Knockout of the non-essential gene SUGCT creates diet-linked, age-related microbiome disbalance with a diabetes-like metabolic syndrome phenotype.

SUGCT (C7orf10) is a mitochondrial enzyme that synthesizes glutaryl-CoA from glutarate in tryptophan and lysine catabolism, but it has not been studied in vivo. Although mutations in Sugct lead to Glutaric Aciduria Type 3 disease in humans, patients remain largely asymptomatic despite high levels of glutarate in the urine. To study the disease mechanism, we generated SugctKO mice and uncovered imbalanced lipid and acylcarnitine metabolism in kidney in addition to changes in the gut microbiome. After SugctKO mice were treated with antibiotics, metabolites were comparable to WT, indicating that the microbiome affects metabolism in SugctKO mice. SUGCT loss of function contributes to gut microbiota dysbiosis, leading to age-dependent pathological changes in kidney, liver, and adipose tissue. This is associated with an obesity-related phenotype that is accompanied by lipid accumulation in kidney and liver, as well as "crown-like" structures in adipocytes. Furthermore, we show that the SugctKO kidney pathology is accelerated and exacerbated by a high-lysine diet. Our study highlights the importance of non-essential genes with no readily detectable early phenotype, but with substantial contributions to the development of age-related pathologies, which result from an interplay between genetic background, microbiome, and diet in the health of mammals.

Toward Allosterically Increased Catalytic Activity of Insulin-Degrading Enzyme against Amyloid Peptides.

The physiological role of insulin-degrading enzyme (IDE) in the intracytosolic clearance of amyloid ß (Aß) and other amyloid-like peptides supports a hypothesis that human IDE hyperactivation could be therapeutically beneficial for the treatment of late-onset Alzheimer's disease (AD). The major challenge standing in the way of this goal is increasing the specific catalytic activity of IDE against the Aß substrate. There were previous indications that the allosteric mode of IDE activity regulation could potentially provide a highly specific path toward degradation of amyloid-like peptides, while not dramatically affecting activity against other substrates. Recently developed theoretical concepts are used here to explore potential allosteric modulation of the IDE activity as a result of single-residue mutations. Five candidates are selected for experimental follow-up and allosteric free energy calculations: Ser137Ala, Lys396Ala, Asp426Ala, Phe807Ala, and Lys898Ala. Our experiments show that three mutations (Ser137Ala, Phe807Ala, and Lys898Ala) decrease the Km of the Aß substrate. Mutation Lys898Ala results in increased catalytic activity of IDE; on the other hand, Lys364Ala does not change the activity and Asp426Ala diminishes it. Quantifying effects of mutations in terms of allosteric free energy, we show that favorable mutations lead to stabilization of the catalytic sites and other function-relevant distal sites as well as increased dynamics of the IDE-N and IDE-C halves that allow efficient substrate entrance and cleavage. A possibility for intramolecular upregulation of IDE activity against amyloid peptides via allosteric mutations calls for further investigations in this direction. Ultimately, we are hopeful it will lead to the development of IDE-based drugs for the treatment of the late-onset form of AD characterized by an overall impairment of Aß clearance.

Genome-wide analysis of mRNAs associated with mouse peroxisomes.

BACKGROUND: RNA is often targeted to be localized to the specific subcellular compartments. Specific localization of mRNA is believed to be an important mechanism for targeting their protein products to the locations, where their function is required. RESULTS: In this study we performed the genome wide transcriptome analysis of peroxisome preparations from the mouse liver using microarrays. We demonstrate that RNA is absent inside peroxisomes, however it is associated at their exterior via the noncovalent contacts with the membrane proteins. We detect enrichment of specific sets of transcripts in two preparations of peroxisomes, purified with different degrees of stringency. Importantly, among these were mRNAs encoding bona fide peroxisomal proteins, such as peroxins and peroxisomal matrix enzymes involved in beta-oxidation of fatty acids and bile acid biosynthesis. The top-most enriched mRNA, whose association with peroxisomes we confirm microscopically was Hmgcs1, encoding 3-hydroxy-3-methylglutaryl-CoA synthase, a crucial enzyme of cholesterol biosynthesis pathway. We observed significant representation of mRNAs encoding mitochondrial and secreted proteins in the peroxisomal fractions. CONCLUSIONS: This is a pioneer genome-wide study of localization of mRNAs to peroxisomes that provides foundation for more detailed dissection of mechanisms of RNA targeting to subcellular compartments.

