Novel functions of a retroposed gene.

Retroposed protein-coding genes are commonly considered to be nonfunctional duplicates. However, they often gain transcriptional capability and have important roles. Amici et al. recently identified novel functions of a retroposed gene. HAPSTR2, a retrocopy of HAPSTR1, encodes a protein that stabilizes the HAPSTR1 protein and functionally buffers its loss.

The Functional Meaning of 5'UTR in Protein-Coding Genes.

As it is well known, messenger RNA has many regulatory regions along its sequence length. One of them is the 5' untranslated region (5'UTR), which itself contains many regulatory elements such as upstream ORFs (uORFs), internal ribosome entry sites (IRESs), microRNA binding sites, and structural components involved in the regulation of mRNA stability, pre-mRNA splicing, and translation initiation. Activation of the alternative, more upstream transcription start site leads to an extension of 5'UTR. One of the consequences of 5'UTRs extension may be head-to-head gene overlap. This review describes elements in 5'UTR of protein-coding transcripts and the functional significance of protein-coding genes 5' overlap with implications for transcription, translation, and disease.

miRNA in head and neck squamous cell carcinomas: promising but still distant future of personalized oncology.

Head and neck squamous cell carcinoma is one of the most common and fatal cancers worldwide. Lack of appropriate preventive screening tests, late detection, and high heterogeneity of these tumors are the main reasons for the unsatisfactory effects of therapy and, consequently, unfavorable outcomes for patients. An opportunity to improve the quality of diagnostics and treatment of this group of cancers are microRNAs (miRNAs) - molecules with a great potential both as biomarkers and therapeutic targets. This review aims to present the characteristics of these short non-coding RNAs (ncRNAs) and summarize the current reports on their use in oncology focused on medical strategies tailored to patients' needs.

The Mediating Role of the Gut Microbiota in the Physical Growth of Children.

Gut microbiota succession overlaps with intensive growth in infancy and early childhood. The multitude of functions performed by intestinal microbes, including participation in metabolic, hormonal, and immune pathways, makes the gut bacterial community an important player in cross-talk between intestinal processes and growth. Long-term disturbances in the colonization pattern may affect the growth trajectory, resulting in stunting or wasting. In this review, we summarize the evidence on the mediating role of gut microbiota in the mechanisms controlling the growth of children.

Promoter switching in response to changing environment and elevated expression of protein-coding genes overlapping at their 5' ends.

Despite the number of studies focused on sense-antisense transcription, the key question of whether such organization evolved as a regulator of gene expression or if this is only a byproduct of other regulatory processes has not been elucidated to date. In this study, protein-coding sense-antisense gene pairs were analyzed with a particular focus on pairs overlapping at their 5' ends. Analyses were performed in 73 human transcription start site libraries. The results of our studies showed that the overlap between genes is not a stable feature and depends on which TSSs are utilized in a given cell type. An analysis of gene expression did not confirm that overlap between genes causes downregulation of their expression. This observation contradicts earlier findings. In addition, we showed that the switch from one promoter to another, leading to genes overlap, may occur in response to changing environment of a cell or tissue. We also demonstrated that in transfected and cancerous cells genes overlap is observed more often in comparison with normal tissues. Moreover, utilization of overlapping promoters depends on particular state of a cell and, at least in some groups of genes, is not merely coincidental.

Not So Dead Genes-Retrocopies as Regulators of Their Disease-Related Progenitors and Hosts.

Retroposition is RNA-based gene duplication leading to the creation of single exon nonfunctional copies. Nevertheless, over time, many of these duplicates acquire transcriptional capabilities. In human in most cases, these so-called retrogenes do not code for proteins but function as regulatory long noncoding RNAs (lncRNAs). The mechanisms by which they can regulate other genes include microRNA sponging, modulation of alternative splicing, epigenetic regulation and competition for stabilizing factors, among others. Here, we summarize recent findings related to lncRNAs originating from retrocopies that are involved in human diseases such as cancer and neurodegenerative, mental or cardiovascular disorders. Special attention is given to retrocopies that regulate their progenitors or host genes. Presented evidence from the literature and our bioinformatics analyses demonstrates that these retrocopies, often described as unimportant pseudogenes, are significant players in the cell's molecular machinery.

Comparative genomics in the search for conserved long noncoding RNAs.

