Structural analysis of temperature-dependent alternative splicing of HsfA2 pre-mRNA from tomato plants

Temperature-dependent alternative splicing was recently demonstrated for intron 2 of the gene coding for heat shock factor HsfA2 of the tomato plant Solanum lycopersicum, but the molecular mechanism regulating the abundance of such temperature-dependent splice variants is still unknown. We report here on regulatory pre-mRNA structures that could function as regulators by controlling the use of splice sites in a temperature-dependent manner. We investigate pre-mRNA structures at the splice sites of intron 2 of the gene coding for HsfA2 from S. lycopersicum using NMR- and CD-spectroscopy as well as in-line probing. The pre-mRNA undergoes conformational changes between two different secondary structures at the 3ʹ splice site of the intron in a temperature-dependent manner. Previously, it was shown that three single nucleotide polymorphisms (SNPs) in intron 2 of the HsfA2 pre-mRNA affect the splicing efficiency of its pre-mRNA and are linked to the thermotolerance in different tomato species. By comparing pre-mRNA fragments of the tomato species S. lycopersicum and S. peruvianum, we show that these SNPs result in substantial structural differences between the pre-mRNAs of the two species.