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Abstract Background Association testing between molecular phenotypes and genomic variants can help to understand how genotype affects phenotype. RNA sequencing provides access to molecular phenotypes such as gene expression and alternative splicing while DNA sequencing or microarray genotyping are the prevailing options to obtain genomic variants. Results We genotype variants for 74 male Braunvieh cattle from both DNA (~ 13-fold coverage) and deep total RNA sequencing from testis, vas deferens, and epididymis tissue (~ 250 million reads per tissue). We show that RNA sequencing can be used to identify approximately 40% of variants (7–10 million) called from DNA sequencing, with over 80% precision. Within highly expressed coding regions, over 92% of expected variants were called with nearly 98% precision. Allele-specific expression and putative post-transcriptional modifications negatively impact variant genotyping accuracy from RNA sequencing and contribute to RNA-DNA differences. Variants called from RNA sequencing detect roughly 75% of eGenes identified using variants called from DNA sequencing, demonstrating a nearly 2-fold enrichment of eQTL variants. We observe a moderate-to-strong correlation in nominal association p-values (Spearman ρ2 ~ 0.6), although only 9% of eGenes have the same top associated variant. Conclusions We find hundreds of thousands of RNA-DNA differences in variants called from RNA and DNA sequencing on the same individuals. We identify several highly significant eQTL when using RNA sequencing variant genotypes which are not found with DNA sequencing variant genotypes, suggesting that using RNA sequencing variant genotypes for association testing results in an increased number of false positives. Our findings demonstrate that caution must be exercised beyond filtering for variant quality or imputation accuracy when analysing or imputing variants called from RNA sequencing.

Abstract Background Several studies suggested that cavefish populations of Astyanax mexicanus settled during the Late Pleistocene. This implies that the cavefish’s most conspicuous phenotypic changes, blindness and depigmentation, and more cryptic characters important for cave life, evolved rapidly. Results Using the published genomes of 47 Astyanax cavefish from la Cueva de El Pachón, El Sótano de la Tinaja, La Cueva Chica and El Sótano de Molino, we searched for putative loss-of-function mutations in previously defined sets of genes, i.e., vision, circadian clock and pigmentation genes. Putative non-functional alleles for four vision genes were identified. Then, we searched genome-wide for putative non-functional alleles in these four cave populations. Among 512 genes with segregating putative non-functional alleles in cavefish that are absent in surface fish, we found an enrichment in visual perception genes. Among cavefish populations, different levels of shared putative non-functional alleles were found. Using a subset of 12 genes for which putative loss-of-function mutations were found, we extend the analysis of shared pseudogenes to 11 cave populations. Using a subset of six genes for which putative loss-of-function mutations were found in the El Sótano del Toro population, where extensive hybridization with surface fish occurs, we found a correlation between the level of eye regression and the amount of putative non-functional alleles. Conclusions We confirm that very few putative non-functional alleles are present in a large set of vision genes, in accordance with the recent origin of Astyanax mexicanus cavefish. Furthermore, the genome-wide analysis indicates an enrichment of putative loss-of-function alleles in genes with vision-related GO-terms, suggesting that visual perception may be the function chiefly impacted by gene losses related to the shift from a surface to a cave environment. The geographic distribution of putative loss-of-function alleles newly suggests that cave populations from Sierra de Guatemala and Sierra de El Abra share a common origin, albeit followed by independent evolution for a long period. It also supports that populations from the Micos area have an independent origin. In El Sótano del Toro, the troglomorphic phenotype is maintained despite massive introgression of the surface genome.