Long noncoding RNAs (lncRNAs) have emerged as prominent regulators of gene expression in eukaryotes. The identification of lncRNA orthologs is essential in efforts to decipher their roles across model organisms, as homologous genes tend to have similar molecular and biological functions. The relatively high sequence plasticity of lncRNA genes compared with protein-coding genes, makes the identification of their orthologs a challenging task. This is why comparative genomics of lncRNAs requires the development of specific and, sometimes, complex approaches. Here, we briefly review current advancements and challenges associated with four levels of lncRNA conservation: genomic sequences, splicing signals, secondary structures and syntenic transcription.

Long-Term Waterlogging as Factor Contributing to Hypoxia Stress Tolerance Enhancement in Cucumber: Comparative Transcriptome Analysis of Waterlogging Sensitive and Tolerant Accessions.

Waterlogging (WL), excess water in the soil, is a phenomenon often occurring during plant cultivation causing low oxygen levels (hypoxia) in the soil. The aim of this study was to identify candidate genes involved in long-term waterlogging tolerance in cucumber using RNA sequencing. Here, we also determined how waterlogging pre-treatment (priming) influenced long-term memory in WL tolerant (WL-T) and WL sensitive (WL-S) i.e., DH2 and DH4 accessions, respectively. This work uncovered various differentially expressed genes (DEGs) activated in the long-term recovery in both accessions. De novo assembly generated 36,712 transcripts with an average length of 2236 bp. The results revealed that long-term waterlogging had divergent impacts on gene expression in WL-T DH2 and WL-S DH4 cucumber accessions: after 7 days of waterlogging, more DEGs in comparison to control conditions were identified in WL-S DH4 (8927) than in WL-T DH2 (5957). Additionally, 11,619 and 5007 DEGs were identified after a second waterlogging treatment in the WL-S and WL-T accessions, respectively. We identified genes associated with WL in cucumber that were especially related to enhanced glycolysis, adventitious roots development, and amino acid metabolism. qRT-PCR assay for hypoxia marker genes i.e., alcohol dehydrogenase (adh), 1-aminocyclopropane-1-carboxylate oxidase (aco) and long chain acyl-CoA synthetase 6 (lacs6) confirmed differences in response to waterlogging stress between sensitive and tolerant cucumbers and effectiveness of priming to enhance stress tolerance.

lncEvo: automated identification and conservation study of long noncoding RNAs.

BACKGROUND: Long noncoding RNAs represent a large class of transcripts with two common features: they exceed an arbitrary length threshold of 200 nt and are assumed to not encode proteins. Although a growing body of evidence indicates that the vast majority of lncRNAs are potentially nonfunctional, hundreds of them have already been revealed to perform essential gene regulatory functions or to be linked to a number of cellular processes, including those associated with the etiology of human diseases. To better understand the biology of lncRNAs, it is essential to perform a more in-depth study of their evolution. In contrast to protein-encoding transcripts, however, they do not show the strong sequence conservation that usually results from purifying selection; therefore, software that is typically used to resolve the evolutionary relationships of protein-encoding genes and transcripts is not applicable to the study of lncRNAs. RESULTS: To tackle this issue, we developed lncEvo, a computational pipeline that consists of three modules: (1) transcriptome assembly from RNA-Seq data, (2) prediction of lncRNAs, and (3) conservation study-a genome-wide comparison of lncRNA transcriptomes between two species of interest, including search for orthologs. Importantly, one can choose to apply lncEvo solely for transcriptome assembly or lncRNA prediction, without calling the conservation-related part. CONCLUSIONS: lncEvo is an all-in-one tool built with the Nextflow framework, utilizing state-of-the-art software and algorithms with customizable trade-offs between speed and sensitivity, ease of use and built-in reporting functionalities. The source code of the pipeline is freely available for academic and nonacademic use under the MIT license at https://gitlab.com/spirit678/lncrna_conservation_nf .

Cancer, Retrogenes, and Evolution.

