Shuhui Song

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.

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.

How many human genes can be defined as housekeeping with current expression data

BackgroundHousekeeping (HK) genes are ubiquitously expressed in all tissue/cell types and constitute a basal transcriptome for the maintenance of basic cellular functions. Partitioning transcriptomes into HK and tissue-specific (TS) genes relatively is fundamental for studying gene expression and cellular differentiation. Although many studies have aimed at large-scale and thorough categorization of human HK genes, a meaningful consensus has yet to be reached.ResultsWe collected two latest gene expression datasets (both EST and microarray data) from public databases and analyzed the gene expression profiles in 18 human tissues that have been well-documented by both two data types. Benchmarked by a manually-curated HK gene collection (HK408), we demonstrated that present data from EST sampling was far from saturated, and the inadequacy has limited the gene detectability and our understanding of TS expressions. Due to a likely over-stringent threshold, microarray data showed higher false negative rate compared with EST data, leading to a significant underestimation of HK genes. Based on EST data, we found that 40.0% of the currently annotated human genes were universally expressed in at least 16 of 18 tissues, as compared to only 5.1% specifically expressed in a single tissue. Our current EST-based estimate on human HK genes ranged from 3,140 to 6,909 in number, a ten-fold increase in comparison with previous microarray-based estimates.ConclusionWe concluded that a significant fraction of human genes, at least in the currently annotated data depositories, was broadly expressed. Our understanding of tissue-specific expression was still preliminary and required much more large-scale and high-quality transcriptomic data in future studies. The new HK gene list categorized in this study will be useful for genome-wide analyses on structural and functional features of HK genes.

Proteomic profiling of rice embryos from a hybrid rice cultivar and its parental lines.

Elite rice hybrids, when compared to their parental lines, exhibit increased yield and other favorable agronomical traits, such as pathogen‐ and water‐stress resistances, which are described as heterosis, and the molecular mechanism of heterosis remains to be elucidated. Since genomic sequences of the paternal (9311) and maternal lines (P64S) of a major rice hybrid variety LYP9 (Liang‐You‐Pei‐Jiu) were acquired recently, we performed a proteomic study on mature embryos of this hybrid triad based on 2‐DE and MALDI‐TOF MS analyses, and identified 54 differentially expressed proteins involved in major biological processes including nutrient reservoir, response to stress, and metabolism. We observed that most of the storage proteins exhibit overdominance and stress‐induced proteins display additivity. We compared proteomic results with transcriptomic data generated from the same embryo samples and found 28 candidate heterosis‐associated genes shared by the two datasets. We further traced back to their genomic structures including protein‐coding and regulatory regions and found that most of these genes have multiple copies in rice genomes as paralogous genes. Based on alignment of coding and regulation regions, we found that most of the differentially expressed genes at both protein and RNA levels are recent gene duplicates (paralogous genes) with relative little difference in protein‐coding regions between orthologous genes (between genes of the two parental genomes) as compared to regulatory regions that harbor numerous indels and base substitutions.

wapRNA: a web-based application for the processing of RNA sequences.

SUMMARY mRNA/miRNA-seq technology is becoming the leading technology to globally profile gene expression and elucidate the transcriptional regulation mechanisms in living cells. Although there are many tools available for analyzing RNA-seq data, few of them are available as easy accessible online web tools for processing both mRNA and miRNA data for the RNA-seq based user community. As such, we have developed a comprehensive web application tool for processing mRNA-seq and miRNA-seq data. Our web tool wapRNA includes four different modules: mRNA-seq and miRNA-seq sequenced from SOLiD or Solexa platform and all the modules were tested on previously published experimental data. We accept raw sequence data with an optional reads filter, followed by mapping and gene annotation or miRNA prediction. wapRNA also integrates downstream functional analyses such as Gene Ontology, KEGG pathway, miRNA targets prediction and comparison of genes or miRNAs different expression in different samples. Moreover, we provide the executable packages for installation on users local server. AVAILABILITY wapRNA is freely available for use at http://waprna.big.ac.cn. The executable packages and the instruction for installation can be downloaded from our web site. CONTACT husn@big.ac.cn; songshh@big.ac.cn. SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Redox-active quinones induces genome-wide DNA methylation changes by an iron-mediated and Tet-dependent mechanism