Abstract Introduction Pseudogenes have been implicated for their role in regulating cellular differentiation and organismal development. However, their role in promoting cancer-associated differentiation has not been well-studied. This study explores the tumour landscape of oesophageal carcinoma to identify pseudogenes that may regulate events of differentiation to promote oncogenic transformation. Materials and method De-regulated differentiation-associated pseudogenes were identified using DeSeq2 followed by ‘InteractiVenn’ analysis to identify their expression pattern. Gene expression dependent and independent enrichment analyses were performed with GSEA and ShinyGO, respectively, followed by quantification of cellular reprogramming, extent of differentiation and pleiotropy using three unique metrics. Stage-specific gene regulatory networks using Bayesian Network Splitting Average were generated, followed by network topology analysis. MEME, STREME and Tomtom were employed to identify transcription factors and miRNAs that play a regulatory role downstream of pseudogenes to initiate cellular reprogramming and further promote oncogenic transformation. The patient samples were stratified based on the expression pattern of pseudogenes, followed by GSEA, mutation analysis and survival analysis using GSEA, MAF and ‘survminer’, respectively. Results Pseudogenes display a unique stage-wise expression pattern that characterizes stage II (SII) ESCA with a high rate of cellular reprogramming, degree of differentiation and pleiotropy. Gene regulatory network and associated topology indicate high robustness, thus validating high pleiotropy observed for SII. Pseudogene-regulated expression of SOX2, FEV, PRRX1 and TFAP2A in SII may modulate cellular reprogramming and promote oncogenesis. Additionally, patient stratification-based mutational analysis in SII signifies APOBEC3A (A3A) as a potential hallmark of homeostatic mutational events of reprogrammed cells which in addition to de-regulated APOBEC3G leads to distinct events of hypermutations. Further enrichment analysis for both cohorts revealed the critical role of combinatorial expression of pseudogenes in cellular reprogramming. Finally, survival analysis reveals distinct genes that promote poor prognosis in SII ESCA and patient-stratified cohorts, thus providing valuable prognostic bio-markers along with markers of differentiation and oncogenesis for distinct landscapes of pseudogene expression. Conclusion Pseudogenes associated with the events of differentiation potentially aid in the initiation of cellular reprogramming to facilitate oncogenic transformation, especially during SII ESCA. Despite a better overall survival of SII, patient stratification reveals combinatorial de-regulation of pseudogenes as a notable marker for a high degree of cellular differentiation with a unique mutational landscape.

Abstract Background Buruli ulcer (BU) disease, caused by Mycobacterium ulcerans (MU), and characterized by necrotic ulcers is still a health problem in Africa and Australia. The genome of the bacterium has several pseudogenes due to recent evolutionary events and environmental pressures. Pseudogenes are genetic elements regarded as nonessential in bacteria, however, they are less studied due to limited available tools to provide understanding of their evolution and roles in MU pathogenicity. Results This study developed a bioinformatic pipeline to profile the pseudogenomes of sequenced MU clinical isolates from different countries. One hundred and seventy-two MU genomes analyzed revealed that pseudogenomes of African strains corresponded to the two African lineages 1 and 2. Pseudogenomes were lineage and location specific and African lineage 1 was further divided into A and B. Lineage 2 had less relaxation in positive selection than lineage 1 which may signify different evolutionary points. Based on the Gil-Latorre model, African MU strains may be in the latter stages of evolutionary adaption and are adapting to an environment rich in metabolic resources with a lower temperature and decreased UV radiation. The environment fosters oxidative metabolism and MU may be less reliant on some secondary metabolites. In-house pseudogenomes from Ghana and Cote d’Ivoire were different from other African strains, however, they were identified as African strains. Conclusion Our bioinformatic pipeline provides pseudogenomic insights to complement other whole genome analyses, providing a better view of the evolution of the genome of MU and suggest an adaptation model which is important in understanding transmission. MU pseudogene profiles vary based on lineage and country, and an apparent reduction in insertion sequences used for the detection of MU which may adversely affect the sensitivity of diagnosis.

ABSTRACTSalmonella enterica serovar Typhi and Paratyphi A are the cause of typhoid and paratyphoid fever in humans, which are systemic life-threatening illnesses. Both serovars are exclusively adapted to the human host, where they can cause life-long persistent infection. A distinct feature of these serovars is the presence of a relatively high number of degraded coding sequences coding for metabolic pathways, most likely a consequence of their adaptation to a single host. As a result of convergent evolution, these serovars shared many of the degraded coding sequences although often affecting different genes in the same metabolic pathway. However, there are several coding sequences that appear intact in one serovar while clearly degraded in the other, suggesting differences in their metabolic capabilities. Here, we examined the functionality of metabolic pathways that appear intact in S. Typhi but that show clear signs of degradation in S. Paratyphi A. We found that, in all cases, the existence of single amino acid substitutions in S. Typhi metabolic enzymes, transporters, or transcription regulators resulted in the inactivation of these metabolic pathways. Thus, the inability of S. Typhi to metabolize Glucose-6-Phosphate or 3-phosphoglyceric acid is due to the silencing of the expression of the genes encoding the transporters for these compounds due to point mutations in the transcriptional regulatory proteins. In contrast, its inability to utilize glucarate or galactarate is due to the presence of point mutations in the transporter and enzymes necessary for the metabolism of these sugars. These studies provide additional support for the concept of adaptive convergent evolution of these two human-adapted S. enterica serovars and highlight a limitation of bioinformatic approaches to predict metabolic capabilities.IMPORTANCESalmonella enterica serovar Typhi and Paratyphi A are the cause of typhoid and paratyphoid fever in humans, which are systemic life-threatening illnesses. Both serovars can only infect the human host, where they can cause life-long persistent infection. Because of their adaptation to the human host, these bacterial pathogens have changed their metabolism, leading to the loss of their ability to utilize certain nutrients. In this study we examined the functionality of metabolic pathways that appear intact in S. Typhi but that show clear signs of degradation in S. Paratyphi A. We found that, in all cases, the existence of single amino acid substitutions in S. Typhi metabolic enzymes, transporters, or transcription regulators resulted in the inactivation of these metabolic pathways. These studies provide additional support for the concept of adaptive convergent evolution of these two human-adapted S. enterica serovars.

