Thidathip Wongsurawat

Quantitative model of R-loop forming structures reveals a novel level of RNA–DNA interactome complexity

R-loop is the structure co-transcriptionally formed between nascent RNA transcript and DNA template, leaving the non-transcribed DNA strand unpaired. This structure can be involved in the hyper-mutation and dsDNA breaks in mammalian immunoglobulin (Ig) genes, oncogenes and neurodegenerative disease related genes. R-loops have not been studied at the genome scale yet. To identify the R-loops, we developed a computational algorithm and mapped R-loop forming sequences (RLFS) onto 66 803 sequences defined by UCSC as ‘known’ genes. We found that ∼59% of these transcribed sequences contain at least one RLFS. We created R-loopDB (http://rloop.bii.a-star.edu.sg/), the database that collects all RLFS identified within over half of the human genes and links to the UCSC Genome Browser for information integration and visualisation across a variety of bioinformatics sources. We found that many oncogenes and tumour suppressors (e.g. Tp53, BRCA1, BRCA2, Kras and Ptprd) and neurodegenerative diseases related genes (e.g. ATM, Park2, Ptprd and GLDC) could be prone to significant R-loop formation. Our findings suggest that R-loops provide a novel level of RNA–DNA interactome complexity, playing key roles in gene expression controls, mutagenesis, recombination process, chromosomal rearrangement, alternative splicing, DNA-editing and epigenetic modifications. RLFSs could be used as a novel source of prospective therapeutic targets.

R-Loops in Proliferating Cells but Not in the Brain: Implications for AOA2 and Other Autosomal Recessive Ataxias

Disruption of the Setx gene, defective in ataxia oculomotor apraxia type 2 (AOA2) leads to the accumulation of DNA/RNA hybrids (R-loops), failure of meiotic recombination and infertility in mice. We report here the presence of R-loops in the testes from other autosomal recessive ataxia mouse models, which correlate with fertility in these disorders. R-loops were coincident in cells showing high basal levels of DNA double strand breaks and in those cells undergoing apoptosis. Depletion of Setx led to high basal levels of R-loops and these were enhanced further by DNA damage both in vitro and in vivo in tissues with proliferating cells. There was no evidence for accumulation of R-loops in the brains of mice where Setx, Atm, Tdp1 or Aptx genes were disrupted. These data provide further evidence for genome destabilization as a consequence of disrupted transcription in the presence of DNA double strand breaks arising during DNA replication or recombination. They also suggest that R-loop accumulation does not contribute to the neurodegenerative phenotype in these autosomal recessive ataxias.

ReproArrayGTS: a cDNA microarray for identification of reproduction-related genes in the giant tiger shrimp Penaeus monodon and characterization of a novel nuclear autoantigenic sperm protein (NASP) gene.

Expressed sequence tags (ESTs) were established from various tissues of the giant tiger shrimp (Penaeus monodon). To simultaneously examine expression patterns of a large number of transcripts in ovaries and testes of P. monodon, a cDNA microarray (ReproArray(GTS)) containing 4992 features amplified from cDNAs of ovary (1920) and testis (3072) EST libraries was constructed and subjected to high-throughput gene expression analysis in four different stages of ovarian development (previtellogenesis, vitellogenesis, early cortical rod and late cortical rod stages). Several transcripts were found to be differentially expressed during P. monodon ovarian development. Among many important reproduction-related genes with differential expression from microarray data, nuclear autoantigenic sperm protein (NASP) was further characterized by RACE-PCR. The full-length cDNA of P. monodon NASP (PmNASP) was 2126 bp in length containing an open reading frame (ORF) of 1812 bp corresponding to a deduced protein of 603 amino acids with 5? and 3?UTRs of 93 and 202 bp (excluding the poly A tail), respectively. Higher PmNASP transcript levels at later stages of ovarian development was consistently confirmed by quantitative real-time PCR. This study indicated that ReproArray(GTS) is effective for high-throughput screening of genes that play important roles in ovarian development of P. monodon.

