Unproductive alternative splicing and nonsense mRNAs: A widespread phenomenon among plant circadian clock genes

Abstract Background Recent mapping of eukaryotic transcriptomes and spliceomes using massively parallel RNA sequencing (RNA-seq) has revealed that the extent of alternative splicing has been considerably underestimated. Evidence also suggests that many pre-mRNAs undergo unproductive alternative splicing resulting in incorporation of in-frame premature termination codons (PTCs). The destinies and potential functions of the PTC-harboring mRNAs remain poorly understood. Unproductive alternative splicing in circadian clock genes presents a special case study because the daily oscillations of protein expression levels require rapid and steep adjustments in mRNA levels. Results We conducted a systematic survey of alternative splicing of plant circadian clock genes using RNA-seq and found that many Arabidopsis thaliana circadian clock-associated genes are alternatively spliced. Results were confirmed using reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR), and/or Sanger sequencing. Intron retention events were frequently observed in mRNAs of the CCA1/LHY-like subfamily of MYB transcription factors. In contrast, the REVEILLE2 (RVE2) transcript was alternatively spliced via inclusion of a "poison cassette exon" (PCE). The PCE type events introducing in-frame PTCs are conserved in some mammalian and plant serine/arginine-rich splicing factors. For some circadian genes such as CCA1 the ratio of the productive isoform (i.e., a representative splice variant encoding the full-length protein) to its PTC counterpart shifted sharply under specific environmental stress conditions. Conclusions Our results demonstrate that unproductive alternative splicing is a widespread phenomenon among plant circadian clock genes that frequently generates mRNA isoforms harboring in-frame PTCs. Because LHY and CCA1 are core components of the plant central circadian oscillator, the conservation of alternatively spliced variants between CCA1 and LHY and for CCA1 across phyla [2] indicates a potential role of nonsense transcripts in regulation of circadian rhythms. Most of the alternatively spliced isoforms harbor in-frame PTCs that arise from full or partial intron retention events. However, a PTC in the RVE2 transcript is introduced through a PCE event. The conservation of AS events and modulation of the relative abundance of nonsense isoforms by environmental and diurnal conditions suggests possible regulatory roles for these alternatively spliced transcripts in circadian clock function. The temperature-dependent expression of the PTC transcripts among members of CCA1/LHY subfamily indicates that alternative splicing may be involved in regulation of the clock temperature compensation mechanism. Reviewers This article was reviewed by Dr. Eugene Koonin, Dr. Chungoo Park (nominated by Dr. Kateryna Makova), and Dr. Marcelo Yanovsky (nominated by Dr. Valerian Dolja).</p

Unproductive alternative splicing and nonsense mRNAs: A widespread phenomenon among plant circadian clock genes

Background: Recent mapping of eukaryotic transcriptomes and spliceomes using massively parallel RNA sequencing (RNA-seq) has revealed that the extent of alternative splicing has been considerably underestimated. Evidence also suggests that many pre-mRNAs undergo unproductive alternative splicing resulting in incorporation of in-frame premature termination codons (PTCs). The destinies and potential functions of the PTC-harboring mRNAs remain poorly understood. Unproductive alternative splicing in circadian clock genes presents a special case study because the daily oscillations of protein expression levels require rapid and steep adjustments in mRNA levels. Results: We conducted a systematic survey of alternative splicing of plant circadian clock genes using RNA-seq and found that many Arabidopsis thaliana circadian clock-associated genes are alternatively spliced. Results were confirmed using reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR), and/or Sanger sequencing. Intron retention events were frequently observed in mRNAs of the CCA1/LHY-like subfamily of MYB transcription factors. In contrast, the REVEILLE2 (RVE2) transcript was alternatively spliced via inclusion of a "poison cassette exon" (PCE). The PCE type events introducing in-frame PTCs are conserved in some mammalian and plant serine/arginine-rich splicing factors. For some circadian genes such as CCA1 the ratio of the productive isoform (i.e., a representative splice variant encoding the full-length protein) to its PTC counterpart shifted sharply under specific environmental stress conditions. Conclusions: Our results demonstrate that unproductive alternative splicing is a widespread phenomenon among plant circadian clock genes that frequently generates mRNA isoforms harboring in-frame PTCs. Because LHY and CCA1 are core components of the plant central circadian oscillator, the conservation of alternatively spliced variants between CCA1 and LHY and for CCA1 across phyla [2] indicates a potential role of nonsense transcripts in regulation of circadian rhythms. Most of the alternatively spliced isoforms harbor in-frame PTCs that arise from full or partial intron retention events. However, a PTC in the RVE2 transcript is introduced through a PCE event. The conservation of AS events and modulation of the relative abundance of nonsense isoforms by environmental and diurnal conditions suggests possible regulatory roles for these alternatively spliced transcripts in circadian clock function. The temperature-dependent expression of the PTC transcripts among members of CCA1/LHY subfamily indicates that alternative splicing may be involved in regulation of the clock temperature compensation mechanism.KEYWORDS: Circadian clock associated 1 (CCA1), Premature termination codon, Alternative splicing, Cassette exon, RNA-seq, Nonsense mRNAs, Reveille 2 (RVE2), Late elongated hypocotyl (LHY), Intron retention, Circadian dock, Arabidopsis thalianaThis is the publisher’s final pdf. The published article is copyrighted by BioMed Central Ltd. and can be found at: http://www.biomedcentral.com