Tysnd1 deficiency in mice interferes with the peroxisomal localization of PTS2 enzymes, causing lipid metabolic abnormalities and male infertility.

Peroxisomes are subcellular organelles involved in lipid metabolic processes, including those of very-long-chain fatty acids and branched-chain fatty acids, among others. Peroxisome matrix proteins are synthesized in the cytoplasm. Targeting signals (PTS or peroxisomal targeting signal) at the C-terminus (PTS1) or N-terminus (PTS2) of peroxisomal matrix proteins mediate their import into the organelle. In the case of PTS2-containing proteins, the PTS2 signal is cleaved from the protein when transported into peroxisomes. The functional mechanism of PTS2 processing, however, is poorly understood. Previously we identified Tysnd1 (Trypsin domain containing 1) and biochemically characterized it as a peroxisomal cysteine endopeptidase that directly processes PTS2-containing prethiolase Acaa1 and PTS1-containing Acox1, Hsd17b4, and ScpX. The latter three enzymes are crucial components of the very-long-chain fatty acids ß-oxidation pathway. To clarify the in vivo functions and physiological role of Tysnd1, we analyzed the phenotype of Tysnd1(-/-) mice. Male Tysnd1(-/-) mice are infertile, and the epididymal sperms lack the acrosomal cap. These phenotypic features are most likely the result of changes in the molecular species composition of choline and ethanolamine plasmalogens. Tysnd1(-/-) mice also developed liver dysfunctions when the phytanic acid precursor phytol was orally administered. Phyh and Agps are known PTS2-containing proteins, but were identified as novel Tysnd1 substrates. Loss of Tysnd1 interferes with the peroxisomal localization of Acaa1, Phyh, and Agps, which might cause the mild Zellweger syndrome spectrum-resembling phenotypes. Our data established that peroxisomal processing protease Tysnd1 is necessary to mediate the physiological functions of PTS2-containing substrates.

HOXD-AS1 is a novel lncRNA encoded in HOXD cluster and a marker of neuroblastoma progression revealed via integrative analysis of noncoding transcriptome.

BACKGROUND: Long noncoding RNAs (lncRNAs) constitute a major, but poorly characterized part of human transcriptome. Recent evidence indicates that many lncRNAs are involved in cancer and can be used as predictive and prognostic biomarkers. Significant fraction of lncRNAs is represented on widely used microarray platforms, however they have usually been ignored in cancer studies. RESULTS: We developed a computational pipeline to annotate lncRNAs on popular Affymetrix U133 microarrays, creating a resource allowing measurement of expression of 1581 lncRNAs. This resource can be utilized to interrogate existing microarray datasets for various lncRNA studies. We found that these lncRNAs fall into three distinct classes according to their statistical distribution by length. Remarkably, these three classes of lncRNAs were co-localized with protein coding genes exhibiting distinct gene ontology groups. This annotation was applied to microarray analysis which identified a 159 lncRNA signature that discriminates between localized and metastatic stages of neuroblastoma. Analysis of an independent patient cohort revealed that this signature differentiates also relapsing from non-relapsing primary tumors. This is the first example of the signature developed via the analysis of expression of lncRNAs solely. One of these lncRNAs, termed HOXD-AS1, is encoded in HOXD cluster. HOXD-AS1 is evolutionary conserved among hominids and has all bona fide features of a gene. Studying retinoid acid (RA) response of SH-SY5Y cell line, a model of human metastatic neuroblastoma, we found that HOXD-AS1 is a subject to morphogenic regulation, is activated by PI3K/Akt pathway and itself is involved in control of RA-induced cell differentiation. Knock-down experiments revealed that HOXD-AS1 controls expression levels of clinically significant protein-coding genes involved in angiogenesis and inflammation, the hallmarks of metastatic cancer. CONCLUSIONS: Our findings greatly extend the number of noncoding RNAs functionally implicated in tumor development and patient treatment and highlight their role as potential prognostic biomarkers of neuroblastomas.

ALEX1 suppresses colony formation ability of human colorectal carcinoma cell lines.

