Targeting alternative splicing by RNAi: from the differential impact on splice variants to triggering artificial pre-mRNA splicing.

Alternative splicing generates multiple transcript and protein isoforms from a single gene and controls transcript intracellular localization and stability by coupling to mRNA export and nonsense-mediated mRNA decay (NMD). RNA interference (RNAi) is a potent mechanism to modulate gene expression. However, its interactions with alternative splicing are poorly understood. We used artificial microRNAs (amiRNAs, also termed shRNAmiR) to knockdown all splice variants of selected target genes in Arabidopsis thaliana. We found that splice variants, which vary by their protein-coding capacity, subcellular localization and sensitivity to NMD, are affected differentially by an amiRNA, although all of them contain the target site. Particular transcript isoforms escape amiRNA-mediated degradation due to their nuclear localization. The nuclear and NMD-sensitive isoforms mask RNAi action in alternatively spliced genes. Interestingly, Arabidopsis SPL genes, which undergo alternative splicing and are targets of miR156, are regulated in the same manner. Moreover, similar results were obtained in mammalian cells using siRNAs, indicating cross-kingdom conservation of these interactions among RNAi and splicing isoforms. Furthermore, we report that amiRNA can trigger artificial alternative splicing, thus expanding the RNAi functional repertoire. Our findings unveil novel interactions between different post-transcriptional processes in defining transcript fates and regulating gene expression.

The cyclin-dependent kinase G group defines a thermo-sensitive alternative splicing circuit modulating the expression of Arabidopsis ATU2AF65A.

The ability to adapt growth and development to temperature variations is crucial to generate plant varieties resilient to predicted temperature changes. However, the mechanisms underlying plant response to progressive increases in temperature have just started to be elucidated. Here, we report that the cyclin-dependent kinase G1 (CDKG1) is a central element in a thermo-sensitive mRNA splicing cascade that transduces changes in ambient temperature into differential expression of the fundamental spliceosome component, ATU2AF65A. CDKG1 is alternatively spliced in a temperature-dependent manner. We found that this process is partly dependent on both the cyclin-dependent kinase G2 (CDKG2) and the interacting co-factor CYCLIN L1 (CYCL1), resulting in two distinct messenger RNAs. The relative abundance of both CDKG1 transcripts correlates with ambient temperature and possibly with different expression levels of the associated protein isoforms. Both CDKG1 alternative transcripts are necessary to fully complement the expression of ATU2AF65A across the temperature range. Our data support a previously unidentified temperature-dependent mechanism based on the alternative splicing (AS) of CDKG1 and regulated by CDKG2 and CYCL1. We propose that changes in ambient temperature affect the relative abundance of CDKG1 transcripts, and this in turn translates into differential CDKG1 protein expression coordinating the AS of ATU2AF65A.

A high quality Arabidopsis transcriptome for accurate transcript-level analysis of alternative splicing.

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.

Unmasking alternative splicing inside protein-coding exons defines exitrons and their role in proteome plasticity.

Alternative splicing (AS) diversifies transcriptomes and proteomes and is widely recognized as a key mechanism for regulating gene expression. Previously, in an analysis of intron retention events in Arabidopsis, we found unusual AS events inside annotated protein-coding exons. Here, we also identify such AS events in human and use these two sets to analyse their features, regulation, functional impact, and evolutionary origin. As these events involve introns with features of both introns and protein-coding exons, we name them exitrons (exonic introns). Though exitrons were detected as a subset of retained introns, they are clearly distinguishable, and their splicing results in transcripts with different fates. About half of the 1002 Arabidopsis and 923 human exitrons have sizes of multiples of 3 nucleotides (nt). Splicing of these exitrons results in internally deleted proteins and affects protein domains, disordered regions, and various post-translational modification sites, thus broadly impacting protein function. Exitron splicing is regulated across tissues, in response to stress and in carcinogenesis. Intriguingly, annotated intronless genes can be also alternatively spliced via exitron usage. We demonstrate that at least some exitrons originate from ancestral coding exons. Based on our findings, we propose a "splicing memory" hypothesis whereby upon intron loss imprints of former exon borders defined by vestigial splicing regulatory elements could drive the evolution of exitron splicing. Altogether, our studies show that exitron splicing is a conserved strategy for increasing proteome plasticity in plants and animals, complementing the repertoire of AS events.

Lost in Translation: Pitfalls in Deciphering Plant Alternative Splicing Transcripts.

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 spliceosome-associated protein Nrl1 suppresses homologous recombination-dependent R-loop formation in fission yeast.

