Project description:Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Cold stress, which adversely affects plants growth and development, regulates the transcription and splicing of plants splicing factors. This affects the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix Arabidopsis tiling arrays to survey the transcriptome under cold treatment conditions.
Project description:Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Cold stress, which adversely affects plants growth and development, regulates the transcription and splicing of plants splicing factors. This affects the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix Arabidopsis tiling arrays to survey the transcriptome under cold treatment conditions. Two-week old Arabidopsis seedlings grown on agar were subjected to 24 hours of cold (4°C) treatment under long day conditions. Control and cold-treated plants were harvested at the same time to ensure that observed differences would not be due to circadian clock effects on transcripts. Total RNA from four biological repeats were used for microarray hybridization.
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: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.