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Ratio-based analysis of differential mRNA processing and expression of a polyadenylation factor mutant pcfs4 using arabidopsis tiling microarray.

Zheng J, Xing D, Wu X, Shen Y, Kroll DM, Ji G, Li QQ - PLoS ONE (2011)

Bottom Line: Quantitative PCR analysis of a set of DPGs confirmed that most of these genes were truly differentially processed in pcfs4 mutant plants.The enriched GO term "regulation of flower development" among PCFS4 targets further indicated the efficacy of the RADPRE pipeline.This simple but effective program is available upon request.

View Article: PubMed Central - PubMed

Affiliation: Department of Automation, Xiamen University, Xiamen, Fujian, China.

ABSTRACT

Background: Alternative polyadenylation as a mechanism in gene expression regulation has been widely recognized in recent years. Arabidopsis polyadenylation factor PCFS4 was shown to function in leaf development and in flowering time control. The function of PCFS4 in controlling flowering time was correlated with the alternative polyadenylation of FCA, a flowering time regulator. However, genetic evidence suggested additional targets of PCFS4 that may mediate its function in both flowering time and leaf development.

Methodology/principal findings: To identify further targets, we investigated the whole transcriptome of a PCFS4 mutant using Affymetrix Arabidopsis genomic tiling 1.0R array and developed a data analysis pipeline, termed RADPRE (Ratio-based Analysis of Differential mRNA Processing and Expression). In RADPRE, ratios of normalized probe intensities between wild type Columbia and a pcfs4 mutant were first generated. By doing so, one of the major problems of tiling array data--variations caused by differential probe affinity--was significantly alleviated. With the probe ratios as inputs, a hierarchy of statistical tests was carried out to identify differentially processed genes (DPG) and differentially expressed genes (DEG). The false discovery rate (FDR) of this analysis was estimated by using the balanced random combinations of Col/pcfs4 and pcfs4/Col ratios as inputs. Gene Ontology (GO) analysis of the DPGs and DEGs revealed potential new roles of PCFS4 in stress responses besides flowering time regulation.

Conclusion/significance: We identified 68 DPGs and 114 DEGs with FDR at 1% and 2%, respectively. Most of the 68 DPGs were subjected to alternative polyadenylation, splicing or transcription initiation. Quantitative PCR analysis of a set of DPGs confirmed that most of these genes were truly differentially processed in pcfs4 mutant plants. The enriched GO term "regulation of flower development" among PCFS4 targets further indicated the efficacy of the RADPRE pipeline. This simple but effective program is available upon request.

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A flow-chart of RADPRE analysis pipeline.A) Preprocessing of data including background correction, across-array normalization, probe filtering and trimming, ratio generation, and log-transformation. B) To identify transcripts with at least one of its exon ratio means not equal to one, a one-sample two-tails T-test was applied to every exon of an annotated transcript with the  hypothesis that the ratio mean of the exon was equal to one. C) For those transcripts identified from the T-test in (B), a one-way ANOVA and F-test was performed for each transcript with its exons as the “level” parameter. Every transcript with the ratio means of all its exons being equal would be a putative DEG target. Otherwise, the transcript would be a direct DPG target. D) A further one-sample two-tails T-test was applied to every one of the putative DEG targets from (C) to test whether the ratio mean of the whole transcript was equal to one. If the ratio mean was not equal to one, the transcript would be a DEG target.
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pone-0014719-g001: A flow-chart of RADPRE analysis pipeline.A) Preprocessing of data including background correction, across-array normalization, probe filtering and trimming, ratio generation, and log-transformation. B) To identify transcripts with at least one of its exon ratio means not equal to one, a one-sample two-tails T-test was applied to every exon of an annotated transcript with the hypothesis that the ratio mean of the exon was equal to one. C) For those transcripts identified from the T-test in (B), a one-way ANOVA and F-test was performed for each transcript with its exons as the “level” parameter. Every transcript with the ratio means of all its exons being equal would be a putative DEG target. Otherwise, the transcript would be a direct DPG target. D) A further one-sample two-tails T-test was applied to every one of the putative DEG targets from (C) to test whether the ratio mean of the whole transcript was equal to one. If the ratio mean was not equal to one, the transcript would be a DEG target.

Mentions: There were six CEL file data (wt1, wt2, wt3, pcfs4.1, pcfs4.2 and pcfs4.3) from the hybridization of Arabidopsis tiling 1.0R array with the targets prepared from three biological replicates of each wild type Col (WT) and the PCFS4 mutant (pcfs4-1) grown in a randomized block design. The suffix number of the file name denotes the block number. The data was analyzed according to the steps shown in Figure 1. The underlying principle, the analysis details and the results of each step are described as follows.


