Huan Wang

Genome-Wide Analysis Uncovers Regulation of Long Intergenic Noncoding RNAs in Arabidopsis

This work identifies 6480 long intergenic noncoding RNAs in Arabidopsis, many of which show organ-specific and stress-responsive expression. The biogenesis of a group of long intergenic noncoding RNAs is coregulated by the RNA processing proteins SERRATE, CBP20, and CBP80. Long intergenic noncoding RNAs (lincRNAs) transcribed from intergenic regions of yeast and animal genomes play important roles in key biological processes. Yet, plant lincRNAs remain poorly characterized and how lincRNA biogenesis is regulated is unclear. Using a reproducibility-based bioinformatics strategy to analyze 200 Arabidopsis thaliana transcriptome data sets, we identified 13,230 intergenic transcripts of which 6480 can be classified as lincRNAs. Expression of 2708 lincRNAs was detected by RNA sequencing experiments. Transcriptome profiling by custom microarrays revealed that the majority of these lincRNAs are expressed at a level between those of mRNAs and precursors of miRNAs. A subset of lincRNA genes shows organ-specific expression, whereas others are responsive to biotic and/or abiotic stresses. Further analysis of transcriptome data in 11 mutants uncovered SERRATE, CAP BINDING PROTEIN20 (CBP20), and CBP80 as regulators of lincRNA expression and biogenesis. RT-PCR experiments confirmed these three proteins are also needed for splicing of a small group of intron-containing lincRNAs.

Genome-wide identification of long noncoding natural antisense transcripts and their responses to light in Arabidopsis

Recent research on long noncoding RNAs (lncRNAs) has expanded our understanding of gene transcription regulation and the generation of cellular complexity. Depending on their genomic origins, lncRNAs can be transcribed from intergenic or intragenic regions or from introns of protein-coding genes. We have recently reported more than 6000 intergenic lncRNAs in Arabidopsis. Here, we systematically identified long noncoding natural antisense transcripts (lncNATs), defined as lncRNAs transcribed from the opposite DNA strand of coding or noncoding genes. We found a total of 37,238 sense-antisense transcript pairs and 70% of annotated mRNAs to be associated with antisense transcripts in Arabidopsis. These lncNATs could be reproducibly detected by different technical platforms, including strand-specific tiling arrays, Agilent custom expression arrays, strand-specific RNA-seq, and qRT-PCR experiments. Moreover, we investigated the expression profiles of sense-antisense pairs in response to light and observed spatial and developmental-specific light effects on 626 concordant and 766 discordant NAT pairs. Genes for a large number of the light-responsive NAT pairs are associated with histone modification peaks, and histone acetylation is dynamically correlated with light-responsive expression changes of NATs.

IAA-Ala Resistant3, an Evolutionarily Conserved Target of miR167, Mediates Arabidopsis Root Architecture Changes during High Osmotic Stress

When plants are subjected to water stress such as drought and high salt, they develop more lateral roots through reducing miRNA167 levels and indirectly increasing IAR3 mRNA levels. The increased IAR3 enzyme converts inactive auxin to the active form, which promotes root branching. This stress adaptation miRNA-mRNA pair is evolutionarily conserved. The functions of microRNAs and their target mRNAs in Arabidopsis thaliana development have been widely documented; however, roles of stress-responsive microRNAs and their targets are not as well understood. Using small RNA deep sequencing and ATH1 microarrays to profile mRNAs, we identified IAA-Ala Resistant3 (IAR3) as a new target of miR167a. As expected, IAR3 mRNA was cleaved at the miR167a complementary site and under high osmotic stress miR167a levels decreased, whereas IAR3 mRNA levels increased. IAR3 hydrolyzes an inactive form of auxin (indole-3-acetic acid [IAA]-alanine) and releases bioactive auxin (IAA), a central phytohormone for root development. In contrast with the wild type, iar3 mutants accumulated reduced IAA levels and did not display high osmotic stress–induced root architecture changes. Transgenic plants expressing a cleavage-resistant form of IAR3 mRNA accumulated high levels of IAR3 mRNAs and showed increased lateral root development compared with transgenic plants expressing wild-type IAR3. Expression of an inducible noncoding RNA to sequester miR167a by target mimicry led to an increase in IAR3 mRNA levels, further confirming the inverse relationship between the two partners. Sequence comparison revealed the miR167 target site on IAR3 mRNA is conserved in evolutionarily distant plant species. Finally, we showed that IAR3 is required for drought tolerance.

