaction takes place. After the second transesterfication reaction, the two exons are ligated together. The intron is liberated and rapidly degraded. The splicing factors are believed to be reused.


Description of rollover illustration:
In the spliceosome, numerous RNA-RNA, RNA-protein and protein-protein interactions must occur. We currently know most about the RNA-RNA interactions, because it has been possible to study these interactions both in vitro and in vivo. The RNA components of the snRNP complexes interact with each other and with the pre-mRNA. First, the U1 snRNA and the U2 snRNA bind to the pre-mRNA at the 5' SS and the branch point, respectively (top figure). The U4 and U6 snRNAs bind to each other before they associate with the pre-mRNA (middle figure). In the spliceosome (bottom figure), the U5 snRNA helps to hold the two exons together. The U6 snRNA is believed to play an important role, close to the reaction center. It appears to replace the U1 snRNA at the 5' SS and also binds to the U2 snRNA. At this stage the U4 snRNA no longer binds to the U6 snRNA.

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