4. The spliceosome contains snRNAs

22.3 Nuclear splicing proceeds through a lariat

Key terms defined in this section
Lariat is an intermediate in RNA splicing in which a circular structure with a tail is created by a 5´-2´ bond.

The mechanism of splicing has been characterized in vitro, using systems in which introns can be removed from RNA precursors. Nuclear extracts can splice purified RNA precursors, which shows that the action of splicing is not linked to the process of transcription. Splicing is also independent of modification of RNA, and can occur to RNAs that are neither capped nor polyadenylated.




Figure 22.6 Splicing occurs in two stages, in which the 5 F exon is separated and then is joined to the 3 F exon.

The stages of splicing in vitro are illustrated in the pathway of Figure 22.6. We discuss the reaction in terms of the individual RNA species that can be identified, but we should remember that in vivo the species containing exons are not released as free molecules, but remain held together by the splicing apparatus (for review see Sharp, 1994).


In the first stage, a cut is made at the 5′ splice site, separating the left exon and the right intron-exon molecule. The left exon takes the form of a linear molecule. The right intron-exon molecule forms a lariat, in which the 5′ terminus generated at the end of the intron becomes linked by a 5′ V2′ bond to a base within the intron. The target base is an A in a sequence that is called the branch site (Reed and Maniatis, 1985).


In the second stage, cutting at the 3′ splice site releases the free intron in lariat form, while the right exon is ligated (spliced) to the left exon. The cleavage and ligation reactions are shown separately in the figure for illustrative purposes, but actually occur as one coordinated transfer.


The lariat is then "debranched" to give a linear excised intron, which is rapidly degraded.


The sequences needed for splicing are the short consensus sequences at the 5and 3splice sites and at the branch site. Together with the knowledge that most of the sequence of an intron can be deleted without impeding splicing, this indicates that there is no demand for specific conformation in the intron (or exon).


The branch site provides the means by which the 3′ splice site is identified. The branch site in yeast is highly conserved, and has the consensus sequence UACUAAC. The branch site in higher eukaryotes is not well conserved, but has a preference for purines or pyrimidines at each position and retains the target A nucleotide (see Figure 22.6) (Zhuang et al., 1989).


The branch site lies 18 V40 nucleotides upstream of the 3′ splice site. Mutations or deletions of the branch site in yeast prevent splicing. In higher eukaryotes, the relaxed constraints in its sequence result in the ability to use related sequences in the vicinity when the authentic branch is deleted. Proximity to the 3′ splice site appears to be important, since the cryptic site is always close to the authentic site. When a cryptic branch sequence is used in this manner, splicing otherwise appears to be normal; and the exons give the same products as wild type. The role of the branch site therefore is to identify the nearest 3splice site as the target for connection to the 5′ splice site (Reed and Maniatis, 1986).


The bond that forms the lariat goes from the 5′ position of the invariant G that was at the 5′ end of the intron to the 2′ position of the invariant A in the branch site. This corresponds to the third A residue in the yeast UACUAAC box.




Figure 22.7 Nuclear splicing occurs by two transesterification reactions in which a free OH end attacks a phosphodiester bond.

The chemical reactions proceed by transesterification: a bond is in effect transferred from one location to another. Figure 22.7 shows that the first step is a nucleophilic attack by the 2′ VOH of the invariant A of the UACUAAC sequence on the 5′ splice site. In the second step, the free 3′ VOH of the exon that was released by the first reaction now attacks the bond at the 3′ splice site. Note that the number of phosphodiester bonds is conserved. There were originally two 5′ V3′ bonds at the exon-intron splice sites; one has been replaced by the 5′ V3′ bond between the exons, and the other has been replaced by the 5′ V2′ bond that forms the lariat (for review see Weiner, 1993).




Reviews
Sharp, P.A. (1994). Split genes and RNA splicing. Cell 77, 805-815.
Weiner, A. (1993). mRNA splicing and autocatalytic introns: distant cousins or the products of chemical determinism. Cell 72, 161-164.

Research
Reed, R. and Maniatis, T. (1985). Intron sequences involved in lariat formation during pre-mRNA splicing. Cell 41, 95-105.
Reed, R. and Maniatis, T. (1986). A role for exon sequences and splice-site proximity in splice-site selection. Cell 46, 681-690.
Zhuang, Y. A., Goldstein, A. M., and Weiner, A. M. (1989). UACUAAC is the preferred branch site for mammalian mRNA splicing. Proc. Nat. Acad. Sci. USA 86, 2752-2756.



Genes VII
Genes VII
ISBN: B000R0CSVM
EAN: N/A
Year: 2005
Pages: 382

flylib.com © 2008-2017.
If you may any questions please contact us: flylib@qtcs.net