This review summarizes the knowledge about retrogenes in the context of cancer and evolution. The retroposition, in which the processed mRNA from parental genes undergoes reverse transcription and the resulting cDNA is integrated back into the genome, results in additional copies of existing genes. Despite the initial misconception, retroposition-derived copies can become functional, and due to their role in the molecular evolution of genomes, they have been named the "seeds of evolution". It is convincing that retrogenes, as important elements involved in the evolution of species, also take part in the evolution of neoplastic tumors at the cell and species levels. The occurrence of specific "resistance mechanisms" to neoplastic transformation in some species has been noted. This phenomenon has been related to additional gene copies, including retrogenes. In addition, the role of retrogenes in the evolution of tumors has been described. Retrogene expression correlates with the occurrence of specific cancer subtypes, their stages, and their response to therapy. Phylogenetic insights into retrogenes show that most cancer-related retrocopies arose in the lineage of primates, and the number of identified cancer-related retrogenes demonstrates that these duplicates are quite important players in human carcinogenesis.

A chromatin-associated splicing isoform of OIP5-AS1 acts in cis to regulate the OIP5 oncogene.

A large portion of the human genome is transcribed into long noncoding RNAs that can range from 200 nucleotides to several kilobases in length. The number of identified lncRNAs is still growing, but only a handful of them have been functionally characterized. However, it is known that the functions of lncRNAs are closely related to their subcellular localization. Cytoplasmic lncRNAs can regulate mRNA stability, affect translation and act as miRNA sponges, while nuclear-retained long noncoding RNAs have been reported to be involved in transcriptional control, chromosome scaffolding, modulation of alternative splicing and chromatin remodelling. Through these processes, lncRNAs have diverse regulatory roles in cell biology and diseases. OIP5-AS1 (also known as Cyrano), a poorly characterized lncRNA expressed antisense to the OIP5 oncogene, is deregulated in multiple cancers. We showed that one of the OIP5-AS1 splicing forms (ENST00000501665.2) is retained in the cell nucleus where it associates with chromatin, thus narrowing down the spectrum of its possible mechanisms of action. Its knockdown with antisense LNA gapmeRs led to inhibited expression of a sense partner, OIP5, strongly suggesting a functional coupling between OIP5 and ENST00000501665.2. A subsequent bioinformatics analysis followed by RAP-MS and RNA Immunoprecipitation experiments suggested its possible mode of action; in particular, we found that ENST00000501665.2 directly binds to a number of nuclear proteins, including SMARCA4, a component of the SWI/SNF chromatin remodelling complex, whose binding motif is located in the promoter of the OIP5 oncogene.

Complex Analysis of Retroposed Genes' Contribution to Human Genome, Proteome and Transcriptome.

Gene duplication is a major driver of organismal evolution. One of the main mechanisms of gene duplications is retroposition, a process in which mRNA is first transcribed into DNA and then reintegrated into the genome. Most gene retrocopies are depleted of the regulatory regions. Nevertheless, examples of functional retrogenes are rapidly increasing. These functions come from the gain of new spatio-temporal expression patterns, imposed by the content of the genomic sequence surrounding inserted cDNA and/or by selectively advantageous mutations, which may lead to the switch from protein coding to regulatory RNA. As recent studies have shown, these genes may lead to new protein domain formation through fusion with other genes, new regulatory RNAs or other regulatory elements. We utilized existing data from high-throughput technologies to create a complex description of retrogenes functionality. Our analysis led to the identification of human retroposed genes that substantially contributed to transcriptome and proteome. These retrocopies demonstrated the potential to encode proteins or short peptides, act as cis- and trans- Natural Antisense Transcripts (NATs), regulate their progenitors' expression by competing for the same microRNAs, and provide a sequence to lncRNA and novel exons to existing protein-coding genes. Our study also revealed that retrocopies, similarly to retrotransposons, may act as recombination hot spots. To our best knowledge this is the first complex analysis of these functions of retrocopies.

Towards a deeper annotation of human lncRNAs.

A substantial fraction of the human transcriptome is composed of the so-called long noncoding RNAs (lncRNAs), yet the available catalogs of known lncRNAs are far from complete. Moreover, functional studies of these RNAs are challenged by several factors, such as their tissue-specific expression and functional heterogeneity, resulting in only ca. 1% of them being well characterized. Here, we describe a set of 41,400 novel lncRNAs discovered with RNA-Seq data from 1463 samples encompassing diverse tissues and cell lines. We utilized publicly available transcriptomic and genomic data to provide their characteristics, such as tissue specificity, cellular abundance, polyA status, cellular localization, evolutionary conservation and transcript stability, which allowed us to speculate on their possible biological roles. We also pinpointed 24 novel lncRNAs as candidates for breast cancer biomarkers. The results bring us closer to a comprehensive annotation of human lncRNAs, though vast amounts of further work are needed to validate the predictions and fully decipher their biology. This article is part of a Special Issue entitled: ncRNA in control of gene expression edited by Kotb Abdelmohsen.