Messenger RNA polyadenylation, the process wherein the primary RNA polymerase II transcript is cleaved and a poly(A) tract added, is a key step in the expression of genes in plants. Moreover, it is a point at which gene expression may be regulated by determining the functionality of the mature mRNA. Polyadenylation is mediated by a complex (the polyadenylation complex, or PAC) that consists of between 15 and 20 subunits. While the general functioning of these subunits may be inferred by extending paradigms established in well-developed eukaryotic models, much remains to be learned about the roles of individual subunits in the regulation of polyadenylation in plants. To gain further insight into this, we conducted a survey of variability in the plant PAC. For this, we drew upon a database of naturally-occurring variation in numerous geographic isolates of Arabidopsis thaliana. For a subset of genes encoding PAC subunits, the patterns of variability included the occurrence of premature stop codons in some Arabidopsis accessions. These and other observations lead us to conclude that some genes purported to encode PAC subunits in Arabidopsis are actually pseudogenes, and that others may encode proteins with dispensable functions in the plant. Many subunits of the PAC showed patterns of variability that were consistent with their roles as essential proteins in the cell. Several other PAC subunits exhibit patterns of variability consistent with selection for new or altered function. We propose that these latter subunits participate in regulatory interactions important for differential usage of poly(A) sites.

The idea that proteins are the main determining factors in the functioning of cells and organisms, and their dysfunctions are the first cause of pathologies, has been predominant in biology and biomedicine until recently. This protein-centered view was too simplistic and failed to explain the physiological and pathological complexity of the cell. About 80% of the human genome is dynamically and pervasively transcribed, mostly as non-protein-coding RNAs (ncRNAs), which competitively interact with each other and with coding RNAs generating a complex RNA network regulating RNA processing, stability, and translation and, accordingly, fine-tuning the gene expression of the cells. Qualitative and quantitative dysregulations of RNA-RNA interaction networks are strongly involved in the onset and progression of many pathologies, including cancers and degenerative diseases. This review will summarize the RNA species involved in the competitive endogenous RNA network, their mechanisms of action, and involvement in pathological phenotypes. Moreover, it will give an overview of the most advanced experimental and computational methods to dissect and rebuild RNA networks.

There are about 14,000 pseudogenes that are mutated or truncated sequences resembling functional parent genes. About two-thirds of pseudogenes are processed, while others are duplicated. Although initially thought dead, emerging studies indicate they have functional and regulatory roles. We study 14-3-3ζ, an adaptor protein that regulates cytokine signaling and inflammatory diseases, including rheumatoid arthritis, cancer, and neurological disorders. To understand how 14-3-3ζ (gene symbol YWHAZ) performs diverse functions, we examined the human genome and identified nine YWHAZ pseudogenes spread across many chromosomes. Unlike the 32 kb exon-to-exon sequence in YWHAZ, all pseudogenes are much shorter and lack introns. Out of six, four YWHAZ exons are highly conserved, but the untranslated region (UTR) shows significant diversity. The putative amino acid sequence of pseudogenes is 78–97% homologous, resulting in striking structural similarities with the parent protein. The OMIM and Decipher database searches revealed chromosomal loci containing pseudogenes are associated with human diseases that overlap with the parent gene. To the best of our knowledge, this is the first report on pseudogenes of the 14-3-3 family protein and their implications for human health. This bioinformatics-based study introduces a new insight into the complexity of 14-3-3ζ’s functions in biology.

