Gökhan Karakülah

mESAdb: microRNA Expression and Sequence Analysis Database

microRNA expression and sequence analysis database (http://konulab.fen.bilkent.edu.tr/mirna/) (mESAdb) is a regularly updated database for the multivariate analysis of sequences and expression of microRNAs from multiple taxa. mESAdb is modular and has a user interface implemented in PHP and JavaScript and coupled with statistical analysis and visualization packages written for the R language. The database primarily comprises mature microRNA sequences and their target data, along with selected human, mouse and zebrafish expression data sets. mESAdb analysis modules allow (i) mining of microRNA expression data sets for subsets of microRNAs selected manually or by motif; (ii) pair-wise multivariate analysis of expression data sets within and between taxa; and (iii) association of microRNA subsets with annotation databases, HUGE Navigator, KEGG and GO. The use of existing and customized R packages facilitates future addition of data sets and analysis tools. Furthermore, the ability to upload and analyze user-specified data sets makes mESAdb an interactive and expandable analysis tool for microRNA sequence and expression data.

NRL-Regulated Transcriptome Dynamics of Developing Rod Photoreceptors.

Gene regulatory networks (GRNs) guiding differentiation of cell types and cell assemblies in the nervous system are poorly understood because of inherent complexities and interdependence of signaling pathways. Here, we report transcriptome dynamics of differentiating rod photoreceptors in the mammalian retina. Given that the transcription factor NRL determines rod cell fate, we performed expression profiling of developing NRL-positive (rods) and NRL-negative (S-cone-like) mouse photoreceptors. We identified a large-scale, sharp transition in the transcriptome landscape between postnatal days 6 and 10 concordant with rod morphogenesis. Rod-specific temporal DNA methylation corroborated gene expression patterns. De novo assembly and alternative splicing analyses revealed previously unannotated rod-enriched transcripts and the role of NRL in transcript maturation. Furthermore, we defined the relationship of NRL with other transcriptional regulators and downstream cognate effectors. Our studies provide the framework for comprehensive system-level analysis of the GRN underlying the development of a single sensory neuron, the rod photoreceptor.

Next generation sequencing technology and genomewide data analysis: Perspectives for retinal research.

The advent of high throughput next generation sequencing (NGS) has accelerated the pace of discovery of disease-associated genetic variants and genomewide profiling of expressed sequences and epigenetic marks, thereby permitting systems-based analyses of ocular development and disease. Rapid evolution of NGS and associated methodologies presents significant challenges in acquisition, management, and analysis of large data sets and for extracting biologically or clinically relevant information. Here we illustrate the basic design of commonly used NGS-based methods, specifically whole exome sequencing, transcriptome, and epigenome profiling, and provide recommendations for data analyses. We briefly discuss systems biology approaches for integrating multiple data sets to elucidate gene regulatory or disease networks. While we provide examples from the retina, the NGS guidelines reviewed here are applicable to other tissues/cell types as well.

MicroRNA exocytosis by large dense-core vesicle fusion

Neurotransmitters and peptide hormones are secreted into outside the cell by a vesicle fusion process. Although non-coding RNA (ncRNA) that include microRNA (miRNA) regulates gene expression inside the cell where they are transcribed, extracellular miRNA has been recently discovered outside the cells, proposing that miRNA might be released to participate in cell-to-cell communication. Despite its importance of extracellular miRNA, the molecular mechanisms by which miRNA can be stored in vesicles and released by vesicle fusion remain enigmatic. Using next-generation sequencing, vesicle purification techniques, and synthetic neurotransmission, we observe that large dense-core vesicles (LDCVs) contain a variety of miRNAs including miR-375. Furthermore, miRNA exocytosis is mediated by the SNARE complex and accelerated by Ca2+. Our results suggest that miRNA can be a novel neuromodulator that can be stored in vesicles and released by vesicle fusion together with classical neurotransmitters.

Regulation of Noncoding Transcriptome in Developing Photoreceptors by Rod Differentiation Factor NRL

Purpose Transcriptome analysis by next generation sequencing allows qualitative and quantitative profiling of expression patterns associated with development and disease. However, most transcribed sequences do not encode proteins, and little is known about the functional relevance of noncoding (nc) transcriptome in neuronal subtypes. The goal of this study was to perform a comprehensive analysis of long noncoding (lncRNAs) and antisense (asRNAs) RNAs expressed in mouse retinal photoreceptors. Methods Transcriptomic profiles were generated at six developmental time points from flow-sorted Nrlp-GFP (rods) and Nrlp-GFP;Nrl−/− (S-cone like) mouse photoreceptors. Bioinformatic analysis was performed to identify novel noncoding transcripts and assess their regulation by rod differentiation factor neural retina leucine zipper (NRL). In situ hybridization (ISH) was used for validation and cellular localization. Results NcRNA profiles demonstrated dynamic yet specific expression signature and coexpression clusters during rod development. In addition to currently annotated 586 lncRNAs and 454 asRNAs, we identified 1037 lncRNAs and 243 asRNAs by de novo assembly. Of these, 119 lncRNAs showed altered expression in the absence of NRL and included NRL binding sites in their promoter/enhancer regions. ISH studies validated the expression of 24 lncRNAs (including 12 previously unannotated) and 4 asRNAs in photoreceptors. Coexpression analysis demonstrated 63 functional modules and 209 significant antisense-gene correlations, allowing us to predict possible role of these lncRNAs in rods. Conclusions Our studies reveal coregulation of coding and noncoding transcripts in rod photoreceptors by NRL and establish the framework for deciphering the function of ncRNAs during retinal development.