Identification of testis-relevant genes using in silico analysis from testis ESTs and cDNA microarray in the black tiger shrimp (Penaeus monodon)

BackgroundPoor reproductive maturation of the black tiger shrimp (Penaeus monodon) in captivity is one of the serious threats to sustainability of the shrimp farming industry. Understanding molecular mechanisms governing reproductive maturation processes requires the fundamental knowledge of integrated expression profiles in gonads of this economically important species. In P. monodon, a non-model species for which the genome sequence is not available, expressed sequence tag (EST) and cDNA microarray analyses can help reveal important transcripts relevant to reproduction and facilitate functional characterization of transcripts with important roles in male reproductive development and maturation.ResultsIn this study, a conventional testis EST library was exploited to reveal novel transcripts. A total of 4,803 ESTs were unidirectionally sequenced and analyzed in silico using a customizable data analysis package, ESTplus. After sequence assembly, 2,702 unique sequences comprised of 424 contigs and 2,278 singletons were identified; of these, 1,133 sequences are homologous to genes with known functions. The sequences were further characterized according to gene ontology categories (41% biological process, 24% molecular function, 35% cellular component). Through comparison with EST libraries of other tissues of P. monodon, 1,579 transcripts found only in the testis cDNA library were identified. A total of 621 ESTs have not been identified in penaeid shrimp. Furthermore, cDNA microarray analysis revealed several ESTs homologous to testis-relevant genes were more preferentially expressed in testis than in ovary. Representatives of these transcripts, homologs of saposin (PmSap) and Dmc1 (PmDmc1), were further characterized by RACE-PCR. The more abundant expression levels in testis than ovary of PmSap and PmDmc1 were verified by quantitative real-time PCR in juveniles and wild broodstock of P. monodon.ConclusionsWithout a genome sequence, a combination of EST analysis and high-throughput cDNA microarray technology can be a useful integrated tool as an initial step towards the identification of transcripts with important biological functions. Identification and expression analysis of saposin and Dmc1 homologs demonstrate the power of these methods for characterizing functionally important genes in P. monodon.

QmRLFS-finder: a model, web server and stand-alone tool for prediction and analysis of R-loop forming sequences

The possible formation of three-stranded RNA and DNA hybrid structures (R-loops) in thousands of functionally important guanine-rich genic and inter-genic regions could suggest their involvement in transcriptional regulation and even development of diseases. Here, we introduce the first freely available R-loop prediction program called Quantitative Model of R-loop Forming Sequence (RLFS) finder (QmRLFS-finder), which predicts RLFSs in nucleic acid sequences based on experimentally supported structural models of RLFSs. QmRLFS-finder operates via a web server or a stand-alone command line tool. This tool identifies and visualizes RLFS coordinates from any natural or artificial DNA or RNA input sequences and creates standards-compliant output files for further annotation and analysis. QmRLFS-finder demonstrates highly accurate predictions of the detected RLFSs, proposing new perspective to further discoveries in R-loop biology, biotechnology and molecular therapy. QmRLFS-finder is freely available at http://rloop.bii.a-star.edu.sg/?pg=qmrlfs-finder.

Genomic Islands as a Marker to Differentiate between Clinical and Environmental Burkholderia pseudomallei

Burkholderia pseudomallei, as a saprophytic bacterium that can cause a severe sepsis disease named melioidosis, has preserved several extra genes in its genome for survival. The sequenced genome of the organism showed high diversity contributed mainly from genomic islands (GIs). Comparative genome hybridization (CGH) of 3 clinical and 2 environmental isolates, using whole genome microarrays based on B. pseudomallei K96243 genes, revealed a difference in the presence of genomic islands between clinical and environmental isolates. The largest GI, GI8, of B. pseudomallei was observed as a 2 sub-GI named GIs8.1 and 8.2 with distinguishable %GC content and unequal presence in the genome. GIs8.1, 8.2 and 15 were found to be more common in clinical isolates. A new GI, GI16c, was detected on chromosome 2. Presences of GIs8.1, 8.2, 15 and 16c were evaluated in 70 environmental and 64 clinical isolates using PCR assays. A combination of GIs8.1 and 16c (positivity of either GI) was detected in 70% of clinical isolates and 11.4% of environmental isolates (P<0.001). Using BALB/c mice model, no significant difference of time to mortality was observed between K96243 isolate and three isolates without GIs under evaluation (P>0.05). Some virulence genes located in the absent GIs and the difference of GIs seems to contribute less to bacterial virulence. The PCR detection of 2 GIs could be used as a cost effective and rapid tool to detect potentially virulent isolates that were contaminated in soil.