ABSTRACT Pseudogenes, once considered "junk DNA" based on the incorrect assumption that the absence of full coding potential means a complete lack of functionality, have recently become a subject of significant interest in the scientific community. Concurrently, it is widely assumed that bacterial genomes are compact and have a high density of coding genes with little room for non-coding genes, including pseudogenes. A key aspect of genome annotation is the correct identification of genes and the distinction between coding genes and pseudogenes, as it directly impacts functional and comparative genomics studies. In this study, we analyzed the genomic data of 4,699 strains of the bacterium Pseudomonas aeruginosa (P. aeruginosa) as they exhibit high variability in the number of annotated pseudogenes. In particular, we looked for correlations between the number of pseudogenes and other genomic and meta-features of the strains. We identified clusters of orthologous genes and pseudogenes and compared cluster size distributions and length homogeneity within clusters. We then mapped and examined orthology relationships between genes and pseudogenes. Additionally, we generated a phylogenetic tree of the strains and found that phylogenetically related strains are more homogeneous in the number of pseudogenes and share a significant amount of pseudogenes. Finally, we delved into clusters of orthologous genes and pseudogenes and quantified their phylogenetic neighborhood, classifying pseudogenes into evolutionary preserved pseudogenes, mis-annotated pseudogenes, or pseudogenes formed by failed horizontal transfer events. This in-depth study provides important insights that can be incorporated into pseudogene annotation pipelines in the future. IMPORTANCE Accurate annotation of genes and pseudogenes is vital for comparative genomics analysis. Recent studies have shown that bacterial pseudogenes have an important role in regulatory processes and can provide insight into the evolutionary history of homologous genes or the genome as a whole. Due to pseudogenes’ nature as non-functional genes, there is no commonly accepted definition of a pseudogene, which poses difficulties in verifying the annotation through experimental methods and resolving discrepancies among different annotation techniques. Our study introduces an in-depth analysis of annotated genes and pseudogenes and insights that can be incorporated into improved pseudogene annotation pipelines in the future.

Background: The promoters of mammalian genes contain clusters of CG dinucleotides known as CpG islands. Most mammalian housekeeping genes predominantly contain CpG islands (CGIs), facilitating gene transcription. Numerous studies have explored the physiological implications of the relationship between CGIs and gene expression. However, the evolutionary implications of this relationship remain largely unexplored. Pseudogenes, in contrast, are genomic remnants that have lost their function over evolutionary time. Methods: In our current research, we employed comparative genomic techniques to demonstrate a correlation between the absence of gene expression due to a lack of CGIs in the gene promoters and pseudogenization. Results: We showed that there is a significant enrichment of tissue-specific genes in the functional orthologs of pseudogenes. We also found a significant correlation between the lack of CGIs and enriched tissue specificity in these functional orthologs of pseudogenes. Conclusions: We inferred that perhaps tissue-specific genes are more prone to the process of pseudogenization. In this way, because of their impact on gene expression, CGIs may affect the fate of a gene. To our knowledge, this is the first study to propose a connection between CGIs, gene expression, and the pseudogenization process and discuss the evolutionary implications of this potential trilogy.

Different species of toothed whales (Odontoceti) exhibit a variety of tooth forms and enamel types. Some odontocetes have highly prismatic enamel with Hunter-Schreger bands, whereas enamel is vestigial or entirely lacking in other species. Different tooth forms and enamel types are associated with alternate feeding strategies that range from biting and grasping prey with teeth in most oceanic and river dolphins to the suction feeding of softer prey items without the use of teeth in many beaked whales. At the molecular level, previous studies have documented inactivating mutations in the enamel-specific genes of some odontocete species that lack complex enamel. At a broader scale, however, it is unclear whether enamel complexity across the full diversity of extant Odontoceti correlates with the relative strength of purifying selection on enamel-specific genes. Here, we employ sequence alignments for seven enamel-specific genes (ACP4, AMBN, AMELX, AMTN, ENAM, KLK4, MMP20) in 62 odontocete species that are representative of all extant families. The sequences for 33 odontocete species were obtained from databases, and sequences for the remaining 29 species were newly generated for this study. We screened these alignments for inactivating mutations (e.g., frameshift indels) and provide a comprehensive catalog of these mutations in species with one or more inactivated enamel genes. Inactivating mutations are rare in Delphinidae (oceanic dolphins) and Platanistidae/Inioidea (river dolphins) that have higher enamel complexity scores. By contrast, mutations are much more numerous in clades such as Monodontidae (narwhal, beluga), Ziphiidae (beaked whales), Physeteroidea (sperm whales), and Phocoenidae (porpoises) that are characterized by simpler enamel or even enamelless teeth. Further, several higher-level taxa (e.g., Hyperoodon, Kogiidae, Monodontidae) possess shared inactivating mutations in one or more enamel genes, which suggests loss of function of these genes in the common ancestor of each clade. We also performed selection (dN/dS) analyses on a concatenation of these genes and used linear regression and Spearman’s rank-order correlation to test for correlations between enamel complexity and two different measures of selection intensity (# of inactivating mutations per million years, dN/dS values). Selection analyses revealed that relaxed purifying selection is especially prominent in physeteroids, monodontids, and phocoenids. Linear regressions and correlation analyses revealed a strong negative correlation between selective pressure (dN/dS values) and enamel complexity. Stronger purifying selection (low dN/dS) is found on branches with more complex enamel and weaker purifying selection (higher dN/dS) occurs on branches with less complex enamel or enamelless teeth. As odontocetes diversified into a variety of feeding modes, in particular, the suction capture of prey, a reduced reliance on the dentition for prey capture resulted in the relaxed selection of genes that are critical to enamel development.

