IDN1 and IDN2 are required for de novo DNA methylation in Arabidopsis thaliana.

DNA methylation is an epigenetic mark affecting genes and transposons. Screening for mutants that fail to establish DNA methylation yielded two we termed "involved in de novo" (idn) 1 and 2. IDN1 encodes DMS3, an SMC-related protein, and IDN2 encodes a previously unknown double-stranded RNA-binding protein with homology to SGS3. IDN1 and IDN2 control de novo methylation and small interfering RNA (siRNA)-mediated maintenance methylation and are components of the RNA-directed DNA methylation pathway

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

Genome scale transcriptome analysis of shoot organogenesis in Populus

<p>Abstract</p> <p>Background</p> <p>Our aim is to improve knowledge of gene regulatory circuits important to dedifferentiation, redifferentiation, and adventitious meristem organization during <it>in vitro </it>regeneration of plants. Regeneration of transgenic cells remains a major obstacle to research and commercial deployment of most taxa of transgenic plants, and woody species are particularly recalcitrant. The model woody species <it>Populus</it>, due to its genome sequence and amenability to <it>in vitro </it>manipulation, is an excellent species for study in this area. The genes recognized may help to guide the development of new tools for improving the efficiency of plant regeneration and transformation.</p> <p>Results</p> <p>We analyzed gene expression during poplar <it>in vitro </it>dedifferentiation and shoot regeneration using an Affymetrix array representing over 56,000 poplar transcripts. We focused on callus induction and shoot formation, thus we sampled RNAs from tissues: prior to callus induction, 3 days and 15 days after callus induction, and 3 days and 8 days after the start of shoot induction. We used a female hybrid white poplar clone (INRA 717-1 B4, <b><it>Populus tremula × P. alba</it></b>) that is used widely as a model transgenic genotype. Approximately 15% of the monitored genes were significantly up-or down-regulated when controlling the false discovery rate (FDR) at 0.01; over 3,000 genes had a 5-fold or greater change in expression. We found a large initial change in expression after the beginning of hormone treatment (at the earliest stage of callus induction), and then a much smaller number of additional differentially expressed genes at subsequent regeneration stages. A total of 588 transcription factors that were distributed in 45 gene families were differentially regulated. Genes that showed strong differential expression included components of auxin and cytokinin signaling, selected cell division genes, and genes related to plastid development and photosynthesis. When compared with data on in vitro callogenesis in <it>Arabidopsis</it>, 25% (1,260) of up-regulated and 22% (748) of down-regulated genes were in common with the genes regulated in poplar during callus induction.</p> <p>Conclusion</p> <p>The major regulatory events during plant cell organogenesis occur at early stages of dedifferentiation. The regulatory circuits reflect the combinational effects of transcriptional control and hormone signaling, and associated changes in light environment imposed during dedifferentiation.</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

Highly specific gene silencing in a monocot species by artificial microRNAs derived from chimeric MIRNA precursors

[EN] Artificial microRNAs (amiRNAs) are used for selective gene silencing in plants. However, current methods to produce amiRNA constructs for silencing transcripts in monocot species are not suitable for simple, cost-effective and large-scale synthesis. Here, a series of expression vectors based on Oryza sativa MIR390 (OsMIR390) precursor was developed for high-throughput cloning and high expression of amiRNAs in monocots. Four different amiRNA sequences designed to target specifically endogenous genes and expressed from OsMIR390-based vectors were validated in transgenic Brachypodium distachyon plants. Surprisingly, amiRNAs accumulated to higher levels and were processed more accurately when expressed from chimeric OsMIR390-based precursors that include distal stem-loop sequences from Arabidopsis thaliana MIR390a (AtMIR390a). In all cases, transgenic plants displayed the predicted phenotypes induced by target gene repression, and accumulated high levels of amiRNAs and low levels of the corresponding target transcripts. Genome-wide transcriptome profiling combined with 5¿-RLM-RACE analysis in transgenic plants confirmed that amiRNAs were highly specific.We thank Goretti Nguyen, Robyn Stevens, Jacob Mreen, Fangfang Ma and Madison Schniers for invaluable technical assistance, and Zacchery R. Smith for his initial contribution to develop the pH7WG2B-OsMIR390-B/c vector. Noah Fahlgren was supported by a USDA AFRI NIFA Postdoctoral Fellowship (MOW-2012-01361). This work was supported by grants from the National Science Foundation (MCB-1231726, MCB-1330562) and National Institutes of Health (AI043288) to James C. Carrington, and from the Department of Energy (DOE DE-SC0006627) to Todd C. Mockler.Carbonell, A.; Fahlgren, N.; Mitchell, S.; Cox, KLJ.; Reilly, KC.; Mockler, TC.; Carrington, JC. (2015). 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Subset of heat-shock transcription factors required for the early response of Arabidopsis to excess light

Sunlight provides energy for photosynthesis and is essential for nearly all life on earth. However, too much or too little light or rapidly fluctuating light conditions cause stress to plants. Rapid changes in the amount of light are perceived as a chang

A multi-organ transcriptome resource for the Burmese Python (Python molurus bivittatus)

<p>Abstract</p> <p>Background</p> <p>Snakes provide a unique vertebrate system for studying a diversity of extreme adaptations, including those related to development, metabolism, physiology, and venom. Despite their importance as research models, genomic resources for snakes are few. Among snakes, the Burmese python is the premier model for studying extremes of metabolic fluctuation and physiological remodelling. In this species, the consumption of large infrequent meals can induce a 40-fold increase in metabolic rate and more than a doubling in size of some organs. To provide a foundation for research utilizing the python, our aim was to assemble and annotate a transcriptome reference from the heart and liver. To accomplish this aim, we used the 454-FLX sequencing platform to collect sequence data from multiple cDNA libraries.</p> <p>Results</p> <p>We collected nearly 1 million 454 sequence reads, and assembled these into 37,245 contigs with a combined length of 13,409,006 bp. To identify known genes, these contigs were compared to chicken and lizard gene sets, and to all Genbank sequences. A total of 13,286 of these contigs were annotated based on similarity to known genes or Genbank sequences. We used gene ontology (GO) assignments to characterize the types of genes in this transcriptome resource. The raw data, transcript contig assembly, and transcript annotations are made available online for use by the broader research community.</p> <p>Conclusion</p> <p>These data should facilitate future studies using pythons and snakes in general, helping to further contribute to the utilization of snakes as a model evolutionary and physiological system. This sequence collection represents a major genomic resource for the Burmese python, and the large number of transcript sequences characterized should contribute to future research in this and other snake species.</p