Alternative splicing (AS) is a key regulatory mechanism that contributes to transcriptome and proteome diversity. As very few genome-wide studies analyzing AS in plants are available, we have performed high-throughput sequencing of a normalized cDNA library which resulted in a high coverage transcriptome map of Arabidopsis. We detect~150,000 splice junctions derived mostly from typical plant introns, including an eightfold increase in the number of U12 introns (2069). Around 61% of multiexonic genes are alternatively spliced under normal growth conditions. Moreover, we provide experimental validation of 540 AS transcripts (from 256 genes coding for important regulatory factors) using highresolution RT-PCR and Sanger sequencing. Intron retention (IR) is the most frequent AS event (~40%), but many IRs have relatively low read coverage and are less well-represented in assembled transcripts. Additionally,~51% of Arabidopsis genes produce AS transcripts which do not involve IR. Therefore, the significance of IR in generating transcript diversity was generally overestimated in previous assessments. IR analysis allowed the identification of a large set of cryptic introns inside annotated coding exons. Importantly, a significant fraction of these cryptic introns are spliced out in frame, indicating a role in protein diversity. Furthermore, we show extensive AS coupled to nonsense-mediated decay in AFC2, encoding a highly conserved LAMMER kinase which phosphorylates splicing factors, thus establishing a complex loop in AS regulation. We provide the most comprehensive analysis of AS to date which will serve as a valuable resource for the plant community to study transcriptome complexity and gene regulation. [Supplemental material is available for this article.]Alternative splicing (AS) is a widespread mechanism which increases transcriptome and proteome complexity and controls developmental programs and responses to the environment in higher eukaryotes. The splicing process, removal of introns and ligation of exons, is performed by a large RNA-protein complex, the spliceosome, consisting of five small nuclear RNAs (snRNAs) and about 180 proteins with different functions (Wahl et al. 2009). Assembly of the spliceosome on introns in a precursor messenger RNA (pre-mRNA) is directed by cis elements and trans-acting factors (Black 2003;Stamm et al. 2005). The cis sequences include the splice sites, branchpoint, and polypyrimidine tract which have degenerate consensus sequences in higher eukaryotes. While many splice sites are selected in all transcripts (constitutive splicing), others are used to various levels, resulting in alternative transcripts. Selection of such alternative splice sites is affected by auxiliary cis elements located within exonic and intronic sequences, termed splicing enhancers and silencers. These elements are binding sites for trans-acting splicing factors, for example, hnRNP and SR proteins. These proteins, in addition to their functions in constitutive splicing, play a key role in AS by inhibition or ...
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.
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 >350 nt. Unexpectedly, although many intron retention transcripts possess NMD features, they are not sensitive to NMD. Finally, we have shown that AS/NMD regulates the abundance of transcripts of many genes important for plant development and adaptation including transcription factors, RNA processing factors and stress response genes.
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.
Alternative splicing generates multiple transcript and protein isoforms from the same gene and thus is important in gene expression regulation. To date, RNA-sequencing (RNA-seq) is the standard method for quantifying changes in alternative splicing on a genome-wide scale. Understanding the current limitations of RNA-seq is crucial for reliable analysis and the lack of high quality, comprehensive transcriptomes for most species, including model organisms such as Arabidopsis, is a major constraint in accurate quantification of transcript isoforms. To address this, we designed a novel pipeline with stringent filters and assembled a comprehensive Reference Transcript Dataset for Arabidopsis (AtRTD2) containing 82,190 non-redundant transcripts from 34 212 genes. Extensive experimental validation showed that AtRTD2 and its modified version, AtRTD2-QUASI, for use in Quantification of Alternatively Spliced Isoforms, outperform other available transcriptomes in RNA-seq analysis. This strategy can be implemented in other species to build a pipeline for transcript-level expression and alternative splicing analyses.
