Arabidopsis IRE1 catalyses unconventional splicing of bZIP60 mRNA to produce the active transcription factor
Item Type Article
Authors Nagashima, Yukihiro;Mishiba, Kei-ichiro;Suzuki, Eiji;Shimada, Yukihisa;Iwata, Yuji;Koizumi, Nozomu
Citation Nagashima Y, Mishiba K, Suzuki E, Shimada Y, Iwata Y, et al.
(2011) Arabidopsis IRE1 catalyses unconventional splicing of bZIP60 mRNA to produce the active transcription factor. Sci Rep 1.
doi:10.1038/srep00029.
Eprint version Publisher's Version/PDF
DOI 10.1038/srep00029
Publisher Springer Nature Journal Scientific Reports
Rights This work is licensed under a Creative Commons Attribution- NonCommercial-ShareALike 3.0 Unported License. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc- sa/3.0/
Download date 2023-12-17 18:34:40
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Link to Item http://hdl.handle.net/10754/325370
Supplementary Information
Arabidopsis IRE1 catalyses unconventional splicing of bZIP60 mRNA to produce the active transcription factor
Yukihiro Nagashima, Kei-ichiro Mishiba, Eiji Suzuki, Yukihisa Shimada, Yuji Iwata and Nozomu Koizumi
Supplementary Figures
Supplementary Fig. S1: Isolation of T-DNA insertion mutants of IRE1 genes.
a, Location of primers used for confirmation of a T-DNA insertion in ire1a and ire1b. A sequence of each primer is listed in Table S3. b, Genotyping of the wild-type, ire1a, ire1b and ire1a/ire1b. PCR
Supplementary Fig. S2: Different tunicamycin sensitivities of seedlings of the wild-type and ire1 mutants. Seeds of the wild-type, ire1a, ire1b and ire1a/ire1b were sown on an MS agar plates with indicated concentrations of tunicamycin. A picture was taken 2 weeks after sowing.
Supplementary Fig. S3: Quantitative RT-PCR analysis of ire1 mutants.
Induction of GPT (a), BiP1 (b) and BiP3 (c) genes by tunicamycin treatment in the wild-type and ire1 mutants. The transcript levels of indicated genes were quantified by quantitative RT-PCR.
Primers used PCR were listed in Supplementary Table S3 online.
Supplementary Fig. S4: Prediction of the secondary structure of bZIP60.
Supplementary Fig. S5: Nucleotide and deduced amino acid sequences of bZIP60u and bZIP60s.
The bZIP domain and TMD are indicated in blue and yellow, respectively. ORF2 in bZIP60s is shown in green. The splicing sites are indicated by triangles.
Supplementary Fig. S6: Alignment of sequences around the splicing sites in plant bZIP60 homologues.
An alignment of the nucleotide sequences of bZIP60 homologues, along with those of human XBP1 and yeast HAC1, is presented. The conserved nucleotides essential for the splicing of HAC1 and XBP1 and those conserved in bZIP60 homologues are highlighted in yellow. The splicing sites are indicated by triangles.
Supplementary Table S1: Expression values of tunicamycin-inducible genes in wild-type and ire1a/ire1b plants. (available at Scientific Reports Online in Excel format)
Supplementary Table S2: Fold induction values of wild-type and ire1a/ire1b and bzip60 mutant plants used in the hierarchical cluster analysis. (available at Scientific Reports Online in Excel format)
Supplementary Table S3: Primers used in this study.
Name Sequence
Primers used for identification of IRE1 insertion mutants Ire1AF GAAGCCACTTGTAACAGATGG Ire1AR AGGGTCTGGGTTTAACAAACGG Ire1BF TTCCTCGGTGCTTGGTGATG Ire1BR GTGCTTCCATCAATAATATC
Ire1BR2 CTAGACGCTTAACCGCTACAAGACGACC T-DNA-1 TCAACGTCGTTCGCCAGGTGCG
T-DNA-2 CACAGATGTAAGTGCAGGTTTACCC Primers used for quantitative PCR
GptQF ACGTTCTGAGACGTAACATG GptQR CAACAATACCCAATGACTCTG Bip1QF TCAGTCCTGAGGAGATTAGTGCT Bip1QR TGCCTTTGAGCATCATTGAA Bip3QF CGAAACGTCTGATTGGAAGAA Bip3QR GGCTTCCCATCTTTGTTCAC Act8QF TCAGCACTTTCCAGCAGATG Act8QR ATGCCTGGACCTGCTTCAT Primers used for probe preparation
bzip60NF CGGCGGTTAGATCGAGAGAGAGG bzip60NR CCATGGTCGCCGCAAGGGTTAAGATTTGG BiP1NF CAAAAAGAGAGATCGTACGCAAAA Bip1NR ACTGATCCTAACTTCGTAGCCTCTT Bip3NF ACAAACGAGATCGAAGAAGAGTTCTC Bip3NR ACCGTCCCCAGTTTCTGCTCTTCGC Sar1NF ATGTTTTTATTCGATTGGTTCTATGGAATC Sar1NR CTACTTGATATACTGAGATAGCC
Primers used for detection of the unconventional splicing
bzip60iF CCAGTCTCTACGTTACTGTTTGCAAAAGGGTAATGGC bzip60iR GCAAATGAAGTTTACTCCCAGAAGCCAAAGCAGGG bzip60sF TGATGATGACGAAGAAGGAGACGATGATG
bzip60sF2 TCGAAGCAGGAGTCTGCTGTTGG bzip60sR AGGGAACCCAACAGCAGACTCC
bzip60sR2 TCATCCATAACCATAAGACCCACATTCAC