Ratio-based analysis of differential mRNA processing and expression of a polyadenylation factor mutant pcfs4 using arabidopsis tiling microarray.

Zheng J, Xing D, Wu X, Shen Y, Kroll DM, Ji G, Li QQ - PLoS ONE (2011)

A flow-chart of RADPRE analysis pipeline.A) Preprocessing of data including background correction, across-array normalization, probe filtering and trimming, ratio generation, and log-transformation. B) To identify transcripts with at least one of its exon ratio means not equal to one, a one-sample two-tails T-test was applied to every exon of an annotated transcript with the  hypothesis that the ratio mean of the exon was equal to one. C) For those transcripts identified from the T-test in (B), a one-way ANOVA and F-test was performed for each transcript with its exons as the “level” parameter. Every transcript with the ratio means of all its exons being equal would be a putative DEG target. Otherwise, the transcript would be a direct DPG target. D) A further one-sample two-tails T-test was applied to every one of the putative DEG targets from (C) to test whether the ratio mean of the whole transcript was equal to one. If the ratio mean was not equal to one, the transcript would be a DEG target.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3045369&req=5

pone-0014719-g001: A flow-chart of RADPRE analysis pipeline.A) Preprocessing of data including background correction, across-array normalization, probe filtering and trimming, ratio generation, and log-transformation. B) To identify transcripts with at least one of its exon ratio means not equal to one, a one-sample two-tails T-test was applied to every exon of an annotated transcript with the hypothesis that the ratio mean of the exon was equal to one. C) For those transcripts identified from the T-test in (B), a one-way ANOVA and F-test was performed for each transcript with its exons as the “level” parameter. Every transcript with the ratio means of all its exons being equal would be a putative DEG target. Otherwise, the transcript would be a direct DPG target. D) A further one-sample two-tails T-test was applied to every one of the putative DEG targets from (C) to test whether the ratio mean of the whole transcript was equal to one. If the ratio mean was not equal to one, the transcript would be a DEG target.
Mentions: There were six CEL file data (wt1, wt2, wt3, pcfs4.1, pcfs4.2 and pcfs4.3) from the hybridization of Arabidopsis tiling 1.0R array with the targets prepared from three biological replicates of each wild type Col (WT) and the PCFS4 mutant (pcfs4-1) grown in a randomized block design. The suffix number of the file name denotes the block number. The data was analyzed according to the steps shown in Figure 1. The underlying principle, the analysis details and the results of each step are described as follows.

Bottom Line: Quantitative PCR analysis of a set of DPGs confirmed that most of these genes were truly differentially processed in pcfs4 mutant plants.The enriched GO term "regulation of flower development" among PCFS4 targets further indicated the efficacy of the RADPRE pipeline.This simple but effective program is available upon request.

View Article: PubMed Central - PubMed

Affiliation: Department of Automation, Xiamen University, Xiamen, Fujian, China.

ABSTRACT

Background: Alternative polyadenylation as a mechanism in gene expression regulation has been widely recognized in recent years. Arabidopsis polyadenylation factor PCFS4 was shown to function in leaf development and in flowering time control. The function of PCFS4 in controlling flowering time was correlated with the alternative polyadenylation of FCA, a flowering time regulator. However, genetic evidence suggested additional targets of PCFS4 that may mediate its function in both flowering time and leaf development.

Methodology/principal findings: To identify further targets, we investigated the whole transcriptome of a PCFS4 mutant using Affymetrix Arabidopsis genomic tiling 1.0R array and developed a data analysis pipeline, termed RADPRE (Ratio-based Analysis of Differential mRNA Processing and Expression). In RADPRE, ratios of normalized probe intensities between wild type Columbia and a pcfs4 mutant were first generated. By doing so, one of the major problems of tiling array data--variations caused by differential probe affinity--was significantly alleviated. With the probe ratios as inputs, a hierarchy of statistical tests was carried out to identify differentially processed genes (DPG) and differentially expressed genes (DEG). The false discovery rate (FDR) of this analysis was estimated by using the balanced random combinations of Col/pcfs4 and pcfs4/Col ratios as inputs. Gene Ontology (GO) analysis of the DPGs and DEGs revealed potential new roles of PCFS4 in stress responses besides flowering time regulation.

Conclusion/significance: We identified 68 DPGs and 114 DEGs with FDR at 1% and 2%, respectively. Most of the 68 DPGs were subjected to alternative polyadenylation, splicing or transcription initiation. Quantitative PCR analysis of a set of DPGs confirmed that most of these genes were truly differentially processed in pcfs4 mutant plants. The enriched GO term "regulation of flower development" among PCFS4 targets further indicated the efficacy of the RADPRE pipeline. This simple but effective program is available upon request.

Show MeSH