Characterization of BPSS1521 (bprD), a Regulator of Burkholderia pseudomallei Virulence Gene Expression in the Mouse Model

The Gram-negative saprophytic bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a severe infectious disease of both humans and animals. Severity of the disease is thought to be dependent on both the health status of the host, including diabetes mellitus and kidney disease, and bacterial-derived factors. To identify the bacterial factors important during an acute infection, gene expression profiles in the spleen, lung, and liver of BALB/c (Th2 prototype) and C57BL/6 mice (Th1 prototype) were determined using DNA microarrays. This analysis identified BPSS1521 (bprD), a predicted transcriptional regulator located in the type III secretion system (T3SS-3) operon, to be up regulated, specifically in C57BL/6 mice. BALB/c mice infected with a bprD mutant showed a shorter time to death and increased inflammation, as determined by histopathological analysis and enumeration of bacteria in the spleen. Elevated numbers of multinucleated giant cells (MNGCs), which is the hallmark of melioidosis, were detected in both the wild-type and the bprD mutants; a similar elevation occurs in melioidosis patients. One striking observation was the increased expression of BPSS1520 (bprC), located downstream of bprD, in the bprD mutant. BprC is a regulator of T6SS-1 that is required for the virulence of B. pseudomallei in murine infection models. Deletion of bprD led to the overexpression of bprC and a decreased time to death. bprD expression was elevated in C57BL/6 —as compared to BALB/c—mice, suggesting a role for BprD in the natural resistance of C57BL/6 mice to B. pseudomallei. Ultimately, this analysis using mice with different immune backgrounds may enhance our understanding of the outcomes of infection in a variety of models.

R-loopDB: a database for R-loop forming sequences (RLFS) and R-loops

R-loopDB (http://rloop.bii.a-star.edu.sg) was originally constructed as a collection of computationally predicted R-loop forming sequences (RLFSs) in the human genic regions. The renewed R-loopDB provides updates, improvements and new options, including access to recent experimental data. It includes genome-scale prediction of RLFSs for humans, six other animals and yeast. Using the extended quantitative model of RLFSs (QmRLFS), we significantly increased the number of RLFSs predicted in the human genes and identified RLFSs in other organism genomes. R-loopDB allows searching of RLFSs in the genes and in the 2 kb upstream and downstream flanking sequences of any gene. R-loopDB exploits the Ensembl gene annotation system, providing users with chromosome coordinates, sequences, gene and genomic data of the 1 565 795 RLFSs distributed in 121 056 genic or proximal gene regions of the covered organisms. It provides a comprehensive annotation of Ensembl RLFS-positive genes including 93 454 protein coding genes, 12 480 long non-coding RNA and 7 568 small non-coding RNA genes and 7 554 pseudogenes. Using new interface and genome viewers of R-loopDB, users can search the gene(s) in multiple species with keywords in a single query. R-loopDB provides tools to carry out comparative evolution and genome-scale analyses in R-loop biology.

Complete genomic and transcriptional landscape analysis using third-generation sequencing: a case study of Saccharomyces cerevisiae CEN.PK113-7D

Abstract Completion of eukaryal genomes can be difficult task with the highly repetitive sequences along the chromosomes and short read lengths of second-generation sequencing. Saccharomyces cerevisiae strain CEN.PK113-7D, widely used as a model organism and a cell factory, was selected for this study to demonstrate the superior capability of very long sequence reads for de novo genome assembly. We generated long reads using two common third-generation sequencing technologies (Oxford Nanopore Technology (ONT) and Pacific Biosciences (PacBio)) and used short reads obtained using Illumina sequencing for error correction. Assembly of the reads derived from all three technologies resulted in complete sequences for all 16 yeast chromosomes, as well as the mitochondrial chromosome, in one step. Further, we identified three types of DNA methylation (5mC, 4mC and 6mA). Comparison between the reference strain S288C and strain CEN.PK113-7D identified chromosomal rearrangements against a background of similar gene content between the two strains. We identified full-length transcripts through ONT direct RNA sequencing technology. This allows for the identification of transcriptional landscapes, including untranslated regions (UTRs) (5′ UTR and 3′ UTR) as well as differential gene expression quantification. About 91% of the predicted transcripts could be consistently detected across biological replicates grown either on glucose or ethanol. Direct RNA sequencing identified many polyadenylated non-coding RNAs, rRNAs, telomere-RNA, long non-coding RNA and antisense RNA. This work demonstrates a strategy to obtain complete genome sequences and transcriptional landscapes that can be applied to other eukaryal organisms.

Case of Microcephaly after Congenital Infection with Asian Lineage Zika Virus, Thailand

We sequenced the virus genomes from 3 pregnant women in Thailand with Zika virus diagnoses. All had infections with the Asian lineage. The woman infected at gestational week 9, and not those infected at weeks 20 and 24, had a fetus with microcephaly. Asian lineage Zika viruses can cause microcephaly.