An autoregulatory loop controlling Arabidopsis HsfA2 expression: role of heat shock-induced alternative splicing

Heat shock transcription factorA2 (HsfA2) is a key regulator in response to heat stress in Arabidopsis (Arabidopsis thaliana), and its heat shock (HS)-induced transcription regulation has been extensively studied. Recently, alternative splicing, a critical posttranscriptional event, has been shown to regulate HS-inducible expression of HsfA2; however, the molecular mechanism remains largely unknown. Here, we demonstrate a new heat stress-induced splice variant, HsfA2-III, is involved in the self-regulation of HsfA2 transcription in Arabidopsis. HsfA2-III is generated through a cryptic 5' splice site in the intron, which is activated by severe heat (42°C-45°C). We confirmed that HsfA2-III encodes a small truncated HsfA2 isoform (S-HsfA2) by an immunoblot assay with anti-S-HsfA2 antiserum. S-HsfA2 has an extra leucine-rich motif next to its carboxyl-terminal truncated DNA-binding domain. The biological significance of S-HsfA2 was further demonstrated by its nuclear localization and heat shock element (HSE)-binding ability. In yeast (Saccharomyces cerevisiae), the leucine-rich motif can inhibit the transcriptional activation activity of S-HsfA2, while it appears not to be required for the truncated DNA-binding domain-mediated binding ability of S-HsfA2-HSE. Further results reveal that S-HsfA2 could bind to the TATA box-proximal clusters of HSE in the HsfA2 promoter to activate its own transcription. This S-HsfA2-modulated HsfA2 transcription is not mediated through homodimer or heterodimer formation with HsfA1d or HsfA1e, which are known transcriptional activators of HsfA2. Altogether, our findings provide new insights into how HS posttranscriptionally regulates HsfA2 expression. Severe HS-induced alternative splicing also occurs in four other HS-inducible Arabidopsis Hsf genes, suggesting that it is a common feature among Arabidopsis Hsfs.