Deep sequencing of small RNAs specifically associated with Arabidopsis AGO1 and AGO4 uncovers new AGO functions

As important components of small RNA (smRNA) pathways, Argonaute (AGO) proteins mediate the interaction of incorporated smRNAs with their targets. Arabidopsis contains 10 AGO proteins with specialized or redundant functions. Among them, AGO1 mainly acts in microRNA (miRNA) and small-interfering RNA (siRNA) pathways for post-transcriptional gene silencing (PTGS), whereas AGO4 regulates transcriptional gene silencing (TGS) via endogenous 24-nucleotide (nt) smRNAs. To fully characterize smRNAs associated with AGO1 and AGO4, we developed a two-step protocol to purify AGO/smRNA complexes from flowers, leaves, roots and seedlings with enhanced purity, and sequenced the smRNAs by Illumina technology. Besides recovering most previously annotated smRNAs, we also identified some additional miRNAs, phased smRNA clusters and small-interfering RNAs derived from the overlapping region of natural antisense transcript pairs (NAT) (nat-siRNAs). We also identified a smRNA distribution feature on miRNA precursors which may help to identify authentic miRNAs. Organ-specific sequencing provided digital expression profiles of all obtained smRNAs, especially miRNAs. The presence and conservation of collateral miRNAs on known miRNA precursors were also investigated. Intriguingly, about 30% of AGO1-associated smRNAs were 24-nt long and unrelated to the 21-nt species. Further analysis showed that DNA-dependent RNA polymerase IV (Pol IV)-dependent smRNAs were mainly 24 nt and associated with AGO4, whereas the majority of the potential Pol V-dependent ones were 21-nt smRNAs and bound to AGO1, suggesting the potential involvement of AGO1 in Pol V-related pathways.

Long noncoding RNA transcriptome of plants.

Since their discovery more than two decades ago, animal long noncoding RNAs (lncRNAs) have emerged as important regulators of many biological processes. Recently, a large number of lncRNAs have also been identified in higher plants, and here, we review their identification, classification and known regulatory functions in various developmental events and stress responses. Knowledge gained from a deeper understanding of this special group of noncoding RNAs may lead to biotechnological improvement of crops. Some possible examples in this direction are discussed.

The response and recovery of the Arabidopsis thaliana transcriptome to phosphate starvation

BackgroundOver application of phosphate fertilizers in modern agriculture contaminates waterways and disrupts natural ecosystems. Nevertheless, this is a common practice among farmers, especially in developing countries as abundant fertilizers are believed to boost crop yields. The study of plant phosphate metabolism and its underlying genetic pathways is key to discovering methods of efficient fertilizer usage. The work presented here describes a genome-wide resource on the molecular dynamics underpinning the response and recovery in roots and shoots of Arabidopsis thaliana to phosphate-starvation.ResultsGenome-wide profiling by micro- and tiling-arrays (accessible from GEO: GSE34004) revealed minimal overlap between root and shoot transcriptomes suggesting two independent phosphate-starvation regulons. Novel gene expression patterns were detected for over 1000 candidates and were classified as either initial, persistent, or latent responders. Comparative analysis to AtGenExpress identified cohorts of genes co-regulated across multiple stimuli. The hormone ABA displayed a dominant role in regulating many phosphate-responsive candidates. Analysis of co-regulation enabled the determination of specific versus generic members of closely related gene families with respect to phosphate-starvation. Thus, among others, we showed that PHR1-regulated members of closely related phosphate-responsive families (PHT1;1, PHT1;7–9, SPX1-3, and PHO1;H1) display greater specificity to phosphate-starvation than their more generic counterparts.ConclusionOur results uncover much larger, staged responses to phosphate-starvation than previously described. To our knowledge, this work describes the most complete genome-wide data on plant nutrient stress to-date.

RNAcentral: an international database of ncRNA sequences

Abstract The field of non-coding RNA biology has been hampered by the lack of availability of a comprehensive, up-to-date collection of accessioned RNA sequences. Here we present the first release of RNAcentral, a database that collates and integrates information from an international consortium of established RNA sequence databases. The initial release contains over 8.1 million sequences, including representatives of all major functional classes. A web portal (http://rnacentral.org) provides free access to data, search functionality, cross-references, source code and an integrated genome browser for selected species.