The plastome (plastid genome) represents an indispensable molecular data source for studying phylogeny and evolution in plants. Although the plastome size is much smaller than that of nuclear genome, and multiple plastome annotation tools have been specifically developed, accurate annotation of plastomes is still a challenging task. Different plastome annotation tools apply different principles and workflows, and annotation errors frequently occur in published plastomes and those issued in GenBank. It is therefore timely to compare available annotation tools and establish standards for plastome annotation. In this review, we review the basic characteristics of plastomes, trends in the publication of new plastomes, the annotation principles and application of major plastome annotation tools, and common errors in plastome annotation. We propose possible methods to judge pseudogenes and RNA-editing genes, jointly consider sequence similarity, customed algorithms, conserved domain or protein structure. We also propose the necessity of establishing a database of reference plastomes with standardized annotations, and put forward a set of quantitative standards for evaluating plastome annotation quality for the scientific community. In addition, we discuss how to generate standardized GenBank annotation flatfiles for submission and downstream analysis. Finally, we prospect future technologies for plastome annotation integrating plastome annotation approaches with diverse evidences and algorithms of nuclear genome annotation tools. This review will help researchers more efficiently use available tools to achieve high-quality plastome annotation, and promote the process of standardized annotation of the plastome.

Background and Objectives: Leprosy remains an important health problem worldwide. It is one of the oldest recorded diseases of humankind. In this study, we expanded the analysis of the geographic distribution of Mycobacterium leprae by investigating SNPs and rpoT genotypes in South Central Coast and Central Highlands clinical isolates, providing insights into the distribution and transmission of leprosy in Vietnam and in this geographic region. Materials and Methods: 27 clinical isolates from the patients, determined the genotypes of M. leprae by SNP and rpoT polymorphism. SNP genotyping was performed by PCR amplification and sequencing, rpoT genotyping by PCR amplification and electrophoresis. Results: All of 27 DNA samples (100%) were positive with RLEP TaqMan PCR (Ct value range is 18-32 on 3 replicates). SNP type 1 was identified in 15 isolates (56%), while SNP type 3 was detected in 12 samples (44%). SNP type 2 and type 4, were not detected. The 6-base repeat region of the rpoT gene was amplified by PCR and analyzed by 4% MetaPhor™ agarose gel electrophoresis. All isolates yielded amplification products of 91-bp, but not 97-bp. Conclusion: This study showed that 56% of isolates belonged to type 1, 44% to type 3. In addition, all samples have the 3-copy hexamer genotype in the rpoT gene.

Many animal species employ sperm nuclear basic proteins (SNBPs) or protamines to package sperm genomes tightly. SNBPs vary across animal lineages and evolve rapidly in mammals. We used a phylogenomic approach to investigate SNBP diversification in Drosophila species. We found that most SNBP genes in Drosophila melanogaster evolve under positive selection except for genes essential for male fertility. Unexpectedly, evolutionarily young SNBP genes are more likely to be critical for fertility than ancient, conserved SNBP genes. For example, CG30056 is dispensable for male fertility despite being one of three SNBP genes universally retained in Drosophila species. We found 19 independent SNBP gene amplification events that occurred preferentially on sex chromosomes. Conversely, the montium group of Drosophila species lost otherwise-conserved SNBP genes, coincident with an X-Y chromosomal fusion. Furthermore, SNBP genes that became linked to sex chromosomes via chromosomal fusions were more likely to degenerate or relocate back to autosomes. We hypothesize that autosomal SNBP genes suppress meiotic drive, whereas sex-chromosomal SNBP expansions lead to meiotic drive. X-Y fusions in the montium group render autosomal SNBPs dispensable by making X-versus-Y meiotic drive obsolete or costly. Thus, genetic conflicts between sex chromosomes may drive SNBP rapid evolution during spermatogenesis in Drosophila species.