Arm protein lost in epithelial cancers, on chromosome X (ALEX; also known as armadillo repeat containing, X-linked [ARMCX]) is a novel subgroup within the armadillo (ARM) family, which has several ARM repeat domains. The biological function of classical ARM family members such as ß-catenin is well understood, but that of the ALEX/ARMCX family members is largely unknown. Here we evaluate the effects of ALEX1 overexpression on in vitro colony formation ability and expression of ALEX1 mRNA in human colorectal tumor. Overexpression of ALEX1 suppressed the anchorage-dependent and -independent colony formation of human colorectal carcinoma cell lines by the study of stable clones of HCT116 cells expressing ALEX1 protein. Bisulfite genomic sequencing revealed that the promoter region of ALEX1 gene was highly methylated in both HCT116 and SW480 cells in comparison with PANC-1 and MCF-7 cells, which express endogenous ALEX1 mRNA, indicating the capability of promoter methylation to silence ALEX1 gene in HCT116 and SW480 cells. Our current findings suggest that overexpression of ALEX1 play a negative role in human colorectal tumorigenesis.

Identification of nucleotide patterns enriched in secreted RNAs as putative cis-acting elements targeting them to exosome nano-vesicles.

BACKGROUND: Exosomes are nanoscale membrane vesicles released by most cells. They are postulated to be involved in cell-cell communication and genetic reprogramming of their target cells. In addition to proteins and lipids, they release RNA molecules many of which are not present in the donor cells implying a highly selective mode of their packaging into these vesicles. Sequence motifs targeting RNA to the vesicles are currently unknown. RESULTS: Ab initio approach was applied for computational identification of potential RNA secretory motifs in the primary sequences of exosome-enriched RNAs (eRNAs). Exhaustive motif analysis for the first time revealed unique sequence features of eRNAs. We discovered multiple linear motifs specifically enriched in secreted RNAs. Their potential function as cis-acting elements targeting RNAs to exosomes is proposed. The motifs co-localized in the same transcripts suggesting combinatorial organization of these secretory signals. We investigated associations of the discovered motifs with other RNA parameters. Secreted RNAs were found to have almost twice shorter half-life times on average, in comparison with cytoplasmic RNAs, and the occurrence of some eRNA-specific motifs significantly correlated with this eRNA feature. Also, we found that eRNAs are highly enriched in long noncoding RNAs. CONCLUSIONS: Secreted RNAs share specific sequence motifs that may potentially function as cis-acting elements targeting RNAs to exosomes. Discovery of these motifs will be useful for our understanding the roles of eRNAs in cell-cell communication and genetic reprogramming of the target cells. It will also facilitate nano-scale vesicle engineering and selective targeting of RNAs of interest to these vesicles for gene therapy purposes.

Human Arm protein lost in epithelial cancers, on chromosome X 1 (ALEX1) gene is transcriptionally regulated by CREB and Wnt/beta-catenin signaling.

The aberrant activation of Wnt signaling is a key process in colorectal tumorigenesis. Canonical Wnt signaling controls transcription of target genes via beta-catenin and T-cell factor/lymphoid enhancer factor family transcription factor complex. Arm protein lost in epithelial cancers, on chromosome X 1 (ALEX1) is a novel member of the Armadillo family which has two Armadillo repeats as opposed to more than six repeats in the classical Armadillo family members. Here we examine cis-regulatory elements and trans-acting factors involved in the transcriptional regulation of the ALEX1 gene. Site-directed mutations of a cyclic AMP response element (CRE) and an E-box impaired the basal activity of human ALEX1 promoter in colorectal and pancreatic cancer cell lines. Moreover, overexpression of CRE-binding protein (CREB) increased the ALEX1 promoter activity in these cell lines, whereas knockdown of CREB expression decreased the expression level of ALEX1 mRNA. Interestingly, luciferase reporter analysis and quantitative real-time RT-PCR demonstrated that the ALEX1 promoter was up-regulated in a CRE-dependent manner by continuous activation of Wnt/beta-catenin signaling induced by a glycogen synthase kinase-3 inhibitor and overexpression of beta-catenin. These results indicate that the CRE and E-box sites are essential cis-regulatory elements for ALEX1 promoter activity, and ALEX1 expression is regulated by CREB and Wntk/beta-catenin signaling.

Predicted mouse peroxisome-targeted proteins and their actual subcellular locations.