CURLY LEAF Regulates Gene Sets Coordinating Seed Size and Lipid Biosynthesis

A histone methyltransferase negatively regulates a set of genes coordinating seed size and lipid biosynthesis pathways in developing Arabidopsis embryos. CURLY LEAF (CLF), a histone methyltransferase of Polycomb Repressive Complex 2 (PRC2) for trimethylation of histone H3 Lys 27 (H3K27me3), has been thought as a negative regulator controlling mainly postgermination growth in Arabidopsis (Arabidopsis thaliana). Approximately 14% to 29% of genic regions are decorated by H3K27me3 in the Arabidopsis genome; however, transcriptional repression activities of PRC2 on a majority of these regions remain unclear. Here, by analysis of transcriptome profiles, we found that approximately 11.6% genes in the Arabidopsis genome were repressed by CLF in various organs. Unexpectedly, approximately 54% of these genes were preferentially repressed in siliques. Further analyses of 118 transcriptome datasets uncovered a group of genes that was preferentially expressed and repressed by CLF in embryos at the mature-green stage. This observation suggests that CLF mediates a large-scale H3K27me3 programming/reprogramming event during embryonic development. Plants of clf-28 produced bigger and heavier seeds with higher oil content, larger oil bodies, and altered long-chain fatty acid composition compared with wild type. Around 46% of CLF-repressed genes were associated with H3K27me3 marks; moreover, we verified histone modification and transcriptional repression by CLF on regulatory genes. Our results suggest that CLF silences specific gene expression modules. Genes operating within a module have various molecular functions, but they cooperate to regulate a similar physiological function during embryo development.

Light-inducible miR163 targets PXMT1 transcripts to promote seed germination and primary root elongation in Arabidopsis

MiR163 targets PXMT1 transcripts at the early stage of light responses to promote seed germination and root development. Expression of many plant microRNAs is responsive to hormones and environmental stimuli, but none has yet been associated with light. Arabidopsis (Arabidopsis thaliana) miR163 is 24 nucleotides in length and targets mRNAs encoding several S-adenosyl-Met-dependent carboxyl methyltransferase family members. Here, we found that miR163 is highly induced by light during seedling de-etiolation as well as seed germination. Under the same condition, its target PXMT1, encoding a methyltransferase that methylates 1,7-paraxanthine, is down-regulated. Light repression of PXMT1 is abolished in a mir163 null mutant, but the repression can be restored to wild-type levels in complementation lines expressing pri-miR163 gene in the mir163 mutant background. During seed germination, miR163 and its target PXMT1 are predominantly expressed in the radicle, and the expression patterns of the two genes are inversely correlated. Moreover, compared with the wild type, mir163 mutant or PXMT1 overexpression line shows delayed seed germination under continuous light, and seedlings develop shorter primary roots with an increased number of lateral roots under long-day condition. Together, our results indicate that miR163 targets PXMT1 mRNA to promote seed germination and modulate root architecture during early development of Arabidopsis seedlings.

The antiphasic regulatory module comprising CDF5 and its antisense RNA FLORE links the circadian clock to photoperiodic flowering

Circadian rhythms of gene expression are generated by the combinatorial action of transcriptional and translational feedback loops as well as chromatin remodelling events. Recently, long noncoding RNAs (lncRNAs) that are natural antisense transcripts (NATs) to transcripts encoding central oscillator components were proposed as modulators of core clock function in mammals (Per) and fungi (frq/qrf). Although oscillating lncRNAs exist in plants, their functional characterization is at an initial stage. By screening an Arabidopsis thaliana lncRNA custom-made array we identified CDF5 LONG NONCODING RNA (FLORE), a circadian-regulated lncRNA that is a NAT of CDF5. Quantitative real-time RT-PCR confirmed the circadian regulation of FLORE, whereas GUS-staining and flowering time evaluation were used to determine its biological function. FLORE and CDF5 antiphasic expression reflects mutual inhibition in a similar way to frq/qrf. Moreover, whereas the CDF5 protein delays flowering by directly repressing FT transcription, FLORE promotes it by repressing several CDFs (CDF1, CDF3, CDF5) and increasing FT transcript levels, indicating both cis and trans function. We propose that the CDF5/FLORE NAT pair constitutes an additional circadian regulatory module with conserved (mutual inhibition) and unique (function in trans) features, able to fine-tune its own circadian oscillation, and consequently, adjust the onset of flowering to favourable environmental conditions.