Evidence for a linear search in bimolecular 3′ splice site AG selection

Abstract

In most eukaryotic introns the 3 splice site is defined by a surprisingly short AG consensus found a variable distance downstream of the branch site. Exactly how the spliceosome determines which AG to use, however, is not well understood. Previously we showed that when the branch site and 3 splice site AG are supplied by separate RNA molecules, there is a strong preference for use of the 5 -most AG in the 3 splice site-containing RNA. Here we show that this apparent 5 33 directionality holds even when this RNA contains four tandem repeats of a 6-nt sequence containing AG. Exactly the same pattern of 3 splice site choice was observed when the same tandem repeats were incorporated into a full-length splicing substrate. When the 3 splice site AG is supplied by a separate RNA, that RNA must be linear with an unobstructed 5 end. Similarly, the branch-containing RNA must be truncated immediately 3 to the polypyrimidine tract. A model is presented that incorporates these observations and reconciles previously proposed mechanisms for 3 splice site selection. P recise excision of introns is an essential step in eukaryotic gene expression. Most introns in the human genome (10 5 -10 6 different sequences) are removed by a single macromolecular machine, the major spliceosome. Within this machine, intron excision occurs in two steps: cleavage at the 5Ј splice site coupled with lariat formation at the branch site, followed by exon ligation at the 3Ј splice site (1, 2). However, the exact mechanisms by which these sites are recognized are not entirely understood. This is particularly true for the 3Ј splice site. In budding yeast, the only conserved sequence defining this key site is a YAG͞ trinucleotide (where Y denotes pyrimidine and ''͞'' denotes the splice site) found a variable distance downstream of the branch site. In mammals the region between the branch site and YAG͞is generally rich in pyrimidines (the polypyrimidine tract or PPT) (1,3,4). Notably, the natural 3Ј splice site in some introns is an AAG͞or GAG͞(5), with the order of preference in a test system being CAG Ϸ UAGϾAAGϾGAG (6). Thus, the sequence defining the exact site of exon ligation is apparently just AG, which could occur randomly every 16 dinucleotides. Given such odds, the spliceosome undoubtedly uses other information to distinguish the correct 3Ј splice site AG from any others nearby. Exactly what that information is, however, and how it is processed by the splicing machinery remain a matter of open debate (see ref. 7 and Discussion).To better study the mechanisms of 3Ј splice site selection by the human spliceosome, we recently developed a bimolecular (trans) assay for exon ligation in HeLa nuclear extracts (8). In this system, spliceosomes assemble and undergo the first chemical step on a 5Ј substrate consisting of a 5Ј exon and intron truncated immediately 3Ј to the PPT (Fig. 1 A). Exon ligation is then initiated by addition of a separate RNA, the 3Ј substrate, containing one or more AGs plus a downstream exon. Previously we found that the only seq...

Four exons of the serotonin receptor 4 gene are associated with multiple distant branch points

“…In introns with distant BPs (dBPs) and PPTs, the 39 ss is located by a scanning process from the BP to the first downstream AG (Smith et al , 1993Liu et al 1997). Similar scanning behavior is observed in bimolecular exon ligation experiments (Chen et al 2000). Consequently, one prerequisite of the region between distant BPs and the 39 ss is that it lacks any other AG dinucleotides leading to its designation as the AG-exclusion zone (AGEZ) (Gooding et al 2006).…”

Systematic Study of Sequence Motifs for RNA trans Splicing in Trypanosoma brucei

“…According to the scanning model, identification of the correct 3ЈSS AG occurs by a linear search mechanism, assuming that the spliceosome begins scanning at the branch point and selects the first downstream AG dinucleotide (39). This model is supported by research with HeLa cells, in which splicing is blocked when hairpin loops are inserted upstream of the AG splice site, possibly preventing movement of the spliceosome (3). With a bimolecular exon ligation assay, it was also shown that the substrate with the 5Ј-most AG is selected among different 3Ј RNA substrates and that no poly(Y) tract was necessary for the second catalytic step (1,3).…”

TheIgHLocus of the Channel Catfish,Ictalurus punctatus, Contains Multiple Constant Region Gene Sequences: Different Genes Encode Heavy Chains of Membrane and Secreted IgD

“…The shift in splice site usage (confirmed by cDNA sequencing) adds two additional amino acids to the exon. This IGHD3 ␦6 splice site reflects the introduction of an AG dinucleotide and the preferential use of the more 5Ј AG as a splice acceptor site (31,32). In toto there are 24 insertions/deletions between these two ␦ genes, with most being short, i.e., 14 are Ͻ5 bp.…”