Abstract
BackgroundAlternative 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 FindingsTo 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/SignificanceWe 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.
Highlights
Polyadenylation is one of the essential processes during the maturation of most mRNAs in eukaryotic cells
We employed a whole genome tiling microarray technology to search the Arabidopsis transcriptome for the differentially processed genes (DPG, defined as those genes whose pre-mRNA processing was altered in the pcfs4 mutant) and differentially expressed genes (DEG, defined as those genes whose steady-state level of mRNA abundance was altered, but the mRNA processing was not affected in the pcfs4 mutant)
Data description There were six CEL file data 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 grown in a randomized block design
Summary
Polyadenylation is one of the essential processes during the maturation of most mRNAs in eukaryotic cells. We reasoned that in addition to FCA, there may be other target(s) of PCFS4 that mediate its function in leaf development, likely through a molecular mechanism similar to the APA of FCA. To explore this possibility, we employed a whole genome tiling microarray technology to search the Arabidopsis transcriptome for the differentially processed genes (DPG, defined as those genes whose pre-mRNA processing was altered in the pcfs mutant) and differentially expressed genes (DEG, defined as those genes whose steady-state level of mRNA abundance was altered, but the mRNA processing was not affected in the pcfs mutant). Genetic evidence suggested additional targets of PCFS4 that may mediate its function in both flowering time and leaf development
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