Xiu-Jie Wang

ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility.

N(6)-methyladenosine (m(6)A) is the most prevalent internal modification of messenger RNA (mRNA) in higher eukaryotes. Here we report ALKBH5 as another mammalian demethylase that oxidatively reverses m(6)A in mRNA in vitro and in vivo. This demethylation activity of ALKBH5 significantly affects mRNA export and RNA metabolism as well as the assembly of mRNA processing factors in nuclear speckles. Alkbh5-deficient male mice have increased m(6)A in mRNA and are characterized by impaired fertility resulting from apoptosis that affects meiotic metaphase-stage spermatocytes. In accordance with this defect, we have identified in mouse testes 1,551 differentially expressed genes that cover broad functional categories and include spermatogenesis-related mRNAs involved in the p53 functional interaction network. The discovery of this RNA demethylase strongly suggests that the reversible m(6)A modification has fundamental and broad functions in mammalian cells.

GOEAST: a web-based software toolkit for Gene Ontology enrichment analysis.

Gene Ontology (GO) analysis has become a commonly used approach for functional studies of large-scale genomic or transcriptomic data. Although there have been a lot of software with GO-related analysis functions, new tools are still needed to meet the requirements for data generated by newly developed technologies or for advanced analysis purpose. Here, we present a Gene Ontology Enrichment Analysis Software Toolkit (GOEAST), an easy-to-use web-based toolkit that identifies statistically overrepresented GO terms within given gene sets. Compared with available GO analysis tools, GOEAST has the following improved features: (i) GOEAST displays enriched GO terms in graphical format according to their relationships in the hierarchical tree of each GO category (biological process, molecular function and cellular component), therefore, provides better understanding of the correlations among enriched GO terms; (ii) GOEAST supports analysis for data from various sources (probe or probe set IDs of Affymetrix, Illumina, Agilent or customized microarrays, as well as different gene identifiers) and multiple species (about 60 prokaryote and eukaryote species); (iii) One unique feature of GOEAST is to allow cross comparison of the GO enrichment status of multiple experiments to identify functional correlations among them. GOEAST also provides rigorous statistical tests to enhance the reliability of analysis results. GOEAST is freely accessible at http://omicslab.genetics.ac.cn/GOEAST/

Draft genome of the wheat A-genome progenitor Triticum urartu

Bread wheat (Triticum aestivum, AABBDD) is one of the most widely cultivated and consumed food crops in the world. However, the complex polyploid nature of its genome makes genetic and functional analyses extremely challenging. The A genome, as a basic genome of bread wheat and other polyploid wheats, for example, T. turgidum (AABB), T. timopheevii (AAGG) and T. zhukovskyi (AAGGAmAm), is central to wheat evolution, domestication and genetic improvement. The progenitor species of the A genome is the diploid wild einkorn wheat T. urartu, which resembles cultivated wheat more extensively than do Aegilops speltoides (the ancestor of the B genome) and Ae. tauschii (the donor of the D genome), especially in the morphology and development of spike and seed. Here we present the generation, assembly and analysis of a whole-genome shotgun draft sequence of the T. urartu genome. We identified protein-coding gene models, performed genome structure analyses and assessed its utility for analysing agronomically important genes and for developing molecular markers. Our T. urartu genome assembly provides a diploid reference for analysis of polyploid wheat genomes and is a valuable resource for the genetic improvement of wheat.

Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation

DNA methylation has important functions in stable, transcriptional gene silencing, immobilization of transposable elements and genome organization. In Arabidopsis, DNA methylation can be induced by double-stranded RNA through the RNA interference (RNAi) pathway, a response known as RNA-directed DNA methylation. This requires a specialized set of RNAi components, including ARGONAUTE4 (AGO4). Here we show that AGO4 binds to small RNAs including small interfering RNAs (siRNAs) originating from transposable and repetitive elements, and cleaves target RNA transcripts. Single mutations in the Asp-Asp-His catalytic motif of AGO4 do not affect siRNA-binding activity but abolish its catalytic potential. siRNA accumulation and non-CpG DNA methylation at some loci require the catalytic activity of AGO4, whereas others are less dependent on this activity. Our results are consistent with a model in which AGO4 can function at target loci through two distinct and separable mechanisms. First, AGO4 can recruit components that signal DNA methylation in a manner independent of its catalytic activity. Second, AGO4 catalytic activity can be crucial for the generation of secondary siRNAs that reinforce its repressive effects.