The formation of RNA-DNA hybrids, referred to as R-loops, can promote genome instability and cancer development. Yet the mechanisms by which R-loops compromise genome instability are poorly understood. Here, we establish roles for the evolutionarily conserved Nrl1 protein in pre-mRNA splicing regulation, R-loop suppression and in maintaining genome stability. nrl1Δ mutants exhibit endogenous DNA damage, are sensitive to exogenous DNA damage, and have defects in homologous recombination (HR) repair. Concomitantly, nrl1Δ cells display significant changes in gene expression, similar to those induced by DNA damage in wild-type cells. Further, we find that nrl1Δ cells accumulate high levels of R-loops, which co-localize with HR repair factors and require Rad51 and Rad52 for their formation. Together, our findings support a model in which R-loop accumulation and subsequent DNA damage sequesters HR factors, thereby compromising HR repair at endogenously or exogenously induced DNA damage sites, leading to genome instability.

Imaging of endogenous messenger RNA splice variants in living cells reveals nuclear retention of transcripts inaccessible to nonsense-mediated decay in Arabidopsis.

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.

Complexity of the alternative splicing landscape in plants.

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.

Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis.

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 high-resolution 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.

AtRTD - a comprehensive reference transcript dataset resource for accurate quantification of transcript-specific expression in Arabidopsis thaliana.

RNA-sequencing (RNA-seq) allows global gene expression analysis at the individual transcript level. Accurate quantification of transcript variants generated by alternative splicing (AS) remains a challenge. We have developed a comprehensive, nonredundant Arabidopsis reference transcript dataset (AtRTD) containing over 74 000 transcripts for use with algorithms to quantify AS transcript isoforms in RNA-seq. The AtRTD was formed by merging transcripts from TAIR10 and novel transcripts identified in an AS discovery project. We have estimated transcript abundance in RNA-seq data using the transcriptome-based alignment-free programmes Sailfish and Salmon and have validated quantification of splicing ratios from RNA-seq by high resolution reverse transcription polymerase chain reaction (HR RT-PCR). Good correlations between splicing ratios from RNA-seq and HR RT-PCR were obtained demonstrating the accuracy of abundances calculated for individual transcripts in RNA-seq. The AtRTD is a resource that will have immediate utility in analysing Arabidopsis RNA-seq data to quantify differential transcript abundance and expression.

Introns of plant pri-miRNAs enhance miRNA biogenesis.

Plant MIR genes are independent transcription units that encode long primary miRNA precursors, which usually contain introns. For two miRNA genes, MIR163 and MIR161, we show that introns are crucial for the accumulation of proper levels of mature miRNA. Removal of the intron in both cases led to a drop-off in the level of mature miRNAs. We demonstrate that the stimulating effects of the intron mostly reside in the 5'ss rather than on a genuine splicing event. Our findings are biologically significant as the presence of functional splice sites in the MIR163 gene appears mandatory for pathogen-triggered accumulation of miR163 and proper regulation of at least one of its targets.

Identification of RNA targets for the nuclear multidomain cyclophilin atCyp59 and their effect on PPIase activity.

AtCyp59 is a multidomain cyclophilin containing a peptidyl-prolyl cis/trans isomerase (PPIase) domain and an evolutionarily highly conserved RRM domain. Deregulation of this class of cyclophilins has been shown to affect transcription and to influence phosphorylation of the C-terminal repeat domain of the largest subunit of the RNA polymerase II. We used a genomic SELEX method for identifying RNA targets of AtCyp59. Analysis of the selected RNAs revealed an RNA-binding motif (G[U/C]N[G/A]CC[A/G]) and we show that it is evolutionarily conserved. Binding to this motif was verified by gel shift assays in vitro and by RNA immunopreciptation assays of AtCyp59 in vivo. Most importantly, we show that binding also occurs on unprocessed transcripts in vivo and that binding of specific RNAs inhibits the PPIase activity of AtCyp59 in vitro. Surprisingly, genome-wide analysis showed that the RNA motif is present in about 70% of the annotated transcripts preferentially in exons. Taken together, the available data suggest that these cyclophilins might have an important function in transcription regulation.

Let there be light: regulation of gene expression in plants.

Gene expression regulation relies on a variety of molecular mechanisms affecting different steps of a messenger RNA (mRNA) life: transcription, processing, splicing, alternative splicing, transport, translation, storage and decay. Light induces massive reprogramming of gene expression in plants. Differences in alternative splicing patterns in response to environmental stimuli suggest that alternative splicing plays an important role in plant adaptation to changing life conditions. In a recent publication, our laboratories showed that light regulates alternative splicing of a subset of Arabidopsis genes encoding proteins involved in RNA processing by chloroplast retrograde signals. The light effect on alternative splicing is also observed in roots when the communication with the photosynthetic tissues is not interrupted, suggesting that a signaling molecule travels through the plant. These results point at alternative splicing regulation by retrograde signals as an important mechanism for plant adaptation to their environment.