BACKGROUND: The import of most intraperoxisomal proteins is mediated by peroxisome targeting signals at their C-termini (PTS1) or N-terminal regions (PTS2). Both signals have been integrated in subcellular location prediction programs. However their present performance, particularly of PTS2-targeting did not seem fitting for large-scale screening of sequences. RESULTS: We modified an earlier reported PTS1 screening method to identify PTS2-containing mouse candidates using a combination of computational and manual annotation. For rapid confirmation of five new PTS2- and two previously identified PTS1-containing candidates we developed the new cell line CHO-perRed which stably expresses the peroxisomal marker dsRed-PTS1. Using CHO-perRed we confirmed the peroxisomal localization of PTS1-targeted candidate Zadh2. Preliminary characterization of Zadh2 expression suggested non-PPARalpha mediated activation. Notably, none of the PTS2 candidates located to peroxisomes. CONCLUSION: In a few cases the PTS may oscillate from "silent" to "functional" depending on its surface accessibility indicating the potential for context-dependent conditional subcellular sorting. Overall, PTS2-targeting predictions are unlikely to improve without generation and integration of new experimental data from location proteomics, protein structures and quantitative Pex7 PTS2 peptide binding assays.

Insulin-Degrading Enzyme in the Fight against Alzheimer's Disease.

After decades of research and clinical trials there is still no cure for Alzheimer's disease (AD). While impaired clearance of amyloid beta (Aß) peptides is considered as one of the major causes of AD, it was recently complemented by a potential role of other toxic amyloidogenic species. Insulin-degrading enzyme (IDE) is the proteolytic culprit of various ß-forming peptides, both extracellular and intracellular. On the basis of demonstrated allosteric activation of IDE against Aß, it is possible to propose a new strategy for the targeted IDE-based cleansing of different toxic aggregation-prone peptides. Consequently, specific allosteric activation of IDE coupled with state-of-the-art compound delivery and CRISP-Cas9 technique of transgene insertion can be instrumental in the fight against AD and related neurodegenerative maladies.

A novel community driven software for functional enrichment analysis of extracellular vesicles data.

Bioinformatics tools are imperative for the in depth analysis of heterogeneous high-throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture "what the community needs in a tool". Here, we describe a novel community-driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines.

Sequence-based discovery of the human and rodent peroxisomal proteome.

BACKGROUND: Peroxisomes are metabolic organelles present in virtually all eukaryotic cells. They contain enzymes involved in hydrogen peroxide-based respiration and lipid metabolism. At present, only a small number of peroxisomal enzymes that are associated with oxidative stress response and metabolic disorders have been characterised biochemically. Therefore, we devised a sequence-based, multistep knowledge discovery strategy to identify potential novel peroxisomal protein candidates in small rodent model organisms and human. METHODS: Screening of 130,629 putative translations of GenBank rodent and primate mRNA sequences was limited to the classical type-1 peroxisomal targeting signal [SA]-K-L. This motif is over-represented among peroxisomal proteins and has a high targeting efficiency. Subsequent steps of identifying co-occurring motifs, secondary structure properties, orthologues and variants, in combination with literature searching and visual inspection by domain experts, aimed at reduction of both false positive and negative validation targets. RESULTS: Our method yielded 117 known peroxisome-targeted proteins and 29 novel candidate proteins. Of special interest were the mouse C530046K17Rik and 1300019N10Rik protein sequences that contain domains associated with enzymatic functions. C530046K17Rik showed no similarity to any known sequence of the animal kingdom, but weak similarity to the possible Leishmania quinone oxidoreductase and a putative cyanobacterium nicotinamide adenine dinucleotide phosphate (NADP)-dependent oxidoreductase. 1300019N10Rik contains two protease-related domains, glutamyl endopeptidase I and trypsin-like serine and cysteine proteases, which may have unique specificities to achieve efficient breakdown of proteins in the peroxisomes. CONCLUSION: One mouse C57BL/6J strain-specific isocitrate dehydrogenase 1 isoform might be suitable to investigate potential phenotypes associated with the deficit of the intraperoxisomal reduced form of NADP (NADPH) and 2-oxoglutarate. Our biological knowledge discovery strategy enabled not only the identification of peroxisomal enzymes already described in the literature, but also the prediction of several novel proteins with possible roles in peroxisomal biochemistry and metabolism that are currently under experimental validation.

Long noncoding RNAs: a potential novel class of cancer biomarkers.

Long noncoding RNAs (lncRNAs) are a novel class of RNA molecules defined as transcripts longer than 200 nucleotides that lack protein coding potential. They constitute a major, but still poorly characterized part of human transcriptome, however, evidence is growing that they are important regulatory molecules involved in various cellular processes. It is becoming increasingly clear that many lncRNAs are deregulated in cancer and some of them can be important drivers of malignant transformation. On the one hand, some lncRNAs can have highly specific expression in particular types of cancer making them a promising tool for diagnosis. The expression of other lncRNAs can correlate with different pathophysiological features of tumor growth and with patient survival, thus making them convenient biomarkers for prognosis. In this review we outline the current state of knowledge about the fast growing field of application of lncRNAs as tumor biomarkers.