DNA methylation has been proven to be a critical epigenetic mark important for various cellular processes. Here, we report that redox-active quinones, a ubiquitous class of chemicals found in natural products, cancer therapeutics and environment, stimulate the conversion of 5mC to 5hmC in vivo, and increase 5hmC in 5751 genes in cells. 5hmC increase is associated with significantly altered gene expression of 3414 genes. Interestingly, in quinone-treated cells, labile iron-sensitive protein ferritin light chain showed a significant increase at both mRNA and protein levels indicating a role of iron regulation in stimulating Tet-mediated 5mC oxidation. Consistently, the deprivation of cellular labile iron using specific chelator blocked the 5hmC increase, and a delivery of labile iron increased the 5hmC level. Moreover, both Tet1/Tet2 knockout and dimethyloxalylglycine-induced Tet inhibition diminished the 5hmC increase. These results suggest an iron-regulated Tet-dependent DNA demethylation mechanism mediated by redox-active biomolecules.

Integrated analysis of miRNA and mRNA expression profiles in response to Cd exposure in rice seedlings

BackgroundIndependent transcriptome profile analyses of miRNAs or mRNAs under conditions of cadmium (Cd) stress have been widely reported in plants. However, a combined analysis of sRNA sequencing expression data with miRNA target expression data to infer the relative activities of miRNAs that regulate gene expression changes resulting from Cd stress has not been reported in rice. To elucidate the roles played by miRNAs in the regulation of changes in gene expression in response to Cd stress in rice (Oryza sativa L.), we simultaneously characterized changes in the miRNA and mRNA profiles following treatment with Cd.ResultsA total of 163 miRNAs and 2,574 mRNAs were identified to be differentially expressed under Cd stress, and the changes in the gene expression profile in the shoot were distinct from those in the root. At the miRNA level, 141 known miRNAs belonging to 48 families, and 39 known miRNAs in 23 families were identified to be differentially expressed in the root and shoot, respectively. In addition, we identified eight new miRNA candidates from the root and five from the shoot that were differentially expressed in response to Cd treatment. For the mRNAs, we identified 1,044 genes in the root and 448 genes in the shoot that were up-regulated, while 572 and 645 genes were down-regulated in the root and shoot, respectively. GO and KEGG enrichment analyses showed that genes encoding secondary, metabolite synthases, signaling molecules, and ABC transporters were significantly enriched in the root, while only ribosomal protein and carotenoid biosynthesis genes were significantly enriched in the shoot. Then 10 known miRNA-mRNA interaction pairs and six new candidate ones, that showed the opposite expression patterns, were identified by aligning our two datasets against online databases and by using the UEA sRNA toolkit respectively.ConclusionsThis study is the first to use high throughput DNA sequencing to simultaneously detect changes in miRNA and mRNA expression patterns in the root and shoot in response to Cd treatment. These integrated high-throughput expression data provide a valuable resource to examine global genome expression changes in response to Cd treatment and how these are regulated by miRNAs.

Transcriptomic profiling of mature embryo from an elite super-hybrid rice LYP9 and its parental lines.

BackgroundThe mature embryo of rice (Oryza sativa, L.) is a synchronized and integrated tissue mass laying the foundation at molecular level for its growth, development, and differentiation toward a developing and ultimately a mature plant. We carried out an EST (expressed-sequence-tags)-based transcriptomic study, aiming at gaining molecular insights into embryonic development of a rice hybrid triad–an elite hybrid rice LYP9 and its parental lines (93-11 and PA64s)–and possible relatedness to heterosis.ResultsWe generated 27,566 high-quality ESTs from cDNA libraries made from mature rice embryos. We classified these ESTs into 7,557 unigenes (2,511 contigs and 5,046 singletons) and 7,250 (95.9%) of them were annotated. We noticed that the high-abundance genes in mature rice embryos belong to two major functional categories, stress-tolerance and preparation-for-development, and we also identified 191 differentially-expressed genes (General Chi-squared test, P-value <= 0.05) between LYP9 and its parental lines, representing typical expression patterns including over-dominance, high- and low-parent dominance, additivity, and under-dominance. In LYP9, the majority of embryo-associated genes were found not only abundantly and specifically enriched but also significantly up-regulated.ConclusionOur results suggested that massively strengthening tissue-(or stage-) characteristic functions may contribute to heterosis rather than a few simple mechanistic explanations at the individual gene level. In addition, the large collection of rice embryonic ESTs provides significant amount of data for future comparative analyses on plant development, especially for the important crops of the grass family.