SyntDB: defining orthologues of human long noncoding RNAs across primates.

SyntDB (http://syntdb.amu.edu.pl/) is a collection of data on long noncoding RNAs (lncRNAs) and their evolutionary relationships in twelve primate species, including humans. This is the first database dedicated to primate lncRNAs, thousands of which are uniquely stored in SyntDB. The lncRNAs were predicted with our computational pipeline using publicly available RNA-Seq data spanning diverse tissues and organs. Most of the species included in SyntDB still lack lncRNA annotations in public resources. In addition to providing users with unique sets of lncRNAs and their characteristics, SyntDB provides data on orthology relationships between the lncRNAs of humans and other primates, which are not available on this scale elsewhere. Keeping in mind that only a small fraction of currently known human lncRNAs have been functionally characterized and that lncRNA conservation is frequently used to identify the most relevant lncRNAs for functional studies, we believe that SyntDB will contribute to ongoing research aimed at deciphering the biological roles of lncRNAs.

Transposable Elements: Classification, Identification, and Their Use As a Tool For Comparative Genomics.

Most genomes are populated by hundreds of thousands of sequences originated from mobile elements. On the one hand, these sequences present a real challenge in the process of genome analysis and annotation. On the other hand, they are very interesting biological subjects involved in many cellular processes. Here we present an overview of transposable elements biodiversity, and we discuss different approaches to transposable elements detection and analyses.

CANTATAdb 2.0: Expanding the Collection of Plant Long Noncoding RNAs.

Long non-coding RNAs (lncRNAs) are a class of potent regulators of gene expression that are found in a wide array of eukaryotes; however, our knowledge about these molecules in plants is very limited. In particular, a number of plant species with important roles in biotechnology, agriculture and basic research still lack comprehensively identified and annotated sets of lncRNAs. To address these shortcomings, we previously created a database of lncRNAs in 10 model species, called CANTATAdb, and now we are expanding this online resource to encompass 39 species, including three algae. The lncRNAs were identified computationally using publicly available RNA sequencing (RNA-Seq) data. Expression values, coding potential calculations and other types of information were used to provide annotations for the identified lncRNAs. The data are freely available for searching, browsing and downloading from an online database called CANTATAdb 2.0 ( http://cantata.amu.edu.pl , http://yeti.amu.edu.pl/CANTATA/ ).

Comparison of Highly and Weakly Virulent Dickeya solani Strains, With a View on the Pangenome and Panregulon of This Species.

Bacteria belonging to the genera Dickeya and Pectobacterium are responsible for significant economic losses in a wide variety of crops and ornamentals. During last years, increasing losses in potato production have been attributed to the appearance of Dickeya solani. The D. solani strains investigated so far share genetic homogeneity, although different virulence levels were observed among strains of various origins. The purpose of this study was to investigate the genetic traits possibly related to the diverse virulence levels by means of comparative genomics. First, we developed a new genome assembly pipeline which allowed us to complete the D. solani genomes. Four de novo sequenced and ten publicly available genomes were used to identify the structure of the D. solani pangenome, in which 74.8 and 25.2% of genes were grouped into the core and dispensable genome, respectively. For D. solani panregulon analysis, we performed a binding site prediction for four transcription factors, namely CRP, KdgR, PecS and Fur, to detect the regulons of these virulence regulators. Most of the D. solani potential virulence factors were predicted to belong to the accessory regulons of CRP, KdgR, and PecS. Thus, some differences in gene expression could exist between D. solani strains. The comparison between a highly and a low virulent strain, IFB0099 and IFB0223, respectively, disclosed only small differences between their genomes but significant differences in the production of virulence factors like pectinases, cellulases and proteases, and in their mobility. The D. solani strains also diverge in the number and size of prophages present in their genomes. Another relevant difference is the disruption of the adhesin gene fhaB2 in the highly virulent strain. Strain IFB0223, which has a complete adhesin gene, is less mobile and less aggressive than IFB0099. This suggests that in this case, mobility rather than adherence is needed in order to trigger disease symptoms. This study highlights the utility of comparative genomics in predicting D. solani traits involved in the aggressiveness of this emerging plant pathogen.

