Supporting Information

Transcription

1 Supporting Information Deng et al /pnas Fig. S1. Predicted structure of Arabidopsis bzip60 RNA. Lowest free energy form (ΔG = (initially ) of bzip60 mrna folded by M-Fold (1). Boxed area is shown in detail in Fig. 1. 1of5

2 1. Zuker M (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res 31: Fig. S2. RT-PCR assay for detecting unspliced or spliced bzip60 mrna. (A) Flanking primers (FP) assay uses primers flanking the intron to detect both the unspliced and the spliced mrna forms that migrate differently on gels. Specific primers overlap the exon/intron boundary to specifically detect the unspliced mrna (SPU assay) or the exon/exon boundary to specifically detect the spliced mrna (SPS assay). (B) Demonstration of the specificity of the SPU and SPS assays using cdnas of unspliced and spliced bzip60 mrna forms as template. Protein extract ATP None - IRE1a + IRE1b + GST none + + IRE1a IRE1b - - Cut site predictions Size markers 90 nt / nt 55 nt 72/69 58/ nt 20 nt 35/ Fig. S3. In vitro RNA cleavage assay. His-tagged C-terminal elements of AtIRE1a and AtIRE1b, containing the kinase and ribonuclease domains, were enriched by using a nickel affinity column. The partially purified enzymes were incubated with a 125-base segment of 32 P-labeled bzip60 mrna. The reactions were carried out for 1 h at 37 C and terminated by RNA extraction. The RNA was loaded onto a sequencing gel, and bands were detected by autoradiography. Figures on right represent interpretations of the origin of each band. 2of5

3 Fig. S4. Detection of bzip60 mrna splicing in various parts of Arabidopsis seedlings. Seedlings were treated with 2 mm DTT for 2 h, separated into leaves, hypocotyls, and roots, and RNA was extracted and analyzed by using FP, SPU, and SPS assays. Actin was used as a control. Fig. S5. Detection of IRE1 transcripts in WT and ire1b mutant seedlings. (A) RNA was extracted from WT and ire1a seedlings and analyzed for the production of full-length IRE1a transcripts. (B)RNA was extracted from WT and ire1b seedlings,which were untreated or treated with 2 mm DTT for 2 h.rna was analyzed by RT-PCR for full-length IRE1b transcripts. Fig. S6. Expression of bzip60 in nontransgenic wild-type and bzip60-1 mutant lines and in transgenic lines bearing various bzip60 cdna constructs. Lanes 1 4 show expression levels of endogenous bzip60 mrna. Lanes 5 16 show expression 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. RT-PCR assay used in these experiments: The bzip60 transgenes are cdna constructs containing only the coding region of the bzip60 gene. The expression of the endogenous bzip60 gene was assessed by using a forward primer around the start codon and a reverse primer located in the 3 UTR. The expression of the transgene and the endogenous gene together was assessed by using a forward primer around the start codon and reverse primer around the stop codon. Although these primers will register the expression of both the endogenous bzip60 gene and the transgene, the expression of the endogenous gene was undetectable in the bzip60 T-DNA mutant background (see lanes 3 and 4); therefore, the primers measured the expression of only the transgene in the bzip60 mutant background. 3of5

4 Fig. S7. Effect of different stresses on bzip60 splicing. bzip60 mrna splicing was analyzed in all cases by the FP assay. (Top) Two-week-old seedlings were treated with 10 μm ABA, 1 mm ethephon, or heat (42 C) for various times as indicated. (Middle) Seedlings were germinated and grown for 10 d on 10 μm ABA or 1 mm ethephon. (Bottom) Seedlings were treated with anoxia, salt stress (100 mm NaCl), or nitrate limitation for various times as indicated. Fig. S8. Effect of heat treatment on bzip60 mrna splicing and BIP3 expression. (A) RNA was extracted from WT and bzip60-1 mutant seedlings after treatment at 42 C for 1 h followed by recovery at room temperature (22 C) for 2 h. (B) RNA was extracted from WT and bzip60-1 seedlings after treatment at 37 C for 2 h. 4of5