Genome Enabled Discovery of Carbon Sequestration Genes in Poplar

The goals of the S.H. Strauss laboratory portion of 'Genome-enabled discovery of carbon sequestration genes in poplar' are (1) to explore the functions of candidate genes using Populus transformation by inserting genes provided by Oakridge National Laboratory (ORNL) and the University of Florida (UF) into poplar; (2) to expand the poplar transformation toolkit by developing transformation methods for important genotypes; and (3) to allow induced expression, and efficient gene suppression, in roots and other tissues. As part of the transformation improvement effort, OSU developed transformation protocols for Populus trichocarpa 'Nisqually-1' clone and an early flowering P. alba clone, 6K10. Complete descriptions of the transformation systems were published (Ma et. al. 2004, Meilan et. al 2004). Twenty-one 'Nisqually-1' and 622 6K10 transgenic plants were generated. To identify root predominant promoters, a set of three promoters were tested for their tissue-specific expression patterns in poplar and in Arabidopsis as a model system. A novel gene, ET304, was identified by analyzing a collection of poplar enhancer trap lines generated at OSU (Filichkin et. al 2006a, 2006b). Other promoters include the pGgMT1 root-predominant promoter from Casuarina glauca and the pAtPIN2 promoter from Arabidopsis root specific PIN2 gene. OSU tested two induction systems, alcohol- and estrogen-inducible, in multiple poplar transgenics. Ethanol proved to be the more efficient when tested in tissue culture and greenhouse conditions. Two estrogen-inducible systems were evaluated in transgenic Populus, neither of which functioned reliably in tissue culture conditions. GATEWAY-compatible plant binary vectors were designed to compare the silencing efficiency of homologous (direct) RNAi vs. heterologous (transitive) RNAi inverted repeats. A set of genes was targeted for post transcriptional silencing in the model Arabidopsis system; these include the floral meristem identity gene (APETALA1 or AP1), auxin response factor gene (ETTIN), the gene encoding transcriptional factor of WD40 family (TRANSPARENTTESTAGLABRA1 or TTG1), and the auxin efflux carrier (PIN-FORMED2 or PIN2) gene. More than 220 transgenic lines of the 1st, 2nd and 3rd generations were analyzed for RNAi suppression phenotypes (Filichkin et. al., manuscript submitted). A total of 108 constructs were supplied by ORNL, UF and OSU and used to generate over 1,881 PCR verified transgenic Populus and over 300 PCR verified transgenic Arabidopsis events. The Populus transgenics alone required Agrobacterium co-cultivations of 124.406 explants

Genome-wide identification of splicing quantitative trait loci (sQTLs) in diverse ecotypes of Arabidopsis thaliana

Alternative splicing (AS) of pre-mRNAs contributes to transcriptome diversity and enables plants to generate different protein isoforms from a single gene and/or fine-tune gene expression during different development stages and environmental changes. Although AS is pervasive, the genetic basis for differential isoform usage in plants is still emerging. In this study, we performed genome-wide analysis in 666 geographically distributed diverse ecotypes of Arabidopsis thaliana to identify genomic regions [splicing quantitative trait loci (sQTLs)] that may regulate differential AS. These ecotypes belong to different microclimatic conditions and are part of the relict and non-relict populations. Although sQTLs were spread across the genome, we observed enrichment for trans-sQTL (trans-sQTLs hotspots) on chromosome one. Furthermore, we identified several sQTL (911) that co-localized with trait-linked single nucleotide polymorphisms (SNP) identified in the Arabidopsis genome-wide association studies (AraGWAS). Many sQTLs were enriched among circadian clock, flowering, and stress-responsive genes, suggesting a role for differential isoform usage in regulating these important processes in diverse ecotypes of Arabidopsis. In conclusion, the current study provides a deep insight into SNPs affecting isoform ratios/genes and facilitates a better mechanistic understanding of trait-associated SNPs in GWAS studies. To the best of our knowledge, this is the first report of sQTL analysis in a large set of Arabidopsis ecotypes and can be used as a reference to perform sQTL analysis in the Brassicaceae family. Since whole genome and transcriptome datasets are available for these diverse ecotypes, it could serve as a powerful resource for the biological interpretation of trait-associated loci, splice isoform ratios, and their phenotypic consequences to help produce more resilient and high yield crop varieties