Regulation of gene expression at the post-transcriptional level is mainly achieved by proteins containing well-defined sequence motifs involved in RNA binding. The most widely spread motifs are the RNA recognition motif (RRM) and the K homology (KH) domain. In this article, we survey the complete Arabidopsis thaliana genome for proteins containing RRM and KH RNA-binding domains. The Arabidopsis genome encodes 196 RRM-containing proteins, a more complex set than found in Caenorhabditis elegans and Drosophila melanogaster. In addition, the Arabidopsis genome contains 26 KH domain proteins. Most of the Arabidopsis RRM-containing proteins can be classified into structural and/or functional groups, based on similarity with either known metazoan or Arabidopsis proteins. Approximately 50% of Arabidopsis RRM-containing proteins do not have obvious homologues in metazoa, and for most of those that are predicted to be orthologues of metazoan proteins, no experimental data exist to confirm this. Additionally, the function of most Arabidopsis RRM proteins and of all KH proteins is unknown. Based on the data presented here, it is evident that among all eukaryotes, only those RNA-binding proteins that are involved in the most essential processes of post-transcriptional gene regulation are preserved in structure and, most probably, in function. However, the higher complexity of RNA-binding proteins in Arabidopsis, as evident in groups of SR splicing factors and poly(A)-binding proteins, may account for the observed differences in mRNA maturation between plants and metazoa. This survey provides a first systematic analysis of plant RNA-binding proteins, which may serve as a basis for functional characterisation of this important protein group in plants.
RNA is structurally very flexible, which provides the basis for its functional diversity. An RNA molecule can often adopt different conformations, which enables the regulation of its function through folding. Proteins help RNAs reach their functionally active conformation by increasing their structural stability or by chaperoning the folding process. Large, dynamic RNA-protein complexes, such as the ribosome or the spliceosome, require numerous proteins that coordinate conformational switches of the RNA components during assembly and during their respective activities.
Light is a source of energy and also a regulator of plant physiological adaptations. We show here that light/dark conditions affect alternative splicing of a subset of Arabidopsis genes preferentially encoding proteins involved in RNA processing. The effect requires functional chloroplasts and is also observed in roots when the communication with the photosynthetic tissues is not interrupted, suggesting that a signaling molecule travels through the plant. Using photosynthetic electron transfer inhibitors with different mechanisms of action we deduce that the reduced pool of plastoquinones initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing and is necessary for proper plant responses to varying light conditions.Light regulates approximately 20% of the transcriptome in Arabidopsis thaliana and rice (1, 2). Alternative splicing has been shown to modulate gene expression during plant development and in response to environmental cues (3). We observed that the alternative splicing of At-RS31 ( Figure 1A), encoding a Ser-Arg-rich splicing factor (4), changed in different light regimes, which led us to investigate how light regulates alternative splicing in plants. Figure 1B). This effect was rapidly reversed when seedlings were placed back in light, with total recovery of the original SI in about 3 hr ( Figure 1C), indicating that the kinetics of the splicing response is slower from light to dark than from dark to light.The light effect is gene-specific ( Figure S1) and is also observed in diurnal cycles under short day conditions ( Figures 1D and S2). Furthermore, three circadian clock mutants behaved like the wild type (WT) in the response of At-RS31 alternative splicing to light/dark ( Figure S3). Changes in At-RS31 splicing are proportional to light intensity both under constant light or in short day grown seedlings ( Figure S4).Both red (660 nm) and blue (470 nm) lights produced similar results as white light ( Figure 1E). Moreover, At-RS31 splicing is not affected in phytochrome and cryptochrome signaling mutants (5, 6) behave as WT seedlings, ruling out photosensory pathways in this light regulation (Figures 1F, S5 and S6).Light-triggered changes in At-RS31 mRNA patterns are not due to differential mRNA degradation. First, the light effect is not observed in the presence of the transcription inhibitor actinomycin D ( Figure 1G). Second, the effects are still observed in upf mutants, defective in the nonsense-mediated mRNA decay (NMD) pathway (7) mRNA1 is the only isoform encoding a full-length At-RS31 protein (9). mRNA3 and mRNA2 are almost fully retained in the nucleus ( Figure S8). mRNA1 levels decrease considerably in dark without significant changes in the total amount of At-RS31 transcripts (Figures 2A and S9) which suggests that alternative splicing is instrumental in the control of mRNA1 cellular levels and, consequently, At-RS31 protein abundance. To assess how interference with At-RS31 alternative splicing regulation could affect Arabidopsis phenotype we ...
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