Figure 6–27 The consensus nucleotide sequences in an RNA molecule that signal the beginning and the end of most introns in humans. The three blocks of nucleotide sequences shown are required to remove an intron sequence. Here A, G, U, and C are the standard RNA nucleotides; R stands for purines (A or G); and Y stands for pyrimidines (C or U). The A highlighted in red forms the branch point of the lariat produced by splicing (see Figure 6–25). Only the GU at the start of the intron and the AG at its end are invariant nucleotides in the splicing consensus sequences. Several different nucleotides can occupy the remaining positions, although the indicated nucleotides are preferred. The distances along the RNA between the three splicing consensus sequences are highly variable; however, the distance between the branch point and 3ʹ splice junction is typically much shorter than that between the 5ʹ splice junction and the branch point.
These snRNPs form the core of the spliceosome, the large assembly of RNA and protein molecules that performs pre-mRNA splicing in the cell. During the splicing reaction, recognition of the 5ʹ splice junction, the branch-point site, and the 3ʹsplice junction is performed largely through base-pairing between the snRNAs and the consensus RNA sequences in the pre-mRNA substrate.
The spliceosome is a complex and dynamic machine. When studied in vitro, a few components of the spliceosome assemble on pre-mRNA and, as the splicing reaction proceeds, new components enter and those that have already performed their tasks are jettisoned (Figure 6–28). However, many scientists believe that, inside the cell, the spliceosome is a preexisting, loose assembly of all the components—capturing, splicing, and releasing RNA as a coordinated unit, and undergoing extensive rearrangements each time a splice is made.
intron portion of a 5˜ 3˜ pre-mRNA transcript
U1, U4 excised intron sequence in the form of a lariat 3˜ (intron RNA will be degraded in the nucleus; snRNPs will exon 1 exon 2 The U1 snRNP forms base pairs with the 5˜ splice junction (see Figure 6–29) and the BBP (branch-point binding protein) and U2AF (U2 auxilliary factor) recognize the branch-point site.
The U2 snRNP displaces BBP and U2AF and forms base pairs with the branch-point site consensus sequence.
The U4/U6•U5 “triple” snRNP enters the reaction. In this triple snRNP, the U4 and U6 snRNAs are held frmly together by base-pair interactions. Subsequent rearrangements break apart the U4/U6 base pairs, allowing U6 to displace U1 at the 5˜ splice junction (see Figure 6–29). This creates the active site that catalyzes the frst phosphoryl-transferase reaction.
Additional RNA–RNA rearrangements create the active site for the second phosphoryl-transferase reaction, which then completes the splice (see Figure 6–25A).
Figure 6–28 The pre-mRNA splicing mechanism. RNA splicing is catalyzed by an assembly of snRNPs (shown as colored circles) plus other proteins (most of which are not shown), which together constitute the spliceosome. The spliceosome recognizes the splicing signals on a pre-mRNA molecule, brings the two ends of the intron together, and provides the enzymatic activity for the two reaction steps required (see Figure 6–25A and Movie 6.5). As indicated, a set of proteins called the exon junction complex (EJC) remains on the spliced mRNA molecule; its subsequent role will be discussed shortly.
The Spliceosome Uses ATP Hydrolysis to Produce a Complex Series of RNA–RNA Rearrangements