BackgroundThe axolotl, Ambystoma mexicanum is a unique biological model for complete tissue regeneration. Is a neotenic endangered species and is highly susceptible to environmental stress, including infectious disease. In contrast to other amphibians, the axolotl is particularly vulnerable to certain viral infections. Like other salamanders, the axolotl genome is one of the largest (32 Gb) and the impact of genome size on Ig loci architecture is unknown. To better understand the immune response in axolotl, we aimed to characterize the immunoglobulin loci of A. mexicanum and compare it with other model vertebrates.MethodsThe most recently published genome sequence of A. mexicanum (V6) was used for alignment-based annotation and manual curation using previously described axolotl Ig sequences or reference sequences from other vertebrates. Gene models were further curated using A. mexicanum spleen RNA-seq data. Human, Xenopus tropicalis, Danio rerio (zebrafish), and eight tetrapod reference genomes were used for comparison.ResultsCanonical A. mexicanum heavy chain (IGH), lambda (IGL), sigma (IGS), and the putative surrogate light chain (SLC) loci were identified. No kappa locus was found. More than half of the IGHV genes and the IGHF gene are pseudogenes and there is no clan I IGHV genes. Although the IGH locus size is proportional to genome size, we found local size restriction in the IGHM gene and the V gene intergenic distances. In addition, there were V genes with abnormally large V-intron sizes, which correlated with loss of gene functionality.ConclusionThe A. mexicanum immunoglobulin loci share the same general genome architecture as most studied tetrapods. Consistent with its large genome, Ig loci are larger; however, local size restrictions indicate evolutionary constraints likely to be imposed by high transcriptional demand of certain Ig genes, as well as the V(D)J recombination over very long genomic distance ranges. The A. mexicanum has undergone an extensive process of Ig gene loss which partially explains a reduced potential repertoire diversity that may contribute to its impaired antibody response.

Abstract Background Naked mole-rats (Heterocephalus glaber, NMRs) and blind mole-rats (Spalax galili, BMRs) are representative subterranean rodents that have evolved many extraordinary traits, including hypoxia tolerance, longevity, and cancer resistance. Although multiple candidate loci responsible for these traits have been uncovered by genomic studies, many of them are limited to functional changes to amino acid sequence and little is known about the contributions of other genetic events. To address this issue, we focused on gene losses (unitary pseudogenes) and systematically analyzed gene losses in NMRs and BMRs, aiming to elucidate the potential roles of pseudogenes in their adaptation to subterranean lifestyle. Results We obtained the pseudogene repertoires in NMRs and BMRs, as well as their respective aboveground relatives, guinea pigs and rats, on a genome-wide scale. As a result, 167, 139, 341, and 112 pseudogenes were identified in NMRs, BMRs, guinea pigs, and rats, respectively. Functional enrichment analysis identified 4 shared and 2 species-specific enriched functional groups (EFGs) in subterranean lineages. Notably, the pseudogenes in these EFGs might be associated with either regressive (e.g., visual system) or adaptive (e.g., altered DNA damage response) traits. In addition, several pseudogenes including TNNI3K and PDE5A might be associated with specific cardiac features observed in subterranean lineages. Interestingly, we observed 20 convergent gene losses in NMRs and BMRs. Given that the functional investigations of these genes are generally scarce, we provided functional evidence that independent loss of TRIM17 in NMRs and BMRs might be beneficial for neuronal survival under hypoxia, supporting the positive role of eliminating TRIM17 function in hypoxia adaptation. Our results also suggested that pseudogenes, together with positively selected genes, reinforced subterranean adaptations cooperatively. Conclusions Our study provides new insights into the molecular underpinnings of subterranean adaptations and highlights the importance of gene losses in mammalian evolution.

Avian sex chromosomes evolved after the divergence of birds and crocodilians from their common ancestor, so they are younger than the better-studied chromosomes of mammals. It has long been recognized that there may have been several stages to the evolution of avian sex chromosomes. For example, the CHD1 undergoes recombination in paleognaths but not neognaths. Genome assemblies have suggested that there may be variation in the timing of barriers to recombination among Neognathae, but there remains little understanding of the extent of this variability. Here, we look at partial sequences of ATP5F1A, which is on the avian Z and W chromosomes. It is known that recombination of this gene has independently ceased in Galliformes, Anseriformes, and at least five neoavian orders, but whether there are other independent cessations of recombination among Neoaves is not understood. We analyzed a combination of data extracted from published chromosomal-level genomes with data collected using PCR and cloning to identify Z and W copies in 22 orders. Our results suggest that there may be at least 19 independent cessations of recombination within Neognathae, and 3 clades that may still be undergoing recombination (or have only recently ceased recombination). Analyses of ATP5F1A protein sequences revealed an increased amino acid substitution rate for W chromosome gametologs, suggesting relaxed purifying selection on the W chromosome. Supporting this hypothesis, we found that the increased substitution rate was particularly pronounced for buried residues, which are expected to be more strongly constrained by purifying selection. This highlights the dynamic nature of avian sex chromosomes, and that this level of variation among clades means they should be a good system to understand sex chromosome evolution.

