New Era in Plant Alternative Splicing Analysis Enabled by Advances in High-Throughput Sequencing (HTS) Technologies (original) (raw)
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Nucleic acids research, 2011
Alternative splicing (AS) coupled to nonsense-mediated decay (NMD) is a post-transcriptional mechanism for regulating gene expression. We have used a high-resolution AS RT-PCR panel to identify endogenous AS isoforms which increase in abundance when NMD is impaired in the Arabidopsis NMD factor mutants, upf1-5 and upf3-1. Of 270 AS genes (950 transcripts) on the panel, 102 transcripts from 97 genes (32%) were identified as NMD targets. Extrapolating from these data around 13% of intron-containing genes in the Arabidopsis genome are potentially regulated by AS/NMD. This cohort of naturally occurring NMD-sensitive AS transcripts also allowed the analysis of the signals for NMD in plants. We show the importance of AS in introns in 5' or 3'UTRs in modulating NMD-sensitivity of mRNA transcripts. In particular, we identified upstream open reading frames overlapping the main start codon as a new trigger for NMD in plants and determined that NMD is induced if 3'-UTRs were >...
Lost in Translation: Pitfalls in Deciphering Plant Alternative Splicing Transcripts
The Plant cell, 2015
Transcript annotation in plant databases is incomplete and often inaccurate, leading to misinterpretation. As more and more RNA-seq data are generated, plant scientists need to be aware of potential pitfalls and understand the nature and impact of specific alternative splicing transcripts on protein production. A primary area of concern and the topic of this article is the (mis)annotation of open reading frames and premature termination codons. The basic message is that to adequately address expression and functions of transcript isoforms, it is necessary to be able to predict their fate in terms of whether protein isoforms are generated or specific transcripts are unproductive or degraded.
The Plant Cell, 2014
Alternative splicing (AS) is an important regulatory process that leads to the creation of multiple RNA transcripts from a single gene. Alternative transcripts often carry premature termination codons (PTCs), which trigger nonsense-mediated decay (NMD), a cytoplasmic RNA degradation pathway. However, intron retention, the most prevalent AS event in plants, often leads to PTC-carrying splice variants that are insensitive to NMD; this led us to question the fate of these special RNA variants. Here, we present an innovative approach to monitor and characterize endogenous mRNA splice variants within living plant cells. This method combines standard confocal laser scanning microscopy for molecular beacon detection with a robust statistical pipeline for sample comparison. We demonstrate this technique on the localization of NMD-insensitive splice variants of two Arabidopsis thaliana genes, RS2Z33 and the SEF factor. The experiments reveal that these intron-containing splice variants remain within the nucleus, which allows them to escape the NMD machinery. Moreover, fluorescence recovery after photobleaching experiments in the nucleoplasm show a decreased mobility of intron-retained mRNAs compared with fully spliced RNAs. In addition, differences in mobility were observed for an mRNA dependent on its origin from an intron-free or an intron-containing gene.
Genome-wide mapping of alternative splicing in Arabidopsis thaliana
Genome Research, 2010
Alternative splicing can enhance transcriptome plasticity and proteome diversity. In plants, alternative splicing can be manifested at different developmental stages, and is frequently associated with specific tissue types or environmental conditions such as abiotic stress. We mapped the Arabidopsis transcriptome at single-base resolution using the Illumina platform for ultrahigh-throughput RNA sequencing (RNA-seq). Deep transcriptome sequencing confirmed a majority of annotated introns and identified thousands of novel alternatively spliced mRNA isoforms. Our analysis suggests that at least ;42% of intron-containing genes in Arabidopsis are alternatively spliced; this is significantly higher than previous estimates based on cDNA/expressed sequence tag sequencing. Random validation confirmed that novel splice isoforms empirically predicted by RNA-seq can be detected in vivo. Novel introns detected by RNA-seq were substantially enriched in nonconsensus terminal dinucleotide splice signals. Alternative isoforms with premature termination codons (PTCs) comprised the majority of alternatively spliced transcripts. Using an example of an essential circadian clock gene, we show that intron retention can generate relatively abundant PTC + isoforms and that this specific event is highly conserved among diverse plant species. Alternatively spliced PTC + isoforms can be potentially targeted for degradation by the nonsense mediated mRNA decay (NMD) surveillance machinery or regulate the level of functional transcripts by the mechanism of regulated unproductive splicing and translation (RUST). We demonstrate that the relative ratios of the PTC + and reference isoforms for several key regulatory genes can be considerably shifted under abiotic stress treatments. Taken together, our results suggest that like in animals, NMD and RUST may be widespread in plants and may play important roles in regulating gene expression.
Alternative splicing in plants – coming of age
Trends in Plant Science, 2012
More than 60% of intron-containing genes undergo alternative splicing (AS) in plants. This number will increase when AS in different tissues, developmental stages, and environmental conditions are explored. Although the functional impact of AS on protein complexity is still understudied in plants, recent examples demonstrate its importance in regulating plant processes. AS also regulates transcript levels and the link with nonsense-mediated decay and generation of unproductive mRNAs illustrate the need for both transcriptional and AS data in gene expression analyses. AS has influenced the evolution of the complex networks of regulation of gene expression and variation in AS contributed to adaptation of plants to their environment and therefore will impact strategies for improving plant and crop phenotypes.