Small RNAs serve as a genetic buffer against genomic shock in Arabidopsis interspecific hybrids and allopolyploids.

Small RNAs, including microRNAs (miRNAs), small interfering RNAs (siRNAs), and trans-acting siRNAs (tasiRNAs), control gene expression and epigenetic regulation. Although the roles of miRNAs and siRNAs have been extensively studied, their expression diversity and evolution in closely related species and interspecific hybrids are poorly understood. Here, we show comprehensive analyses of miRNA expression and siRNA distributions in two closely related species Arabidopsis thaliana and Arabidopsis arenosa, a natural allotetraploid Arabidopsis suecica, and two resynthesized allotetraploid lines (F1 and F7) derived from A. thaliana and A. arenosa. We found that repeat- and transposon-associated siRNAs were highly divergent between A. thaliana and A. arenosa. A. thaliana siRNA populations underwent rapid changes in F1 but were stably maintained in F7 and A. suecica. The correlation between siRNAs and nonadditive gene expression in allopolyploids is insignificant. In contrast, miRNA and tasiRNA sequences were conserved between species, but their expression patterns were highly variable between the allotetraploids and their progenitors. Many miRNAs tested were nonadditively expressed (deviating from the mid-parent value, MPV) in the allotetraploids and triggered unequal degradation of A. thaliana or A. arenosa targets. The data suggest that small RNAs produced during interspecific hybridization or polyploidization serve as a buffer against the genomic shock in interspecific hybrids and allopolyploids: Stable inheritance of repeat-associated siRNAs maintains chromatin and genome stability, whereas expression variation of miRNAs leads to changes in gene expression, growth vigor, and adaptation.

Activation of the imprinted Dlk1-Dio3 region correlates with pluripotency levels of mouse stem cells

Low reprogramming efficiency and reduced pluripotency have been the two major obstacles in induced pluripotent stem (iPS) cell research. An effective and quick method to assess the pluripotency levels of iPS cells at early stages would significantly increase the success rate of iPS cell generation and promote its applications. We have identified a conserved imprinted region of the mouse genome, the Dlk1-Dio3 region, which was activated in fully pluripotent mouse stem cells but repressed in partially pluripotent cells. The degree of activation of this region was positively correlated with the pluripotency levels of stem cells. A mammalian conserved cluster of microRNAs encoded by this region exhibited significant expression differences between full and partial pluripotent stem cells. Several microRNAs from this cluster potentially target components of the polycomb repressive complex 2 (PRC2) and may form a feedback regulatory loop resulting in the expression of all genes and non-coding RNAs encoded by this region in full pluripotent stem cells. No other genomic regions were found to exhibit such clear expression changes between cell lines with different pluripotency levels; therefore, the Dlk1-Dio3 region may serve as a marker to identify fully pluripotent iPS or embryonic stem cells from partial pluripotent cells. These findings also provide a step forward toward understanding the operating mechanisms during reprogramming to produce iPS cells and can potentially promote the application of iPS cells in regenerative medicine and cancer therapy.

LEAFY COTYLEDON1 Is a Key Regulator of Fatty Acid Biosynthesis in Arabidopsis

In plants, fatty acids are de novo synthesized predominantly in plastids from acetyl-coenzyme A. Although fatty acid biosynthesis has been biochemically well studied, little is known about the regulatory mechanisms of the pathway. Here, we show that overexpression of the Arabidopsis (Arabidopsis thaliana) LEAFY COTYLEDON1 (LEC1) gene causes globally increased expression of fatty acid biosynthetic genes, which are involved in key reactions of condensation, chain elongation, and desaturation of fatty acid biosynthesis. In the plastidial fatty acid synthetic pathway, over 58% of known enzyme-coding genes are up-regulated in LEC1-overexpressing transgenic plants, including those encoding three subunits of acetyl-coenzyme A carboxylase, a key enzyme controlling the fatty acid biosynthesis flux. Moreover, genes involved in glycolysis and lipid accumulation are also up-regulated. Consistent with these results, levels of major fatty acid species and lipids were substantially increased in the transgenic plants. Genetic analysis indicates that the LEC1 function is partially dependent on ABSCISIC ACID INSENSITIVE3, FUSCA3, and WRINKLED1 in the regulation of fatty acid biosynthesis. Moreover, a similar phenotype was observed in transgenic Arabidopsis plants overexpressing two LEC1-like genes of Brassica napus. These results suggest that LEC1 and LEC1-like genes act as key regulators to coordinate the expression of fatty acid biosynthetic genes, thereby representing promising targets for genetic improvement of oil production plants.

