Heat induces the splicing by IRE1 of a mRNA encoding a transcription factor involved in the unfolded protein response in Arabidopsis

Abstract

Adverse environmental conditions produce endoplasmic reticulum (ER) stress in plants. In response to heat or ER stress agents, Arabidopsis seedlings mitigate stress damage by activating ER-associated transcription factors and a RNA splicing factor, IRE1b. IRE1b splices the mRNA-encoding bZIP60, a basic leucine-zipper domain containing transcription factor associated with the unfolded protein response in plants. bZIP60 is required for the up-regulation of BINDING PROTEIN3 (BIP3) in response to ER stress, and loss-of-function mutations in IRE1b or point mutations in the splicing site of bZIP60 mRNA are defective in BIP3 induction. These findings demonstrate that bZIP60 in plants is activated by RNA splicing and afford opportunities for monitoring and modulating stress responses in plants.

Fig. 1.

Twin hairpin loop structure at splicing site in bZIP60 mRNA. (A) Each of the two loops contains three conserved bases (highlighted). Solid block arrows indicate predicted cleavage sites. (Equivalent cleavage sites indicated by open block arrows.) Base substitution mutations are indicated with blue arrows. For constructs with a single point mutation (1PM), the G at nucleotide position 672 in loop 2 has been substituted by a C. This base substitution is not a coding change for bZIP60 mRNA. Constructs with two point mutations (2PM) involve the base substitution in loop 2 and another one in loop 1 in which the G at nucleotide position 652 has been substituted by a U. The second base substitution changes the coding capacity of bZIP60 mRNA; however, the change is a conservative one, exchanging a hydrophobic amino acid for another, a V to a L. (B) Splicing assay using primers immediately flanking splice site (Upper). Electrophoretic gel band pattern of RT-PCR products from RNA sample taken from untreated seedlings or seedlings treated for 2 h with 2 μg/mL tunicamycin. Interpretation of the various RT-PCR products (labeled 1–2) based on reamplication of cDNA from individual bands (Lower). (C) Partial sequence of cDNA derived from unspliced and spliced forms of AtbZIP60 mRNA shown in B. Arrows indicate splice sites inferred from the sequence of the spliced mRNA. Underlined segment of the amino acid sequence derived from the unspliced form is a predicted transmembrane domain. Amino acid sequence in red predicted from the spliced RNA form represents differences in the sequence predicted from the unspliced form.

Fig. 2.

Induction of bZIP60 splicing by treatment with DTT. (A) Seedlings were transferred to liquid LS medium containing 2 mM DTT and incubated for the times indicated. RNA samples were analyzed for the presence of unspliced (U) and spliced (S) bZIP60 mRNA by RT-PCR using the flanking primers (FP) assay or the specific primers assay using primers for unspliced bZIP60 mRNA (SPU) or for spliced mRNA (SPS). Amplification with primers for actin mRNA was used as a control. (B) Effects of T-DNA mutations in IRE1a and IRE1b on splicing of bZIP60 mRNA in seedlings. Seedlings were treated with 2 mM DTT and RNA was extracted 2 h later. bZIP60 mRNA splicing was analyzed by SPU and SPS assays for the presence of unspliced and spliced RNA forms, respectively. (C) Diagrams illustrating the predicted structure of proteins derived from the unspliced and the spliced forms of bZIP60 mRNAs. (D) Localization of bZIP60 proteins derived from unspliced (Top and Middle) and spliced mRNAs (Bottom). Constructs encoding GFP-tagged forms of bZIP60 were introduced by biolistics into tobacco BY-2 cells. RFP-HDEL was used as an ER marker, and RFP alone was used to mark both nuclei and cytoplasm.

Fig. 3.

In vivo splicing analysis of the transgenic plants. (A) Splicing of bZIP60 mRNA was induced by treatment of seedlings with 2 mM DTT for 2 h. Splicing is demonstrated by use of the RT-PCR assay with splice specific primers (SPS assay). Lanes 1–4 show splicing of endogenous bZIP60 mRNA. Lanes 5–16 show splicing of RNA derived from bZIP60 transgenes in the background of bzip60-1. Transgenes bear no point mutations (0PM) or one or two point mutations (1PM or 2PM) in conserved bases in the splicing loops of bZIP60 mRNA as illustrated in Fig. 1A. Two different transgenic lines were tested for each transgene. (B) BIP3 induction after treatment with 2 mM DTT for 2 h in bzip60-2, ire1a, and ire1b mutants. RNA analyzed by RT-PCR in which actin gene expression served as a control. (C) Complementation of bzip60-1 by cDNA-encoding bZIP60 mRNA without point mutations (0PM). Complementation is demonstrated by the support of BIP3 expression as analyzed by using a RT-PCR assay. Lack of complementation by constructs bearing one or two point mutations (1PM or 2PM) in conserved bases in the splicing loops of bZIP60 mRNA. Two different transgenic lines were tested for each transgene. Actin gene expression served as a control.

Fig. 4.

Heat induction of bZIP60 mRNA splicing. Arabidopsis seedlings were heat shocked at 42 °C for various times and, in the instances as indicated, were allowed to recover at room temperature (22 °C). RNA extracted from seedlings was analyzed by the FP assay for unspliced and forms of bZIP60 mRNA or by the SPU assay for unspliced forms and the SPS assay for spliced forms of mRNA.