How does temperature affect splicing events? Isoform switching of splicing factors regulates splicing of LATE ELONGATED HYPOCOTYL (LHY)

Abstract

One of the ways in which plants can respond to temperature is via alternative splicing (AS). Previous work showed that temperature changes affected the splicing of several circadian clock gene transcripts. Here, we investigated the role of RNA‐binding splicing factors (SFs) in temperature‐sensitive AS of the clock gene LATE ELONGATED HYPOCOTYL (LHY). We characterized, in wild type plants, temperature‐associated isoform switching and expression patterns for SF transcripts from a high‐resolution temperature and time series RNA‐seq experiment. In addition, we employed quantitative RT‐PCR of SF mutant plants to explore the role of the SFs in cooling‐associated AS of LHY. We show that the splicing and expression of several SFs responds sufficiently, rapidly, and sensitively to temperature changes to contribute to the splicing of the 5′UTR of LHY. Moreover, the choice of splice site in LHY was altered in some SF mutants. The splicing of the 5′UTR region of LHY has characteristics of a molecular thermostat, where the ratio of transcript isoforms is sensitive to temperature changes as modest as 2 °C and is scalable over a wide dynamic range of temperature. Our work provides novel insight into SF‐mediated coupling of the perception of temperature to post‐transcriptional regulation of the clock.

Global spatial analysis of Arabidopsis natural variants implicates 5′UTR splicing of LATE ELONGATED HYPOCOTYL in responses to temperature

“…We inspected the LHY locus (At1g01060) for SNPs and focused our attention on a subset of five SNPs (Figure S1, Figure a, and Table ) located within the 5′UTR region because the balance between constitutive splicing and retention of the 5′UTR intron 1 in the LHY pre‐mRNA is scalable with temperature transitions (James, Syed, Bordage, et al, ; James et al, ). Sequence base calls, including uncalled N's, and latitude–longitude coordinates for each of the five SNP positions for 1,135 accessions were obtained (see Section 2).…”

“…PTBs contribute to the temperature‐dependent splicing of LHY (James et al, ) and as such PTB would be predicted to interact specifically with LHY mRNA. To test this, we used RNA‐EMSA binding reactions with recombinant PTB1 protein and an in vitro transcribed LHY mRNA fragment comprising exons 1 through 5 and the intervening introns (representing the Col‐0 G/G/U/G/C haplotype) to ask whether PTB1 bound LHY pre‐mRNA.…”

“…We based this analysis of 1,001 genomes accessions on our recent observations of the dynamic responses of LHY AS to temperature, and in particular, the “molecular thermostat” properties of 5′UTR splicing—an apparently adaptive response to temperature such that I1R splicing is most prominent during temperature transitions (and not “steady state” temperatures), akin to fluctuations in temperatures that are a hallmark of natural climatic conditions (James et al, ). We focused our attention on five SNPs in the 5′UTR region—two exonic and three intronic—that cluster as four haplotypes.…”

“…Conceptually, the regulation of LHY therefore represents an interesting model of how the clock adapts to (a) fluctuations and (b) longer term changes in temperature that are analogous in nature to unpredictable everyday changes and longer term (conceivably seasonal) changes in temperature, respectively. The LHY I1R event is of particular interest, because switching between fully spliced (FS) and I1R isoforms with temperature is rapid and reversible (James, Syed, Bordage, et al, ), has characteristics of a thermometer in that it is sensitive to temperature changes as modest as 2 °C, and is scalable and reversible over a wide dynamic range of temperature (James et al, ).…”

“…Pre‐mRNA processing is simultaneous, and mechanistically coupled, to transcription with splicing factors (SFs) recruited either constitutively or on an “as needed” basis to intron‐containing genes (Bentley, ). The complexity of temperature signalling has recently been augmented with studies revealing cooling associated “splicing of the splicing factors” (Verhage et al, ; James et al, ), for example, for temperature‐associated isoform switching of the polypyrimidine (pY) tract‐binding (PTB) proteins and U2 auxiliary factor 65A (James et al, ) both of which compete for interaction with pY‐rich sequences thereby influencing efficiency of splicing (Simpson et al, ).…”