More than 5% of alternatively spliced internal exons in the human genome are derived from Alu elements in a process termed exonization. Alus are comprised of two homologous arms separated by an internal polypyrimidine tract (PPT). In most exonizations, splice sites are selected from within the same arm. We hypothesized that the internal PPT may prevent selection of a splice site further downstream. Here, we demonstrate that this PPT enhanced the selection of an upstream 5′ splice site (5′ss), even in the presence of a stronger 5′ss downstream. Deletion of this PPT shifted selection to the stronger downstream 5′ss. This enhancing effect depended on the strength of the downstream 5′ss, on the efficiency of base-pairing to U1 snRNA, and on the length of the PPT. This effect of the PPT was mediated by the binding of TIA proteins and was dependent on the distance between the PPT and the upstream 5′ss. A wide-scale evolutionary analysis of introns across 22 eukaryotes revealed an enrichment in PPTs within ∼20 nt downstream of the 5′ss. For most metazoans, the strength of the 5′ss inversely correlated with the presence of a downstream PPT, indicative of the functional role of the PPT. Finally, we found that the proteins that mediate this effect, TIA and U1C, and in particular their functional domains, are highly conserved across evolution. Overall, these findings expand our understanding of the role of TIA1/TIAR proteins in enhancing recognition of exons, in general, and Alu exons, in particular.
Human genes are composed of functional regions, termed exons, separated by non-functional regions, termed introns. Intronic sequences may gradually accumulate mutations and subsequently become recognized by the splicing machinery as exons, a process termed exonization. Alu elements are prone to undergo exonization: more than 5% of alternatively spliced internal exons in the human genome originate from Alu elements. A typical Alu element is ∼300 nucleotides long, consisting of two arms separated by a polypyrimdine tract (PPT). Interestingly, in most cases, exonization occurs almost exclusively within either the right arm or the left, not both. Here we found that the PPT between the two arms serves as a binding site for TIA proteins and prevents the exon selection process from expanding into downstream regions. To obtain a wider overview of TIA function, we performed a cross-evolutionary analysis within 22 eukaryotes of this protein and of U1C, a protein known to interact with it, and found that functional regions of both these proteins were highly conserved. These findings highlight the pivotal role of TIA proteins in 5′ splice-site selection of Alu exons and exon recognition in general.