Genome-Wide Detection of Condition-Sensitive Alternative Splicing in Arabidopsis Roots

Abstract

Iron (Fe) deficiency is a world-wide nutritional disorder in both plants and humans, resulting from its restricted bioavailability for plants and, subsequently, low Fe concentration in edible plant parts. Plants have evolved sophisticated mechanisms to alleviate Fe deficiency, with the aim of recalibrating metabolic fluxes and maintaining cellular Fe homeostasis. To analyze condition-sensitive changes in precursor mRNA (pre-mRNA) splicing pattern, we mapped the transcriptome of Fe-deficient and Fe-sufficient Arabidopsis (Arabidopsis thaliana) roots using the RNA sequencing technology and a newly developed software toolbox, the Read Analysis & Comparison Kit in Java (RACKJ). In alternatively spliced genes, stress-related Gene Ontology categories were overrepresented, while housekeeping cellular functions were mainly transcriptionally controlled. Fe deficiency increased the complexity of the splicing pattern and triggered the differential alternative splicing of 313 genes, the majority of which had differentially retained introns. Several genes with important functions in Fe acquisition and homeostasis were both differentially expressed and differentially alternatively spliced upon Fe deficiency, indicating a complex regulation of gene activity in Fe-deficient conditions. A comparison with a data set for phosphate-deficient plants suggests that changes in splicing patterns are nutrient specific and not or not chiefly caused by stochastic fluctuations. In sum, our analysis identified extensive posttranscriptional control, biasing the abundance and activity of proteins in a condition-dependent manner. The production of a mixture of functional and nonfunctional transcripts may provide a means to fine-tune the abundance of transcripts with critical importance in cellular Fe homeostasis. It is assumed that differential gene expression and nutrient deficiency-induced changes in pre-mRNA splicing represent parallel, but potentially interacting, regulatory mechanisms.The removal of introns from the immature mRNA by a process called "pre-mRNA splicing" occurs in the vast majority of eukaryotic protein-coding genes. Pre-mRNA splicing is catalyzed by elaborate ribonucleoprotein megadalton complexes referred to as spliceosomes (Wahl et al., 2009). Multiple mRNA isoforms can be generated from a single gene locus by alternative splicing, potentially producing functionally distinct protein isoforms (Hsu and Hertel, 2009). In mammals, alternative splicing dramatically increases protein diversity, yielding proteins that differ in function, activity, binding properties, or subcellular localization, and is believed to account for the multiexonic gene expression diversity, generating an estimated 100,000 proteins encoded by 25,000 genes in humans (Modrek and Lee, 2002;Kelemen et al., 2013).In humans, more than 95% of the intron-containing genes are alternatively spliced (Pan et al., 2008). Alternative splicing is less prominent in plants, and its importance has been debated. Recent estimates based on RNA sequencing (RNA-seq) data sug...

Genome-Wide Analysis of Heat-Sensitive Alternative Splicing in Physcomitrella patens

“…Most of these reads were potentially from alternatively spliced transcripts. Reads representing each type of AS event were counted by using the analysis tool RACKJ Li et al, 2013; http://rackj.sourceforge. net/).…”

The conundrum of discordant protein and mRNA expression. Are plants special?

“…Splicing patterns appear to be more complex during acclimation to nutrient deficiencies or light exposure than under constant conditions, resulting in significantly induced intron retention features (Li et al, 2013; Wu et al, 2014). The reasons for the different forms and consequences of alternative splicing in animals and plants are currently unknown.…”

Genome-Wide Analysis of Alternative Splicing in Zea mays: Landscape and Genetic Regulation

“…Many alternatively spliced isoforms are known to have tissuepreferential expression patterns (Emrich et al, 2007), and differential AS has been demonstrated to play important roles in development (Rühl et al, 2012;Staiger and Brown, 2013) as well as stress responses (Li et al, 2013;Staiger and Brown, 2013;Cui et al, 2014). Genes encoding regulatory proteins are much more likely to have alternatively spliced isoforms, highlighting the importance of AS for gene expression networks (Duque, 2011).…”