Organization of the multiaminoacyl-tRNA synthetase complex and the cotranslational protein folding.

Aminoacyl-tRNA synthetases (ARSs) play an essential role in the protein synthesis by catalyzing an attachment of their cognate amino acids to tRNAs. Unlike their prokaryotic counterparts, ARSs in higher eukaryotes form a multiaminoacyl-tRNA synthetase complex (MARS), consisting of the subset of ARS polypeptides and three auxiliary proteins. The intriguing feature of MARS complex is the presence of only nine out of twenty ARSs, specific for Arg, Asp, Gln, Glu, Ile, Leu, Lys, Met, and Pro, regardless of the organism, cell, or tissue types. Although existence of MARSs complex in higher eukaryotes has been already known for more than four decades, its functional significance remains elusive. We found that seven of the nine corresponding amino acids (Arg, Gln, Glu, Ile, Leu, Lys, and Met) together with Ala form a predictor of the protein α-helicity. Remarkably, all amino acids (besides Ala) in the predictor have the highest possible number of side-chain rotamers. Therefore, compositional bias of a typical α-helix can contribute to the helix's stability by increasing the entropy of the folded state. It also appears that position-specific α-helical propensity, specifically periodic alternation of charged and hydrophobic residues in the helices, may well be provided by the structural organization of the complex. Considering characteristics of MARS complex from the perspective of the α-helicity, we hypothesize that specific composition and structure of the complex represents a functional mechanism for coordination of translation with the fast and correct folding of amphiphilic α-helices.

Contrasting expression patterns of coding and noncoding parts of the human genome upon oxidative stress.

Oxidative stress (OS) is caused by an imbalance between pro- and anti-oxidant reactions leading to accumulation of reactive oxygen species within cells. We here investigate the effect of OS on the transcriptome of human fibroblasts. OS causes a rapid and transient global induction of transcription characterized by pausing of RNA polymerase II (PolII) in both directions, at specific promoters, within 30 minutes of the OS response. In contrast to protein-coding genes, which are commonly down-regulated, this novel divergent, PolII pausing-phenomenon leads to the generation of thousands of long noncoding RNAs (lncRNAs) with promoter-associated antisense lncRNAs transcripts (si-paancRNAs) representing the major group of stress-induced transcripts. OS causes transient dynamics of si-lncRNAs in nucleus and cytosol, leading to their accumulation at polysomes, in contrast to mRNAs, which get depleted from polysomes. We propose that si-lncRNAs represent a novel component of the transcriptional stress that is known to determine the outcome of immediate-early and later cellular stress responses and we provide insights on the fate of those novel mature lncRNA transcripts by showing that their association with polysomal complexes is significantly increased in OS.

Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the International Society for Extracellular Vesicles.

Secreted membrane-enclosed vesicles, collectively called extracellular vesicles (EVs), which include exosomes, ectosomes, microvesicles, microparticles, apoptotic bodies and other EV subsets, encompass a very rapidly growing scientific field in biology and medicine. Importantly, it is currently technically challenging to obtain a totally pure EV fraction free from non-vesicular components for functional studies, and therefore there is a need to establish guidelines for analyses of these vesicles and reporting of scientific studies on EV biology. Here, the International Society for Extracellular Vesicles (ISEV) provides researchers with a minimal set of biochemical, biophysical and functional standards that should be used to attribute any specific biological cargo or functions to EVs.

Exosomes secreted by human cells transport largely mRNA fragments that are enriched in the 3'-untranslated regions.

UNLABELLED: Small secreted membrane vesicles called exosomes have recently attracted a great interest after the discovery that they transfer mRNA that can be translated into protein in recipient cells. Surprisingly, we found that for the majority of exosomal mRNAs only a fraction of their corresponding probes is detectable on the expression microarrays. Exosomal mRNA fragmentation is characterized with a specific structural pattern. The closer to the 3'-end of the transcript the fragments are localized, the larger fraction among the secreted RNAs they constitute. Since the 3'-ends of transcripts contain elements conferring subcellular localization of mRNA and are rich in miRNA-binding sites, exosomal RNA may act as competing RNA to regulate stability, localization and translation activity of mRNAs in recipient cells. REVIEWERS: This article was reviewed by Neil Smalheiser and Sandor Pongor.