Prediction and Identification of Herpes Simplex Virus 1-Encoded MicroRNAs

ABSTRACT MicroRNAs (miRNAs) are key regulators of gene expression in higher eukaryotes. Recently, miRNAs have been identified from viruses with double-stranded DNA genomes. To attempt to identify miRNAs encoded by herpes simplex virus 1 (HSV-1), we applied a computational method to screen the complete genome of HSV-1 for sequences that adopt an extended stem-loop structure and display a pattern of nucleotide divergence characteristic of known miRNAs. Using this method, we identified 11 HSV-1 genomic loci predicted to encode 13 miRNA precursors and 24 miRNA candidates. Eight of the HSV-1 miRNA candidates were predicted to be conserved in HSV-2. The precursor and the mature form of one HSV-1 miRNA candidate, which is encoded ∼450 bp upstream of the transcription start site of the latency-associated transcript (LAT), were detected during infection of Vero cells by Northern blot hybridization. These RNAs, which behave as late gene products, are not predicted to be conserved in HSV-2. Additionally, small RNAs, including some that are roughly the expected size of precursor miRNAs, were detected using probes for miRNA candidates derived from sequences encoding the 8.3-kilobase LAT, from sequences complementary to UL15 mRNA, and from the region between ICP4 and US1. However, no species the size of typical mature miRNAs were detected using these probes. Three of these latter miRNA candidates were predicted to be conserved in HSV-2. Thus, HSV-1 encodes at least one miRNA. We hypothesize that HSV-1 miRNAs regulate viral and host gene expression.

Nuclear m6A Reader YTHDC1 Regulates mRNA Splicing

The regulatory role of N(6)-methyladenosine (m(6)A) and its nuclear binding protein YTHDC1 in pre-mRNA splicing remains an enigma. Here we show that YTHDC1 promotes exon inclusion in targeted mRNAs through recruiting pre-mRNA splicing factor SRSF3 (SRp20) while blocking SRSF10 (SRp38) mRNA binding. Transcriptome assay with PAR-CLIP-seq analysis revealed that YTHDC1-regulated exon-inclusion patterns were similar to those of SRSF3 but opposite of SRSF10. In vitro pull-down assay illustrated a competitive binding of SRSF3 and SRSF10 to YTHDC1. Moreover, YTHDC1 facilitates SRSF3 but represses SRSF10 in their nuclear speckle localization, RNA-binding affinity, and associated splicing events, dysregulation of which, as the result of YTHDC1 depletion, can be restored by reconstitution with wild-type, but not m(6)A-binding-defective, YTHDC1. Our findings provide the direct evidence that m(6)A reader YTHDC1 regulates mRNA splicing through recruiting and modulating pre-mRNA splicing factors for their access to the binding regions of targeted mRNAs.

FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis

The role of Fat Mass and Obesity-associated protein (FTO) and its substrate N6-methyladenosine (m6A) in mRNA processing and adipogenesis remains largely unknown. We show that FTO expression and m6A levels are inversely correlated during adipogenesis. FTO depletion blocks differentiation and only catalytically active FTO restores adipogenesis. Transcriptome analyses in combination with m6A-seq revealed that gene expression and mRNA splicing of grouped genes are regulated by FTO. M6A is enriched in exonic regions flanking 5′- and 3′-splice sites, spatially overlapping with mRNA splicing regulatory serine/arginine-rich (SR) protein exonic splicing enhancer binding regions. Enhanced levels of m6A in response to FTO depletion promotes the RNA binding ability of SRSF2 protein, leading to increased inclusion of target exons. FTO controls exonic splicing of adipogenic regulatory factor RUNX1T1 by regulating m6A levels around splice sites and thereby modulates differentiation. These findings provide compelling evidence that FTO-dependent m6A demethylation functions as a novel regulatory mechanism of RNA processing and plays a critical role in the regulation of adipogenesis.