Project description:To analyze context-sensitive changes in pre-mRNA splicing pattern and gene expression, we mapped the transcriptome of iron-deficient and iron-sufficient Arabidopsis roots using the RNA-seq technology. RNA-seq data were analyzed with a newly developed software package, RACKJ (Read Analysis & Comparison Kit in Java). Subsets of 460 and 1480 genes were found to be either differentially expressed or affected in their splicing patterns upon iron deficiency. The two groups showed a small, random overlap, indicating that alternative splicing and differential gene expression represent parallel, but potentially interacting regulatory mechanisms. The majority (~95%%) of the context-sensitive alternative splicing events was due to differentially retained introns. Intron retention was mostly repressed in iron-deficient plants, while exon skipping did not show a clear trend in either direction. A comparison with a similar data set for phosphate-deficient plants supported stress-repressed intron retention and revealed nutrient-specific changes of splicing patterns. It is concluded that iron and phosphate deficiency increases splicing fidelity for a subset of transcripts, probably as a means of acclimation to nutrient shortage.
Project description:The shoots and roots of a plant respond differently to osmotic stress, as they have distinct functions and anatomical structures. Under conditions of high solute concentration, such as in saline soils or drought, water uptake by the roots is reduced, resulting in cellular dehydration. In this study, we performed transcriptional profiling of roots of Arabidopsis under osmotic stress conditions such as high salinity and drought using mRNA-Seq for the assessment of gene expression changes in roots of Arabidopsis. mRNA-Seq analysis showed that many differentially expressed genes showed differential expressions under both salt stress and drought stress conditions in roots and were distinct from aerial parts. We confirmed 68 transcription factor genes which is involved in osmotic stress signal transduction in roots and are connected tightly. Interestingly, well-known ABA-dependent and/or -independent osmotic stress-responsive genes were less increased in roots, indicating that osmotic stress response in roots might be regulated by stress pathways other than well-known pathways. We identified 26 osmotic stress-responsive genes, which have alternative splicing variant isoforms, showed distinct expression in roots under osmotic stress conditions from the mRNA-Seq analysis. Quantitative RT-PCR confirmed that alternative splicing variants, such as ANNAT4, MAGL6, TRM19, and CAD9, have differential expressions in roots under osmotic stress conditions, indicating that alternative splicing is an important regulatory mechanism in osmotic stress response in roots. Taken together, our study suggest that many transcription factor families are involved in osmotic stress response in roots and tightly connected each other. In addition, alternative splicing and function of alternative splicing variant isoforms are also important in osmotic stress response in roots. To understand the alternative splicing mechanism in roots, further study is necessary.
Project description:We identified PRP4 kinase-A (PRP4ka) in a forward genetic screen based on an alternatively-spliced GFP reporter gene in Arabidopsis thaliana (Arabidopsis). Prp4 kinase, which was the first spliceosome-associated kinase shown to regulate splicing in fungi and mammals, has not yet been studied in plants. Analysis of RNA-seq data from the prp4ka mutant revealed widespread perturbations in alternative splicing. A quantitative iTRAQ-based phosphoproteomics investigation of the mutant identified phosphorylation changes in several serine/arginine-rich proteins, which regulate constitutive and alternative splicing, as well as other splicing-related factors. The results demonstrate the importance of PRP4ka in alternative splicing and suggest that PRP4ka may influence alternative splicing patterns by phosphorylating a subset of splicing regulators.
Project description:In all living organisms, regulation of gene expression is fundamental for survival and adaptation. Gene expression can be modulated at various steps, including at the level of RNA processing. During the last few years, the importance of alternative splicing of mRNAs in controlling plant development and stress responses were emerged and highlighted its importance. Recently, an other type of alternative splicing has been reported which leads to the generation of circular RNAs (circRNAs), a novel class on endogenous noncoding RNAs. Several functions of circular RNAs have been proven or proposed, including functioning as microRNA or RNA-binding protein decoys, playing regulatory roles in gene expression or affecting transcriptional control via special RNA-RNA interactions. Despite the widening knowledge of circRNAs and their functional aspects in the animal kingdom, relatively little is known about circRNAs in plants. In order to detect and classify circRNAs in Arabidopsis thaliana, we created a workflow that includes generation of Illumina libraries enriched for circRNAs and a comparison of biocomputational tools developed for detecting endogenous circular RNAs in other species. With the power of high-throughput sequencing and evaluation of algorithms, high-fidelity candidates were subjected for an analysis of their functional role in plant development and stress-related responses, especially regarding the role of splicing, including alternative splicing events, splice site preference and strength variances and transcript composition and to comprehend the role of RNA processing in stress response. Here we present an approach combining bioinformatic tools and molecular techniques to investigate the adaptability of detection methods of circRNAs from other species to plant circular RNAs, and based on our high-fidelity results identify and understand the characteristics of circRNAs in Arabidopsis thaliana.
Project description:A silencing signal in plants with an RNA specificity determinant moves through plasmodesmata and the phloem. To identify the mobile RNA we grafted Arabidopsis thaliana shoots to roots that would be a recipient for the silencing signal. Using high throughput sequencing as a sensitive detection method and mutants to block small RNA (sRNA) biogenesis in either source or recipient tissue, we detected endogenous and transgene specific sRNA that moved across the graft union. Surprisingly we found that the mobile endogenous sRNAs account for a substantial proportion of the sRNA in roots and we provide evidence that 24nt mobile sRNAs direct epigenetic modifications in the genome of the recipient cells. Mobile sRNA thus represents a mechanism for transmitting the specification of epigenetic modification and could affect genome defence and responses to external stimuli that have persistent effects in plants. Keywords: Small RNA Analysis, Epigenetics
Project description:Purpose: Circadian clock in plants temporally coordinates biological processes throughout the day synchronizing gene expression with environmental changes. Here, we examined the genome-wide circadian and diurnal control of Arabidopsis transcriptome using high throughout RNA-seq approach. Methods: Transcriptional and posttranscritional profiles were identified and characterized for Arabidopsis seedlings grown under continuous light or long-day condition (16 h light/8 h dark) for one day (each condition has two biological replicates). Results: We show that rhythmic posttranscriptional regulation is also a significant factor for genome-wide profile of circadian plant transcriptome. Two major posttranscriptioal mechanisms alternative splicing (AS) and alternative polyadenylation (APA) show circadian rhythmicity, resulting from the oscillation in the genes invovled in AS and APA. Conclusions: Arabidopsis circadian clock not only controls the transcription of genes, but also affects their posttranscriptional regulation through regulating AS and APA.