Efficiency of gene silencing in \u3ci\u3eArabidopsis\u3c/i\u3e: direct inverted repeats vs. transitive RNAi vectors

We investigated the efficiency of RNA interference (RNAi) in Arabidopsis using transitive and homologous inverted repeat (hIR) vectors. hIR constructs carry self-complementary intron-spliced fragments of the target gene whereas transitive vectors have the target sequence fragment adjacent to an intron-spliced, inverted repeat of heterologous origin. Both transitive and hIR constructs facilitated specific and heritable silencing in the three genes studied (AP1 , ETTIN and TTG1 ). Both types of vectors produced a phenotypic series that phenocopied reduction of function mutants for the respective target gene. The hIR yielded up to fourfold higher proportions of events with strongly manifested reduction of function phenotypes compared to transitive RNAi. We further investigated the efficiency and potential off-target effects of AP1 silencing by both types of vectors using genome-scale microarrays and quantitative RT-PCR. The depletion of AP1 transcripts coincided with reduction of function phenotypic changes among both hIR and transitive lines and also showed similar expression patterns among differentially regulated genes. We did not detect significant silencing directed against homologous potential off-target genes when constructs were designed with minimal sequence similarity. Both hIR and transitive methods are useful tools in plant biotechnology and genomics. The choice of vector will depend on specific objectives such as cloning throughput, number of events and degree of suppression required

Alternative splicing of barley clock genes in response to low temperature:evidence for alternative splicing conservation

Alternative splicing (AS) is a regulated mechanism that generates multiple transcripts from individual genes. It is widespread in eukaryotic genomes and provides an effective way to control gene expression. At low temperatures, AS regulates Arabidopsis clock genes through dynamic changes in the levels of productive mRNAs. We examined AS in barley clock genes to assess whether temperature-dependent AS responses also occur in a monocotyledonous crop species. We identify changes in AS of various barley core clock genes including the barley orthologues of Arabidopsis AtLHY and AtPRR7 which showed the most pronounced AS changes in response to low temperature. The AS events modulate the levels of functional and translatable mRNAs, and potentially protein levels, upon transition to cold. There is some conservation of AS events and/or splicing behaviour of clock genes between Arabidopsis and barley. In addition, novel temperature-dependent AS of the core clock gene HvPPD-H1 (a major determinant of photoperiod response and AtPRR7 orthologue) is conserved in monocots. HvPPD-H1 showed a rapid, temperature-sensitive isoform switch which resulted in changes in abundance of AS variants encoding different protein isoforms. This novel layer of low temperature control of clock gene expression, observed in two very different species, will help our understanding of plant adaptation to different environments and ultimately offer a new range of targets for plant improvement

Global Profiling of Rice and Poplar Transcriptomes Highlights Key Conserved Circadian-Controlled Pathways and cis-Regulatory Modules

Circadian clocks provide an adaptive advantage through anticipation of daily and seasonal environmental changes. In plants, the central clock oscillator is regulated by several interlocking feedback loops. It was shown that a substantial proportion of the Arabidopsis genome cycles with phases of peak expression covering the entire day. Synchronized transcriptome cycling is driven through an extensive network of diurnal and clock-regulated transcription factors and their target cis-regulatory elements. Study of the cycling transcriptome in other plant species could thus help elucidate the similarities and differences and identify hubs of regulation common to monocot and dicot plants.Using a combination of oligonucleotide microarrays and data mining pipelines, we examined daily rhythms in gene expression in one monocotyledonous and one dicotyledonous plant, rice and poplar, respectively. Cycling transcriptomes were interrogated under different diurnal (driven) and circadian (free running) light and temperature conditions. Collectively, photocycles and thermocycles regulated about 60% of the expressed nuclear genes in rice and poplar. Depending on the condition tested, up to one third of oscillating Arabidopsis-poplar-rice orthologs were phased within three hours of each other suggesting a high degree of conservation in terms of rhythmic gene expression. We identified clusters of rhythmically co-expressed genes and searched their promoter sequences to identify phase-specific cis-elements, including elements that were conserved in the promoters of Arabidopsis, poplar, and rice.Our results show that the cycling patterns of many circadian clock genes are highly conserved across poplar, rice, and Arabidopsis. The expression of many orthologous genes in key metabolic and regulatory pathways is diurnal and/or circadian regulated and phased to similar times of day. Our results confirm previous findings in Arabidopsis of three major classes of cis-regulatory modules within the plant circadian network: the morning (ME, GBOX), evening (EE, GATA), and midnight (PBX/TBX/SBX) modules. Identification of identical overrepresented motifs in the promoters of cycling genes from different species suggests that the core diurnal/circadian cis-regulatory network is deeply conserved between mono- and dicotyledonous species