Recurrent epidemics of cholera denote robust adaptive mechanisms of Vibrio cholerae for ecological shifting and persistence despite variable stress conditions. Tracking the evolution of pathobiological traits requires comparative genomic studies of isolates from endemic areas. Here, we investigated the genetic differentiation among V. cholerae clinical and environmental isolates by highlighting the genomic divergence associated with gene decay, genome plasticity, and the acquisition of virulence and adaptive traits. The clinical isolates showed high phylogenetic relatedness due to a higher frequency of shared orthologs and fewer gene variants in contrast to the evolutionarily divergent environmental strains. Divergence of the environmental isolates is linked to extensive genomic rearrangements in regions containing mobile genetic elements resulting in numerous breakpoints, relocations, and insertions coupled with the loss of virulence determinants acf, zot, tcp, and ctx in the genomic islands. Also, four isolates possessed the CRISPR-Cas systems with spacers specific for Vibrio phages and plasmids. Genome synteny and homology analysis of the CRISPR-Cas systems suggest horizontal acquisition. The marked differences in the distribution of other phage and plasmid defense systems such as Zorya, DdmABC, DdmDE, and type-I Restriction Modification systems among the isolates indicated a higher propensity for plasmid or phage disseminated traits in the environmental isolates. Our results reveal that V. cholerae strains undergo extensive genomic rearrangements coupled with gene acquisition, reflecting their adaptation during ecological shifts and pathogenicity.

ABSTRACT In Colombia Aedes aegypti is present in 80% of the country up to 2,300 m; however, little is known of its genetic relations within a country context and, hence, within a broader context, for example, America. The aforementioned, herein, analyzed the gene flow within a context of the Americas, its directionality and genetic diversity of the mitochondrial lineages in the A. aegypti populations for Colombia. This called for the use of the sequences for A. aegypti available of the mitochondrial ND4 gene in the GenBank for Colombia and the American continent. No presence was found of nuclear mitochondrial pseudogenes (NUMTs) for Colombia. It is estimated that in Colombia the gene flow of the A. aegypti populations is occurring from the southeast and northeast toward the center of the country. In comparison with the mitochondrial sequences for America, the vector’s haplotypes in Colombia suggest connections between the populations of mosquitoes from the south with those from the north of the continent. The gene flow model at continental scale suggests bidirectional connections between the populations from the north of the continent with those from the south, while at South American scale it proposes the gene flow in all the directions with respect to the Colombian. The Colombian A. aegypti vector monitoring and control strategies must pay special attention to the vector’s points of entry into Colombia related with Peru, Venezuela, Brazil, Mexico, and North America to avoid the entry of populations with characteristics like resistance to insecticides or vector competition.

The internal transcribed spacer (ITS) is one of the most extensively sequenced molecular markers in plant systematics due to its generally concerted evolution. While non-concerted evolution has been found in some plant taxa, such information is missing in Lycium. Molecular studies of six species and two variants of the genus Lycium revealed high levels of intra- and inter-individual polymorphism in the ITS, indicating non-concerted evolution. All genomic DNA ITS paralogues were identified as putative pseudogenes or functional paralogues through a series of comparisons of sequence features, including length and substitution variation, GC content, secondary structure stability, and the presence of conserved motifs in the 5.8S gene, and the rate of evolution. Approximately, 60% of ITS pseudogenes could be easily detected. Based on phylogenetic analysis, all pseudogenes were highly distinct from their corresponding functional copies, tended to evolve neutrally, and clustered randomly together in the evolutionary tree. The results probably suggest that this ITS non-concerted evolution is related to the recent divergence between tandem repeats within the Lycium genome and hybridization between species. Our study complements those of pseudogenes in plant taxa and provides a theoretical basis for the phylogeny and genetic origin of the genus Lycium while having important implications for the use of ITS molecular markers for phylogenetic reconstruction.

Pseudogenes have been considered as non-functional genes. However, peptides and long non-coding RNAs produced by pseudogenes are expressed in different tumors. Moreover, the dysregulation of pseudogenes is associated with cancer, and their expressions are higher in tumors compared to normal tissues. Recent studies show that pseudogenes can influence the liquid phase condensates formation. Liquid phase separation involves regulating different epigenetic stages, including transcription, chromatin organization, 3D DNA structure, splicing, and post-transcription modifications like m6A. Several membrane-less organelles, formed through the liquid phase separate, are also involved in the epigenetic regulation, and their defects are associated with cancer development. However, the association between pseudogenes and liquid phase separation remains unrevealed. The current study sought to investigate the relationship between pseudogenes and liquid phase separation in cancer development, as well as their therapeutic implications.