Hypermethylation of TRIM59 and KLF14 Influences Cell Death Signaling in Familial Alzheimer's Disease.

Epigenetic mechanisms play an important role in the development and progression of various neurodegenerative diseases. Abnormal methylation of numerous genes responsible for regulation of transcription, DNA replication, and apoptosis has been linked to Alzheimer's disease (AD) pathology. We have recently performed whole transcriptome profiling of familial early-onset Alzheimer's disease (fEOAD) patient-derived fibroblasts. On this basis, we demonstrated a strong dysregulation of cell cycle checkpoints and DNA damage response (DDR) in both fibroblasts and reprogrammed neurons. Here, we show that the aging-correlated hypermethylation of KLF14 and TRIM59 genes associates with abnormalities in DNA repair and cell cycle control in fEOAD. Based on the resulting transcriptome networks, we found that the hypermethylation of KLF14 might be associated with epigenetic regulation of the chromatin organization and mRNA processing followed by hypermethylation of TRIM59 likely associated with the G2/M cell cycle phase and p53 role in DNA repair with BRCA1 protein as the key player. We propose that the hypermethylation of KLF14 could constitute a superior epigenetic mechanism for TRIM59 hypermethylation. The methylation status of both genes affects genome stability and might contribute to proapoptotic signaling in AD. Since this study combines data obtained from various tissues from AD patients, it reinforces the view that the genetic methylation status in the blood may be a valuable predictor of molecular processes occurring in affected tissues. Further research is necessary to define a detailed role of TRIM59 and KLF4 in neurodegeneration of neurons.

Overactive BRCA1 Affects Presenilin 1 in Induced Pluripotent Stem Cell-Derived Neurons in Alzheimer's Disease.

The BRCA1 protein, one of the major players responsible for DNA damage response has recently been linked to Alzheimer's disease (AD). Using primary fibroblasts and neurons reprogrammed from induced pluripotent stem cells (iPSC) derived from familial AD (FAD) patients, we studied the role of the BRCA1 protein underlying molecular neurodegeneration. By whole-transcriptome approach, we have found wide range of disturbances in cell cycle and DNA damage response in FAD fibroblasts. This was manifested by significantly increased content of BRCA1 phosphorylated on Ser1524 and abnormal ubiquitination and subcellular distribution of presenilin 1 (PS1). Accordingly, the iPSC-derived FAD neurons showed increased content of BRCA1(Ser1524) colocalized with degraded PS1, accompanied by an enhanced immunostaining pattern of amyloid-ß. Finally, overactivation of BRCA1 was followed by an increased content of Cdc25C phosphorylated on Ser216, likely triggering cell cycle re-entry in FAD neurons. This study suggests that overactivated BRCA1 could both influence PS1 turnover leading to amyloid-ß pathology and promote cell cycle re-entry-driven cell death of postmitotic neurons in AD.

Transcriptional interference by small transcripts in proximal promoter regions.

Proximal promoter regions (PPR) are heavily transcribed yielding different types of small RNAs. The act of transcription within PPRs might regulate downstream gene expression via transcriptional interference (TI). For analysis, we investigated capped and polyadenylated small RNA transcripts within PPRs of human RefSeq genes in eight different cell lines. Transcripts of our datasets overlapped with experimentally determined transcription factor binding sites (TFBS). For TFBSs intersected by these small RNA transcripts, we established negative correlation of sRNA expression levels and transcription factor (TF) DNA binding affinities; suggesting that the transcripts acted via TI. Accordingly, datasets were designated as TFbiTrs (TF-binding interfering transcripts). Expression of most TFbiTrs was restricted to certain cell lines. This facilitated the analysis of effects related to TFbiTr expression for the same RefSeq genes across cell lines. We consistently uncovered higher relative TF/DNA binding affinities and concomitantly higher expression levels for RefSeq genes in the absence of TFbiTrs. Analysis of corresponding chromatin landscapes supported these results. ChIA-PET revealed the participation of distal enhancers in TFbiTr transcription. Enhancers regulating TFbiTrs, in effect, act as repressors for corresponding downstream RefSeq genes. We demonstrate the significant impact of TI on gene expression using selected small RNA datasets.