Splicing in Caenorhabditis elegans Does Not Require an AG at the 3' Splice Acceptor Site

Abstract

The dinucleotide AG, found at the 3' end of virtually all eukaryotic pre-mRNA introns, is thought to be essential for splicing. Reduction-of-function mutations in two Caenorhabditis elegans genes, the receptor tyrosine kinase gene let-23 and the collagen gene dpy-10, both alter the AG at the end of a short (ca. 50-nucleotide) intron to AA. The in vivo effects of these mutations were studied by sequencing polymerase chain reaction-amplified reverse-transcribed RNA isolated from the two mutants. As expected, we find transcripts that splice to a cryptic AG, skip an exon, and retain an unspliced intron. However, we also find significant levels of splicing at the mutated 3' splice site (AA) and at nearby non-AG dinucleotides. Our results indicate that for short C. elegans introns an AG is not required for splicing at either the correct 3' splice site or incorrect sites. Analysis of a splice site mutant involving a longer, 316-nucleotide C. elegans intron indicates that an AG is also not required there for splicing. We hypothesize that elements besides the invariant AG, e.g., an A-U-rich region, a UUUC motif, and/or a potential branch point sequence, are directing the selection of the 3' splice site and that in wild-type genes these elements cooperate so that proper splicing occurs.

Purifying selection against spurious splicing signals contributes to the base composition evolution of the polypyrimidine tract

“…U2AF1 recognizes and binds to the 3′-AG motif after scanning the region downstream of the branch point, i.e., the polypyrimidine tract (Smith et al, 1993). Mutational analyses showed these motifs can serve as the 3′ splice site in C. elegans (Aroian et al, 1993;Zhang & Blumenthal, 1996). Thus observing them as the most under-represented dimers in asymmetry scores still supports the effect of avoidance of premature splicing in the nucleotide composition evolution of the 3′ region of short introns.…”

Purifying selection against spurious splicing signals contributes to the base composition evolution of the polypyrimidine tract

“…Their introns are exceptionally short, most under 60 nucleotides, and seem to lack an obvious polypyrimidine tract. Additionally, they use splicing signals not containing AG more often than other species (Aroian et al, 1993; Riddle et al, 1997).…”

“…At the dimer level, the most under-represented motif was AG for all species, except C. elegans . In C. elegans AG is not a requirement for maximum splicing efficiency, but AA or AC dinucleotides are also used as a 3’ splice signal (Aroian et al, 1993; Riddle et al, 1997). Thus, observing AA as the most under-represented dimer in asymmetry scores still supports that the nucleotide composition of the 3’ region of short introns evolved to avoid premature splicing.…”

“…Additionally, even though AG is highly conserved, they use splicing signals not containing AG more often than other species (Riddle et al, 1997). It has been demonstrated that AG is not obligatory for the 3’ splice site recognition, since mutations from G to A or to C did not affect splicing (Aroian et al, 1993; Zhang & Blumenthal, 1996). The fungal genomes have also relatively short and few introns (in the hundreds), while other eukaryotes contain thousands of introns (Neuveglise et al, 2011).…”

“…In C. elegans AA was the first and AC the third most under-represented dimers. The mutational analyses showed these motifs can serve as the 3’ splice site in C. elegans (Aroian et al, 1993; Zhang & Blumenthal, 1996). Thus observing them as the most under-represented dimers in asymmetry scores still supports the effect of avoidance of premature splicing in the nucleotide composition evolution of the 3’ region of short introns.…”