Characterization of RNA in exosomes secreted by human breast cancer cell lines using next-generation sequencing.

Exosomes are nanosized (30-100 nm) membrane vesicles secreted by most cell types. Exosomes have been found to contain various RNA species including miRNA, mRNA and long non-protein coding RNAs. A number of cancer cells produce elevated levels of exosomes. Because exosomes have been isolated from most body fluids they may provide a source for non-invasive cancer diagnostics. Transcriptome profiling that uses deep-sequencing technologies (RNA-Seq) offers enormous amount of data that can be used for biomarkers discovery, however, in case of exosomes this approach was applied only for the analysis of small RNAs. In this study, we utilized RNA-Seq technology to analyze RNAs present in microvesicles secreted by human breast cancer cell lines. Exosomes were isolated from the media conditioned by two human breast cancer cell lines, MDA-MB-231 and MDA-MB-436. Exosomal RNA was profiled using the Ion Torrent semiconductor chip-based technology. Exosomes were found to contain various classes of RNA with the major class represented by fragmented ribosomal RNA (rRNA), in particular 28S and 18S rRNA subunits. Analysis of exosomal RNA content revealed that it reflects RNA content of the donor cells. Although exosomes produced by the two cancer cell lines shared most of the RNA species, there was a number of non-coding transcripts unique to MDA-MB-231 and MDA-MB-436 cells. This suggests that RNA analysis might distinguish exosomes produced by low metastatic breast cancer cell line (MDA-MB-436) from that produced by highly metastatic breast cancer cell line (MDA-MB-231). The analysis of gene ontologies (GOs) associated with the most abundant transcripts present in exosomes revealed significant enrichment in genes encoding proteins involved in translation and rRNA and ncRNA processing. These GO terms indicate most expressed genes for both, cellular and exosomal RNA. For the first time, using RNA-seq, we examined the transcriptomes of exosomes secreted by human breast cancer cells. We found that most abundant exosomal RNA species are the fragments of 28S and 18S rRNA subunits. This limits the number of reads from other RNAs. To increase the number of detectable transcripts and improve the accuracy of their expression level the protocols allowing depletion of fragmented rRNA should be utilized in the future RNA-seq analyses on exosomes. Present data revealed that exosomal transcripts are representative of their cells of origin and thus could form basis for detection of tumor specific markers.

Role of genomic architecture in the expression dynamics of long noncoding RNAs during differentiation of human neuroblastoma cells.

BACKGROUND: Mammalian genomes are extensively transcribed producing thousands of long non-protein-coding RNAs (lncRNAs). The biological significance and function of the vast majority of lncRNAs remain unclear. Recent studies have implicated several lncRNAs as playing important roles in embryonic development and cancer progression. LncRNAs are characterized with different genomic architectures in relationship with their associated protein-coding genes. Our study aimed at bridging lncRNA architecture with dynamical patterns of their expression using differentiating human neuroblastoma cells model. RESULTS: LncRNA expression was studied in a 120-hours timecourse of differentiation of human neuroblastoma SH-SY5Y cells into neurons upon treatment with retinoic acid (RA), the compound used for the treatment of neuroblastoma. A custom microarray chip was utilized to interrogate expression levels of 9,267 lncRNAs in the course of differentiation. We categorized lncRNAs into 19 architecture classes according to their position relatively to protein-coding genes. For each architecture class, dynamics of expression of lncRNAs was studied in association with their protein-coding partners. It allowed us to demonstrate positive correlation of lncRNAs with their associated protein-coding genes at bidirectional promoters and for sense-antisense transcript pairs. In contrast, lncRNAs located in the introns and downstream of the protein-coding genes were characterized with negative correlation modes. We further classified the lncRNAs by the temporal patterns of their expression dynamics. We found that intronic and bidirectional promoter architectures are associated with rapid RA-dependent induction or repression of the corresponding lncRNAs, followed by their constant expression. At the same time, lncRNAs expressed downstream of protein-coding genes are characterized by rapid induction, followed by transcriptional repression. Quantitative RT-PCR analysis confirmed the discovered functional modes for several selected lncRNAs associated with proteins involved in cancer and embryonic development. CONCLUSIONS: This is the first report detailing dynamical changes of multiple lncRNAs during RA-induced neuroblastoma differentiation. Integration of genomic and transcriptomic levels of information allowed us to demonstrate specific behavior of lncRNAs organized in different genomic architectures. This study also provides a list of lncRNAs with possible roles in neuroblastoma.