Evolutionarily emerged G tracts between the polypyrimidine tract and 3′ AG are splicing silencers enriched in genes involved in cancer

Abstract

BackgroundThe 3′ splice site (SS) at the end of pre-mRNA introns has a consensus sequence (Y)nNYAG for constitutive splicing of mammalian genes. Deviation from this consensus could change or interrupt the usage of the splice site leading to alternative or aberrant splicing, which could affect normal cell function or even the development of diseases. We have shown that the position “N” can be replaced by a CA-rich RNA element called CaRRE1 to regulate the alternative splicing of a group of genes.ResultsTaking it a step further, we searched the human genome for purine-rich elements between the -3 and -10 positions of the 3′ splice sites of annotated introns. This identified several thousand such 3′SS; more than a thousand of them contain at least one copy of G tract. These sites deviate significantly from the consensus of constitutive splice sites and are highly associated with alterative splicing events, particularly alternative 3′ splice and intron retention. We show by mutagenesis analysis and RNA interference that the G tracts are splicing silencers and a group of the associated exons are controlled by the G tract binding proteins hnRNP H/F. Species comparison of a group of the 3′SS among vertebrates suggests that most (~87%) of the G tracts emerged in ancestors of mammals during evolution. Moreover, the host genes are most significantly associated with cancer.ConclusionWe call these elements together with CaRRE1 regulatory RNA elements between the Py and 3′AG (REPA). The emergence of REPA in this highly constrained region indicates that this location has been remarkably permissive for the emergence of de novo regulatory RNA elements, even purine-rich motifs, in a large group of mammalian genes during evolution. This evolutionary change controls alternative splicing, likely to diversify proteomes for particular cellular functions.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1143) contains supplementary material, which is available to authorized users.

Evolutionary Emergence of a Novel Splice Variant with an Opposite Effect on the Cell Cycle

“…Splicing reporters with 3=SS of different species or human G-tract mutant were constructed as described previously (13,29) (see Fig. 2B).…”

“…The cells were grown and transfected as described previously (29). We used 0.4 g of expression plasmids for HeLa cells in 24-well plates.…”

“…Five days after first transduction, cells were harvested for Western blot analysis, RNA extraction, or immunostaining. The knockdown of hnRNP H/F was carried out as described previously (19,29).…”

“…We have identified G tracts between the Py and 3= AG in more than a thousand human genes, including PRMT5 (for protein arginine methyltransferase 5). We call elements at this location REPA (regulatory elements between the Py and 3=AG) (29). These REPA G tracts appear to have mostly emerged in mammalian ancestors to act as splicing silencers involving hnRNP H and F (H/F).…”

Novel alternative splicing variants of <i>ACOX1</i> and their differential expression patterns in goats

“…These transcripts contribute to diverse biological processes, such as cell differentiation (Kianianmomeni et al, 2014;Cotter et al, 2015), disease development (Sohail et al, 2014) and the adipocyte metabolism (Fiszbein et al, 2017). Previous studies reported that the splicing variants of PPARG (PPARG1 and PPARG2) could affect the lipogenesis of mammary tissue via the upregulated expressions of key lipogenic gene networks (Shi et al, 2014).…”