Nucleic Acids Research, 2013
Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control system that recognizes and degrades transcripts containing NMD cis elements in their 3 0 untranslated region (UTR). In yeasts, unusually long 3 0 UTRs act as NMD cis elements, whereas in vertebrates, NMD is induced by introns located >50 nt downstream from the stop codon. In vertebrates, splicing leads to deposition of exon junction complex (EJC) onto the mRNA, and then 3 0 UTR-bound EJCs trigger NMD. It is proposed that this intron-based NMD is vertebrate specific, and it evolved to eliminate the misproducts of alternative splicing. Here, we provide evidence that similar EJC-mediated intron-based NMD functions in plants, suggesting that this type of NMD is evolutionary conserved. We demonstrate that in plants, like in vertebrates, introns located >50 nt from the stop induces NMD. We show that orthologs of all core EJC components are essential for intronbased plant NMD and that plant Partner of Y14 and mago (PYM) also acts as EJC disassembly factor. Moreover, we found that complex autoregulatory circuits control the activity of plant NMD. We demonstrate that expression of suppressor with morphogenic effect on genitalia (SMG)7, which is essential for long 3 0 UTR-and intron-based NMD, is regulated by both types of NMD, whereas expression of Barentsz EJC component is downregulated by intron-based NMD.
The Plant Journal, 2009
Approximately 20% of plant genes possess upstream open-reading frames (uORFs). The effect of uORFs on gene expression has mainly been studied at the translational level. Very little is known about the impact of plant uORFs on transcript content through the nonsense-mediated mRNA decay (NMD) pathway, which degrades transcripts bearing premature termination codons (PTCs). Here we examine the impact of the uORF of the Arabidopsis AtMHX gene on transcript accumulation. The suggestion that this uORF exposes transcripts containing it to NMD is supported by (i) the increase in transcript levels upon eliminating the uORF from constructs containing it, (ii) experiments with a modified uORF-peptide, which excluded peptide-specific degradation mechanisms, (iii) the increase in levels of the native AtMHX transcript upon treatment with cycloheximide, which inhibits translation and blocks NMD, and (iv) the sensitivity of transcripts containing the uORF of AtMHX to the presence of introns. We also showed that introns can increase NMD efficiency not only in transcripts having relatively short 3' untranslated regions (UTRs), but also in uORF-containing transcripts. AtMHX transcript levels were almost unaltered in mutants of the NMD factors UPF3 and UPF1. Possible reasons, including the existence of a NMD-compensatory mechanism, are discussed. Interestingly, the levels of UPF3 transcript were higher in upf1 mutants, suggesting a compensatory mechanism that links weak function of the NMD machinery to increased expression of UPF3. Our findings highlight that uORFs, which are abundant in plants, can not only inhibit translation but also strongly affect transcript accumulation.
Complexity of the Alternative Splicing Landscape in Plants
The Plant Cell, 2013
Alternative splicing (AS) of precursor mRNAs (pre-mRNAs) from multiexon genes allows organisms to increase their coding potential and regulate gene expression through multiple mechanisms. Recent transcriptome-wide analysis of AS using RNA sequencing has revealed that AS is highly pervasive in plants. Pre-mRNAs from over 60% of intron-containing genes undergo AS to produce a vast repertoire of mRNA isoforms. The functions of most splice variants are unknown. However, emerging evidence indicates that splice variants increase the functional diversity of proteins. Furthermore, AS is coupled to transcript stability and translation through nonsense-mediated decay and microRNA-mediated gene regulation. Widespread changes in AS in response to developmental cues and stresses suggest a role for regulated splicing in plant development and stress responses. Here, we review recent progress in uncovering the extent and complexity of the AS landscape in plants, its regulation, and the roles of AS in gene regulation. The prevalence of AS in plants has raised many new questions that require additional studies. New tools based on recent technological advances are allowing genome-wide analysis of RNA elements in transcripts and of chromatin modifications that regulate AS. Application of these tools in plants will provide significant new insights into AS regulation and crosstalk between AS and other layers of gene regulation.
Plant Journal, 2007
Alternative splicing (AS) increases the proteomic and functional capacity of genomes through the generation of alternative mRNA transcripts from the same gene. AS is now estimated to occur in a third of Arabidopsis and rice genes, and includes genes involved in the control of growth and development, responses to stress and signalling. Regulation of AS reflects the interactions between positive and negative cis sequences in the precursor messenger RNA and a range of trans-acting factors. The levels and activities of these factors differ in different cells and growth conditions. To identify changes in AS in multiple genes simultaneously, we have established a reproducible RT-PCR panel that can analyse 96 alternative splicing events and accurately measure the ratio of alternatively spliced products. This procedure detected statistically significant changes in AS in different plant organs, in plants grown under different light and day-length conditions, and in plants overexpressing splicing factors. The system provides a convenient, medium-throughput means of monitoring changes in AS in multiple genes. It can readily be applied to much larger or targeted sets of gene transcripts to generate information on the significance and regulation of AS in plant growth and development, specific processes and responses to external stimuli.