Scanning and Competition between AGs Are Involved in 3' Splice Site Selection in Mammalian Introns

Abstract

In mammalian intron splicing, the mechanism by which the 3' splice site AG is accurately and efficiently identified has remained unresolved. We have previously proposed that the 3' splice site in mammalian introns is located by a scanning mechanism for the first AG downstream of the branch point-polypyrimidine tract. We now present experiments that lend further support to this model while identifying conditions under which competition can occur between adjacent AGs. The data show that the 3' splice site is identified as the first AG downstream from the branch point by a mechanism that has all the characteristics expected of a 5'-to-3' scanning process that starts from the branch point rather than the pyrimidine tract. Failure to recognize the proximal AG may arise, however, from extreme proximity to the branch point or sequestration within a hairpin. Once an AG has been encountered, the spliceosome can still see a limited stretch of downstream RNA within which an AG more competitive than the proximal one may be selected. Proximity to the branch point is a major determinant of competition, although steric effects render an AG less competitive in close proximity (approximately 12 nucleotides). In addition, the nucleotide preceding the AG has a striking influence upon competition between closely spaced AGs. The order of competitiveness, CAG congruent to UAG > AAG > GAG, is similar to the nucleotide preference at this position in wild-type 3' splice sites. Thus, 3' splice site selection displays properties of both a scanning process and competition between AGs based on immediate sequence context. This refined scanning model, incorporating elements of competition, is the simplest interpretation that is consistent with all of the available data.

m6A modification of U6 snRNA modulates usage of two major classes of pre-mRNA 5’ splice site

“…This results in a continuous helix stack, involving the interaction between U6 A43 and 5’SS A +4 , that forms the receptor onto which the 3’SS docks. Our data are consistent with a model in which a strong interaction between U6 and the 5’SS +4 position stabilises this receptor, enabling 3’SSs distal to the BP to compete more efficiently with proximal 3’SSs that are favoured by scanning (Smith et al, 1993). A more stable receptor could allow the ATPase Prp22, which proof reads exon ligation (Mayas et al, 2006), to promote more efficient sampling and usage of distal 3’SSs.…”

“…However, competition with downstream 3’SSs can occur within a short range, such as in NAGNAG acceptors. Downstream 3’SSs in fio1 -sensitive alternative 3’SS pairs were more likely to have a cytosine as the —3 position than upstream 3’SSs (Figure 7B-C), a feature that has been shown to increase 3’SS competitiveness (Bradley et al, 2012; Smith et al, 1993). However, this appears to reflect differences in the background rate of C -3 at upstream and downstream 3’SSs, rather than a change in motif competitiveness in fio1-3 (Figure 7 – figure supplement 1D).…”

“…3’SS choice involves scanning downstream from the branchpoint to the first available 3’SS motif (Smith et al, 1993). However, competition with downstream 3’SSs can occur within a short range, such as in NAGNAG acceptors.…”

“…In fio1-3, at 20ºC, there is a 12.6% shift in favour of the downstream CAG\\ 3'SS (Figure 7 -figure supplement 1C). 3'SS choice involves scanning downstream from the branchpoint to the first available 3'SS motif (Smith et al, 1993). However, competition with downstream 3'SSs can occur within a short range, such as in NAGNAG acceptors.…”

A broad analysis of splicing regulation in yeast using a large library of synthetic introns

“…Hence it was suggested that the 3'SS is recognized through a scanning mechanism and that any HAG (i.e. [A/C/T]AG) site could be recognized [31]. However, a vast majority of 3'SS of S. cerevisiae natural introns are in fact YAG (i.e.…”