5 Table S1. Primer List of primers used in this study Sequence Primers used in bzip mrna splicing assays bzip60f4 GAAGGAGACGATGATGCTGTGGCT b60ub1 Gcagggattccaacaagagcacag b60sb2 AGCAGGGAACCCAACAGCAGACT b60rt3 CCAAAGCAGGGAACCCAACAGcag Flanking primers (FP) assay bzip60f4/b60rt3 for both of unspliced form and spliced forms Specific primers (SP) assays bzip60f4/b60ub1 SPU assay for unspliced RNA forms bzip60f4/b60sb2 SPSP assay for spliced RNA forms RT-PCR analysis for the gene expression IRE1bf/IRE1bR for full length IRE1B IRE1bf/1bexLP for upstream segment of IRE1B 1bseqF/IRE1bR For downstream segment of IRE1B Ire1bF aaggcgcgccggagatctccataaaccctaatc Ire1bR TGactagtGAATACAGTGGTCTTAGAGTAC 1bexLP TTTGAATGTTTGTCTGAGGCC 1bseqF ACGGAGACAATTATGAGAGGGACG IRE1acdelF/IRE1aT-R for full length IRE1A IREaCdelF TAGAATTCATGCCGCCGAGATGTCCTTT IREaT-R ATGCGGCCGCTTAGATGATGTCGCATTTGAAG B60mF/b60mR for full length BZIP60 b60mf gaaggcgcgccgaccatggcggaggaatttgg b60mr GTGactagtCGCCGCAAGGGTTAAGATTTGG BIP3F/BIP3R for BIP3 BIP3F TTCGACCCGAAACGTCTGATTGGA bip3 GCTTGCCTCTGCGCATCATTGAAA ACTQF/ACTQB for ACTIN ACTQF GGAACTGGAATGGTGAAGGCTG ACTQB CGATTGGATACTTCAGAGTGAGGA Primers used for in vitro transcription (T7 promoter is underlined) and resulting transcripts (5 to 3 sequence) bzip60 T7bZIP-F AAATTAATACGACTCACTATAGGGATACTACCATGATGTCGAAGC bzip8r TGCAATCCGATGCATCGGATTGCAGCAAATGAAGTTTACTCCCAG GGGATACTACCATGATGTCGAAGCAGGAGTCTGCTGTGCTCTTGTT GGAATCCCTGCTGTTGGGTTCCCTGCTTTGGCTTCTGGGAGTAAACT TCATTTGCTGCAATCCGATGCATCGGATTGCA Size markers T7-F AAATTAATACGACTCACTATAGGG MRK20-R GCTTCGACATCATGGTAGTATCCCTATAGTGAGTCGTATTAATTT GGGATACTACCATGATGTCGAAGC MRK40-R AGCACAGCAGACTCCTGCTTCGACATCATGGTAGTAT CCCTATAGTGAGTCGTATTAATTT GGGATACTACCATGATGTCGAAGCAGGAGTCTGCTGTGCT MRK55-R AGGGATTCCAACAAGAGCACAGCAGACTCCTGCTTCGACATCATGGTA GTATCCCTATAGTGAGTCGTATTAATTT GGGATACTACCATGATGTCGAAGCAGGAGTCTGCTGTGCTCTTGTTGGAATCCCT MRK70-R AGGGAACCCAACAGCAGGGATTCCAACAAGAGCACAGCAGACTCCTGC TTCGACATCATGGTAGTATCCCTATAGTGAGTCGTATTAATTT GGGATACTACCATGATGTCGAAGCAGGAGTCTGCTGTG CTCTTGTTGGAATCCCTGCTGTTGGGTTCCCT MRK90-R TTACTCCCAGAAGCCAAAGCAGGGAACCCAACAGCAGGGATTCC AACAAGAGCACAGCAGACTCCTGCTTCGACATCATGGTAGTATC CCTATAGTGAGTCGTATTAATTT AGCACAGCAGACTCCTGCTTCGACATCATGGTAG TATCCCTATAGTGAGTCGTATTAATTT 5of5