Human GC-AG alternative intron isoforms with weak donor sites show enhanced consensus at acceptor exon positions - PubMed (original) (raw)

Human GC-AG alternative intron isoforms with weak donor sites show enhanced consensus at acceptor exon positions

T A Thanaraj et al. Nucleic Acids Res. 2001.

Abstract

It has been previously observed that the intrinsically weak variant GC donor sites, in order to be recognized by the U2-type spliceosome, possess strong consensus sequences maximized for base pair formation with U1 and U5/U6 snRNAs. However, variability in signal strength is a fundamental mechanism for splice site selection in alternative splicing. Here we report human alternative GC-AG introns (for the first time from any species), and show that while constitutive GC-AG introns do possess strong signals at their donor sites, a large subset of alternative GC-AG introns possess weak consensus sequences at their donor sites. Surprisingly, this subset of alternative isoforms shows strong consensus at acceptor exon positions 1 and 2. The improved consensus at the acceptor exon can facilitate a strong interaction with U5 snRNA, which tethers the two exons for ligation during the second step of splicing. Further, these isoforms nearly always possess alternative acceptor sites and exhibit particularly weak polypyrimidine tracts characteristic of AG-dependent introns. The acceptor exon nucleotides are part of the consensus required for the U2AF(35)-mediated recognition of AG in such introns. Such improved consensus at acceptor exons is not found in either normal or alternative GT-AG introns having weak donor sites or weak polypyrimidine tracts. The changes probably reflect mechanisms that allow GC-AG alternative intron isoforms to cope with two conflicting requirements, namely an apparent need for differential splice strength to direct the choice of alternative sites and a need for improved donor signals to compensate for the central mismatch base pair (C-A) in the RNA duplex of U1 snRNA and the pre-mRNA. The other important findings include (i) one in every twenty alternative introns is a GC-AG intron, and (ii) three of every five observed GC-AG introns are alternative isoforms.

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Figures

Figure 1

Figure 1

Illustration of common subsequences, at the donor and acceptor sites, that allow for different interpretations of the gene–transcript alignment.

Figure 2

Figure 2

Sequence logos at splice sites from normal and alternative GC-AG intron isoforms. The ‘RNA structure logo’ program (42) was used to derive the logos.

Figure 3

Figure 3

Sequence logos at splice sites for the three categories of GT-AG normal intron isoforms. Categorization is in terms of donor site strength as assessed by our SPC program (see text). SPC Major introns are those in which the donor sites could be validated by a single major rule of SPC. SPC Minor donor sites could still be validated by SPC but not by the single major rule. SPC Negative donor sites could not be validated by any of the SPC rules.

Figure 4

Figure 4

Sequence logos at splice sites for the three categories of GT-AG alternative intron isoforms. Used are only those isoforms that use alternative donor and acceptor sites [corresponding to (Alt, Alt) in Table 1].

Figure 5

Figure 5

Sequence logos at splice sites for the three categories of GC-AG alternative intron isoforms. In some of the SPC Major introns, only the donor site is alternative. Almost all of the SPC Minor and SPC Negative introns use alternative donor and acceptor sites.

Figure 6

Figure 6

Interaction of the Loop 1 of U5 snRNA with regions from the 5′ and 3′ exons. Bases that occurred in ≥70% of the cases in a group are shown in upper case; those that occurred in 35–70% of cases are shown in lower case (see Table 4 for actual values). Watson–Crick base pairs are indicated by a vertical line; non-Watson–Crick base pairs are indicated by a center dot.

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