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A global profiling of uncapped mRNAs under cold stress reveals specific decay patterns and endonucleolytic cleavages in Brachypodium distachyon

Jingyu Zhang, Zhiwei Mao and Kang Chong*

Author Affiliations

Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing 100093, China

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Genome Biology 2013, 14:R92  doi:10.1186/gb-2013-14-8-r92

Published: 30 August 2013

Additional files

Additional file 1:

Supplemental Figures S1, S2, S3, S5, S6, S7, and corresponding legends. Figure S1. Cold tolerance and vernalization responsiveness assessment of two Brachypodium diploid genotypes, ABR5 and BD21. (a) Seeds for ABR5 and BD21 were germinated and grown for 2 weeks under the growth condition described above. Then the seedlings were treated with -5°C for 6h, 12h, 18h, 24h, and recovered for 10 days. Survival rate was investigated after cold treatment and recovery. Error bars represent standard error. Each time point consisted of 10 to 15 plants, and the experiment was repeated three times.(b) Seeds for ABR5 and BD21 were treated with °C for 1, 2, 3, 4, 5, and 6 weeks as vernalization treatment and grown in a growth chamber at 24°C, with 6 h/8 h (light/dark) photoperiod and approximately 5,000 lux light intensity. Flowering time was measured by the number of days after germination until the first flower bud appears. Error bars represent standard error. Each time point consisted of 10 to 15 plants, and the experiment was repeated three times. Figure S2. Reproducibility of PARE libraries. Scatter plot showing the reproducibility between PARE libraries and their biological repeats. The reads from DW (a) and DC (b) library was mapped to each transcript and the sum of total reads was plotted against corresponding reads from their biological replicate libraries, DW replicate and DC replicate, respectively. Figure S3. Real-time RT-PCR validation of gene expression data provided by RNA-Seq analysis. Total RNA was isolated from 12-day-old Brachypodium seedlings with (WC) and without (WO) cold treatment. Real-time PCR analysis was performed as described by Dai et al. (Plant Physiol 2007, 143:1739). The SuperScripts II reverse transcriptase (Invitrogen, Carlsbad, CA, USA) was used for reverse transcription and SYBR Green PCR Master mix (Applied Biosystems, Foster City, CA, USA) was used for quantitative PCR. The amplification of a Brachypodium Tubulin gene was used as an internal control to normalize all data. The normalized mRNA levels in the WO samples were arbitrarily set to 1. Quantitative PCR reactions were repeated three times. Error bars represent the standard error. Figure S5. Overall mRNA stability estimation through quantification of mRNA abundance before and after cordycepin (transcription inhibitor) treatment. The middle part of the primary leaves from 12-day-old Brachypodium seedlings with (WC) and without (WO) cold treatment (4°C, 24h) were used. Cordycepin treatment was performed as described (Plant J 2002, 31:601). The 3-cm leaf segments were incubated with 600µM cordycepin solution with regular shaking at 22°C for 7h. Then tissue samples were harvested at regular intervals (1h, 2h, and 4h) and quickly frozen in liquid nitrogen. Total RNA was isolated with mirVana miRNA Isolation Kit (Ambion, Austin, TX, USA). Two microgram of total RNA was used for reverse transcription with SuperScript II reverse transcriptase (Invitrogen). The obtained cDNAs were quantified and used as template for quantitative PCR using SYBR Green PCR Master mix (Applied Biosystems, Foster City, USA). For both WO and WC samples, mRNA abundance for the time zero was arbitrarily set to 1 and mRNA abundance for the other time points were shown as the relative value of time zero. Quantitative PCR reactions were repeated three times. Error bars indicate standard deviation. Figure S6. mRNA features correlated with different degradation patterns. The mRNA length, UTR length, GC content, and the number of introns were analyzed for type I-IV mRNAs. N: no conserved motif (E-value <0.001) was identified; Y: conserved motifs were identified and the motifs for Y1, Y2, Y3, and Y4 were indicated below the table. The x-axis represented nucleotide position. The y-axis represented the information content measured in bits. The overall height of each stack indicated the sequence conservation at that position, and the height of a letter within the stack indicated the relative frequency of corresponding nucleotide in the motif. Figure S7. Conserved motifs identified in the 5' and 3' UTRs for subgroups of type I, II, III, and IV genes. I, II, III, and IV: different mRNA decay patterns classified according to the variation tend of uncapped transcript/transcript abundance in cold stress. D: uncapped transcript abundance indicated by PARE reads; R: transcript abundance indicated by RNA-Seq reads; N: no conserved motif (E-value <0.001) was identified. Conserved motifs were analyzed with MEME software (Nucleic Acids Res. 2006, 34:W369) and identified conserved motifs with E-value <0.001 were shown in the table. Figure S8. Endonucleolytic cleavages identified in the DW and DC library. Transcripts with endonucleolytic cleavages shared different conserved motifs. Conserved motifs, at the endonucleolytic cleavage sites, were marked with gray background. Cleavage sites were indicated by the asterisk. The putative functions of all the transcripts with endonucleolytic cleavages were shown. The majority of the detected endonucleolytic cleavages were also found in corresponding biological replicate PARE libraries. Underlined gene names denoted endonucleolytic cleavages that were not detected in corresponding biological replicate PARE libraries.EW: endonucleolytic cleavage groups only found in the DW library;EC: endonucleolytic cleavage groups only found in the DC library;GONG: endonucleolytic cleavage groups found in both DW and DC library.

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Additional file 2:

Supplemental Tables S1 to S6 and corresponding captions. Table S1. Summary of PARE analysis. Table S2. Top 20 genes in DW and DC library. Table S3. Top 10 upregulated and downregulated genes in PARE analysis. Table S4. Enrichment analysis with Fisher's exact test. Table S5. Predicted cold-responsive Brachypodium miRNAs and their putative targets. Table S6. The conservation of motifs identified at the endonucleolytic cleavage sites.

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Additional file 3:

Supplemental Figures S4 and corresponding legend. Figures S4. Relationship between mRNA decay pattern and gene function. Gene Ontology (GO) analysis was performed for the type I, II, III, and IV genes using agriGO[109] which organizes information for molecular function, biological process, and cellular component categories.type I: (a), molecular function; (b),biological process; (c) cellular component;type II: (d), molecular function; (e),biological process; (f) cellular component;type III: (g), molecular function; (h),biological process; (i) cellular component;type IV: (j), molecular function; (k),biological process; (l) cellular component.

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