Alternative splicing and nonsense-mediated decay modulate expression of important regulatory genes in Arabidopsis.

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.

Implementing a rational and consistent nomenclature for serine/arginine-rich protein splicing factors (SR proteins) in plants.

Growing interest in alternative splicing in plants and the extensive sequencing of new plant genomes necessitate more precise definition and classification of genes coding for splicing factors. SR proteins are a family of RNA binding proteins, which function as essential factors for constitutive and alternative splicing. We propose a unified nomenclature for plant SR proteins, taking into account the newly revised nomenclature of the mammalian SR proteins and a number of plant-specific properties of the plant proteins. We identify six subfamilies of SR proteins in Arabidopsis thaliana and rice (Oryza sativa), three of which are plant specific. The proposed subdivision of plant SR proteins into different subfamilies will allow grouping of paralogous proteins and simple assignment of newly discovered SR orthologs from other plant species and will promote functional comparisons in diverse plant species.

A high-resolution single-molecule sequencing-based Arabidopsis transcriptome using novel methods of Iso-seq analysis.

BACKGROUND: Accurate and comprehensive annotation of transcript sequences is essential for transcript quantification and differential gene and transcript expression analysis. Single-molecule long-read sequencing technologies provide improved integrity of transcript structures including alternative splicing, and transcription start and polyadenylation sites. However, accuracy is significantly affected by sequencing errors, mRNA degradation, or incomplete cDNA synthesis. RESULTS: We present a new and comprehensive Arabidopsis thaliana Reference Transcript Dataset 3 (AtRTD3). AtRTD3 contains over 169,000 transcripts-twice that of the best current Arabidopsis transcriptome and including over 1500 novel genes. Seventy-eight percent of transcripts are from Iso-seq with accurately defined splice junctions and transcription start and end sites. We develop novel methods to determine splice junctions and transcription start and end sites accurately. Mismatch profiles around splice junctions provide a powerful feature to distinguish correct splice junctions and remove false splice junctions. Stratified approaches identify high-confidence transcription start and end sites and remove fragmentary transcripts due to degradation. AtRTD3 is a major improvement over existing transcriptomes as demonstrated by analysis of an Arabidopsis cold response RNA-seq time-series. AtRTD3 provides higher resolution of transcript expression profiling and identifies cold-induced differential transcription start and polyadenylation site usage. CONCLUSIONS: AtRTD3 is the most comprehensive Arabidopsis transcriptome currently. It improves the precision of differential gene and transcript expression, differential alternative splicing, and transcription start/end site usage analysis from RNA-seq data. The novel methods for identifying accurate splice junctions and transcription start/end sites are widely applicable and will improve single-molecule sequencing analysis from any species.

Light regulates alternative splicing outcomes via the TOR kinase pathway.

For plants, light is the source of energy and the most relevant regulator of growth and adaptations to the environment by inducing changes in gene expression at various levels, including alternative splicing. Light-triggered chloroplast retrograde signals control alternative splicing in Arabidopsis thaliana. Here, we provide evidence that light regulates the expression of a core set of splicing-related factors in roots. Alternative splicing responses in roots are not directly caused by light but are instead most likely triggered by photosynthesized sugars. The target of rapamycin (TOR) kinase plays a key role in this shoot-to-root signaling pathway. Knocking down TOR expression or pharmacologically inhibiting TOR activity disrupts the alternative splicing responses to light and exogenous sugars in roots. Consistently, splicing decisions are modulated by mitochondrial activity in roots. In conclusion, by activating the TOR pathway, sugars act as mobile signals to coordinate alternative splicing responses to light throughout the whole plant.

Regulation of plant gene expression by alternative splicing.

AS (alternative splicing) is a post-transcriptional process which regulates gene expression through increasing protein complexity and modulating mRNA transcript levels. Regulation of AS depends on interactions between trans-acting protein factors and cis-acting signals in the pre-mRNA (precursor mRNA) transcripts, termed 'combinatorial' control. Dynamic changes in AS patterns reflect changes in abundance, composition and activity of splicing factors in different cell types and in response to cellular or environmental cues. Whereas the SR protein family of splicing factors is well-studied in plants, relatively little is known about other factors influencing the regulation of AS or the consequences of AS on mRNA levels and protein function. To address fundamental questions on AS in plants, we are exploiting a high-resolution RT (reverse transcription)-PCR system to analyse multiple AS events simultaneously. In the present paper, we describe the current applications and development of the AS RT-PCR panel in investigating the roles of splicing factors, cap-binding proteins and nonsense-mediated decay proteins on AS, and examining the extent of AS in genes involved in the same developmental pathway or process.