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

Abstract

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.

Transcriptomic and splicing changes underlying tomato responses to combined water and nutrient stress

“…Consistent with this report, we observed severe up-regulation of HsfA2 in roots of both genotypes. In addition, HsfA2 was recently shown to be alternatively spliced in response to heat stress, with isoform abundance being temperature-dependent (Hu et al, 2020;Broft et al, 2022). The two main variants, HsfA2-II and HsfA2-Ia, characterized by splicing or retention of intron 2 respectively, were detected with different abundance in wild or modern tomato accessions.…”

Surviving a Double-Edged Sword: Response of Horticultural Crops to Multiple Abiotic Stressors

“…Recent studies have further proposed significant roles of post-transcriptional gene expression regulation in mediating the response of horticultural crops to abiotic stresses, which mainly involve pre-mRNA splicing and processing, regulation of mRNA stability and its degradation process, etc. For instance, pre-mRNA alternative splicing can modulate the responses of tomato against high-temperature stress [165]. The Cys2/His2 (C2H2)-type zinc finger protein MdZAT5 targets drought-responsive RNAs and microR-NAs to control apple's tolerance to drought [166].…”

Relevance and Regulation of Alternative Splicing in Plant Heat Stress Response: Current Understanding and Future Directions

“…Differences in the splicing pattern of HsfA2 between haplotypes from modern cultivars and wild tomato species have been attributed to three intronic singly nucleotide polymorphisms (SNPs) ( Hu et al, 2020b ). Based on nuclear magnetic resonance (NMR) analysis, the presence of SNPs alter the local RNA structure and thus might affect the binding of splicing regulators ( Broft et al, 2022 ). Interestingly, while increased temperature did not affect the structure of the 5′-end of the intron, a 3′-end intronic region shows a temperature dependent RNA fold, yielding a highly stable structure under high temperature ( Broft et al, 2022 ).…”

“…Based on nuclear magnetic resonance (NMR) analysis, the presence of SNPs alter the local RNA structure and thus might affect the binding of splicing regulators ( Broft et al, 2022 ). Interestingly, while increased temperature did not affect the structure of the 5′-end of the intron, a 3′-end intronic region shows a temperature dependent RNA fold, yielding a highly stable structure under high temperature ( Broft et al, 2022 ). It is hypothesized, that the stable structure unfolds a single stranded region that might facilitate the binding of splicing silencers.…”