PlanTEnrichment: A tool for enrichment analysis of transposable elements in plants

Transposable elements (TEs) are mobile DNA sequences that play roles in gene regulation, and have a potential to influence the expression of nearby genes by functioning as cis-regulatory sequences. However, bioinformatics tools facilitating analysis of the associations between TEs and nearby genes in plants are still lacking. We therefore reanalyzed the comprehensive annotation data of gene models and TEs of 11 plant species available in Ensembl Plants database, and built an up-to-date, unique tool called PlanTEnrichment, enabling enrichment analysis of TEs located within the upstream regions of a given gene list. PlanTEnrichment takes, for example, a group of differentially expressed genes under a particular biological condition as input and returns the list of TEs associated with those genes, along with their calculated enrichment scores and statistical significances. PlanTEnrichment is freely available at http://tools.ibg.deu.edu.tr/plantenrichment/ and is likely to substantially enhance our understanding of the role of TEs in diverse biological processes.

Differential Expression of Hyperhydricity Responsive Peach MicroRNAs

Hyperhydricity is a syndrome that causes morpho-physiological malformations in tissue culture plantlets. Micro-RNAs (miRNA) are small non-coding RNAs that play important regulatory roles in plant development, stress response, and adaptation to environmental conditions. In this study, differential expression analysis indicated that miRNAs play an underlying role in the responses to the hyperhydricity syndrome in peach Prunus persica (L.) leaves. 24 known and three novel potential miRNAs were characterized in hyperhydric and non-hyperhydric transcriptome libraries. The miRNA-target transcript analyses indicated that transport, plant cuticle development, intracellular part, and stress response are regulated by miRNAs in hyperhydric leaves. It is also suggested that miR5021 and miRnovel2 might play critical regulatory roles in hyperhydricity regarding miRNA-based response to stress. This study went one step further to advance understanding of molecular miRNA-based regulatory mechanisms regarding responses to hyperhydricity in peach.

Barley Long Non-Coding RNAs and Their Tissue-Specific Co-expression Pattern with Coding-Transcripts

Long non-coding RNAs (lncRNA) with non-protein or small peptide-coding potential transcripts are emerging regulatory molecules. With the advent of next-generation sequencing technologies and novel bioinformatics tools, a tremendous number of lncRNAs has been identified in several plant species. Recent reports demonstrated roles of plant lncRNAs such as development and environmental response. Here, we reported a genome-wide discovery of ~8,000 barley lncRNAs and measured their expression pattern upon excessive boron (B) treatment. According to the tissue-based comparison, leaves have a greater number of B-responsive differentially expressed lncRNAs than the root. Functional annotation of the coding transcripts, which were co-expressed with lncRNAs, revealed that molecular function of the ion transport, establishment of localization, and response to stimulus significantly enriched only in the leaf. On the other hand, 32 barley endogenous target mimics (eTM) as lncRNAs, which potentially decoy the transcriptional suppression activity of 18 miRNAs, were obtained. Presented data including identification, expression measurement, and functional characterization of barley lncRNAs suggest that B-stress response might also be regulated by lncRNA expression via cooperative interaction of miRNA-eTM-coding target transcript modules.

RTFAdb: A database of computationally predicted associations between retrotransposons and transcription factors in the human and mouse genomes

In recent years, retrotransposons have gained increasing attention as a source of binding motifs for transcription factors (TFs). Despite the substantial roles of these mobile genetic elements in the regulation of gene expression, a comprehensive resource enabling the investigation of retrotransposon species that are bound by TFs is still lacking. Herein, I introduce for the first time a novel database called RTFAdb, which allows exploring computationally predicted associations between retrotransposons and TFs in diverse cell lines and tissues of human and mouse. My database, using over 3.000 TF ChIP-seq binding profiles collected from human and mouse samples, makes possible searching more than 1.500 retrotransposon species in the binding sites of a total of 596 TFs. RTFAdb is freely available at http://tools.ibg.deu.edu.tr/rtfa/ and has the potential to offer novel insights into mammalian transcriptional networks by providing an additional layer of information regarding the regulatory roles of retrotransposons.

C-Reactive Protein and Platelet-Lymphocyte Ratio as Potential Tumor Markers in Low-Alpha-Fetoprotein Hepatocellular Carcinoma

The hepatocellular carcinoma (HCC) tumor marker alpha-fetoprotein (AFP) is only elevated in about half of the HCC patients, limiting its usefulness in following the effects of therapy or screening. New markers are needed. It has been previously noted that the inflammation markers C-reactive protein (CRP) and platelet-lymphocyte ratio (PLR) are prognostically important and may reflect HCC aggressiveness. We therefore examined these 2 markers in a low-AFP HCC cohort and found that for HCCs > 2 cm, both markers significantly rise with an increasing maximum tumor diameter (MTD). We calculated the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and Youden index value for each marker, and their area-under-the-curve values for each MTD group. Patients were dichotomized into 2 groups based on the CRP and PLR from the receiver-operating characteristic curve analysis. In the logistic regression models of the 4 different MTD patient groups, CRP and PLR levels were statistically significant to estimate MTD in univariate logistic regression models of MTD groups > 2 cm. CRP and PLR were then combined, and the combination was statistically significant to estimate MTD groups of 3-, 4-, and 5-cm cutoffs. CRP and PLR thus have potential as tumor markers for low-AFP HCC patients, and possibly for screening.