Mechanism Analysis of Acid Tolerance Response of Bifidobacterium longum subsp. longum BBMN 68 by Gene Expression Profile Using RNA-Sequencing

To analyze the mechanism of the acid tolerance response (ATR) in Bifidobacterium longum subsp. longum BBMN68, we optimized the acid-adaptation condition to stimulate ATR effectively and analyzed the change of gene expression profile after acid-adaptation using high-throughput RNA-Seq. After acid-adaptation at pH 4.5 for 2 hours, the survival rate of BBMN68 at lethal pH 3.5 for 120 min was increased by 70 fold and the expression of 293 genes were upregulated by more than 2 fold, and 245 genes were downregulated by more than 2 fold. Gene expression profiling of ATR in BBMN68 suggested that, when the bacteria faced acid stress, the cells strengthened the integrity of cell wall and changed the permeability of membrane to keep the H+ from entering. Once the H+ entered the cytoplasm, the cells showed four main responses: First, the F0F1-ATPase system was initiated to discharge H+. Second, the ability to produce NH3 by cysteine-cystathionine-cycle was strengthened to neutralize excess H+. Third, the cells started NER-UVR and NER-VSR systems to minimize the damage to DNA and upregulated HtpX, IbpA, and γ-glutamylcysteine production to protect proteins against damage. Fourth, the cells initiated global response signals ((p)ppGpp, polyP, and Sec-SRP) to bring the whole cell into a state of response to the stress. The cells also secreted the quorum sensing signal (AI-2) to communicate between intraspecies cells by the cellular signal system, such as two-component systems, to improve the overall survival rate. Besides, the cells varied the pathways of producing energy by shifting to BCAA metabolism and enhanced the ability to utilize sugar to supply sufficient energy for the operation of the mechanism mentioned above. Based on these reults, it was inferred that, during industrial applications, the acid resistance of bifidobacteria could be improved by adding BCAA, γ-glutamylcysteine, cysteine, and cystathionine into the acid-stress environment.

Differential gene expression in an elite hybrid rice cultivar (Oryza sativa, L) and its parental lines based on SAGE data

BackgroundIt was proposed that differentially-expressed genes, aside from genetic variations affecting protein processing and functioning, between hybrid and its parents provide essential candidates for studying heterosis or hybrid vigor. Based our serial analysis of gene expression (SAGE) data from an elite Chinese super-hybrid rice (LYP9) and its parental cultivars (93-11 and PA64s) in three major tissue types (leaves, roots and panicles) at different developmental stages, we analyzed the transcriptome and looked for candidate genes related to rice heterosis.ResultsBy using an improved strategy of tag-to-gene mapping and two recently annotated genome assemblies (93-11 and PA64s), we identified 10,268 additional high-quality tags, reaching a grand total of 20,595 together with our previous result. We further detected 8.5% and 5.9% physically-mapped genes that are differentially-expressed among the triad (in at least one of the three stages) with P-values less than 0.05 and 0.01, respectively. These genes distributed in 12 major gene expression patterns; among them, 406 up-regulated and 469 down-regulated genes (P < 0.05) were observed. Functional annotations on the identified genes highlighted the conclusion that up-regulated genes (some of them are known enzymes) in hybrid are mostly related to enhancing carbon assimilation in leaves and roots. In addition, we detected a group of up-regulated genes related to male sterility and 442 down-regulated genes related to signal transduction and protein processing, which may be responsible for rice heterosis.ConclusionWe improved tag-to-gene mapping strategy by combining information from transcript sequences and rice genome annotation, and obtained a more comprehensive view on genes that related to rice heterosis. The candidates for heterosis-related genes among different genotypes provided new avenue for exploring the molecular mechanism underlying heterosis.