Mitochondrial DNA (mtDNA) translocated into the nuclear genome (numt), when co-analysed with genuine mtDNA, could plague phylogeographic studies. To evaluate numt-related biases in population genetics parameters in birds, which are prone to accumulating numts, we targeted the mitochondrial mt-cytb gene. We looked at 13 populations of Audubon's shearwater (Puffinus lherminieri), including five mitochondrial lineages. mt-cytb homologue and paralogue (numt) sequences were determined by Sanger sequencing with and without prior exonuclease digestion of nuclear DNA. Numts formed monophyletic clades corresponding to three of the five mitochondrial lineages tested (the remaining two forming a paraphyletic group). Nineteen percent of numt alleles fell outside of their expected mitochondrial clade, a pattern consistent with multiple translocation events, incomplete lineage sorting (ILS), and/or introgression. When co-analysing mt-cytb paralogues and homologues, excluding individuals with ambiguities underestimates genetic diversity (4%) and differentiation (11%) among least-sampled populations. Removing ambiguous sites drops the proportion of inter-lineage genetic variance by 63%. While co-analysing numts with mitochondrial sequences can lead to severe bias and information loss in bird phylogeographic studies, the separate analysis of genuine mitochondrial loci and their nuclear paralogues can shed light on numt molecular evolution, as well as evolutionary processes such as ILS and introgression.

Paralogs and pseudogenes are abundant within the human genome, and can mediate non-allelic homologous recombination (NAHR) or gene conversion events. The ATAD3 locus contains three paralogs situated in tandem, and is therefore prone to NAHR-mediated deletions and duplications associated with severe neurological phenotypes. To study this locus further, we aimed to generate biallelic loss-of-function variants in ATAD3A by CRISPR/Cas9 genome editing. Unexpectedly, two of the generated clones underwent gene conversion, as evidenced by replacement of the targeted sequence of ATAD3A by a donor sequence from its paralog ATAD3B. We highlight the complexity of CRISPR/Cas9 design, end-product formation, and recombination repair mechanisms for CRISPR/Cas9 delivery as a nucleic acid molecular therapy when targeting genes that have paralogs or pseudogenes, and advocate meticulous evaluation of resultant clones in model organisms. In addition, we suggest that endogenous gene conversion may be used to repair missense variants in genes with paralogs or pseudogenes.

Abstract Background Given the vast range of molecular mechanisms giving rise to breast cancer, it is unlikely universal cures exist. However, by providing a more precise prognosis for breast cancer patients through integrative models, treatments can become more individualized, resulting in more successful outcomes. Specifically, we combine gene expression, pseudogene expression, miRNA expression, clinical factors, and pseudogene-gene functional networks to generate these models for breast cancer prognostics. Establishing a LASSO-generated molecular gene signature revealed that the increased expression of genes STXBP5, GALP and LOC387646 indicate a poor prognosis for a breast cancer patient. We also found that increased CTSLP8 and RPS10P20 and decreased HLA-K pseudogene expression indicate poor prognosis for a patient. Perhaps most importantly we identified a pseudogene-gene interaction, GPS2-GPS2P1 (improved prognosis) that is prognostic where neither the gene nor pseudogene alone is prognostic of survival. Besides, miR-3923 was predicted to target GPS2 using miRanda, PicTar, and TargetScan, which imply modules of gene-pseudogene-miRNAs that are potentially functionally related to patient survival. Results In our LASSO-based model, we take into account features including pseudogenes, genes and candidate pseudogene-gene interactions. Key biomarkers were identified from the features. The identification of key biomarkers in combination with significant clinical factors (such as stage and radiation therapy status) should be considered as well, enabling a specific prognostic prediction and future treatment plan for an individual patient. Here we used our PseudoFuN web application to identify the candidate pseudogene-gene interactions as candidate features in our integrative models. We further identified potential miRNAs targeting those features in our models using PseudoFuN as well. From this study, we present an interpretable survival model based on LASSO and decision trees, we also provide a novel feature set which includes pseudogene-gene interaction terms that have been ignored by previous prognostic models. We find that some interaction terms for pseudogenes and genes are significantly prognostic of survival. These interactions are cross-over interactions, where the impact of the gene expression on survival changes with pseudogene expression and vice versa. These may imply more complicated regulation mechanisms than previously understood. Conclusions We recommend these novel feature sets be considered when training other types of prognostic models as well, which may provide more comprehensive insights into personalized treatment decisions.

Abstract Background The Salmonella enterica subsp. diarizonae serovar 61:k:1,5,(7) (SASd) has been found to be host-adapted to sheep, with a high prevalence in sheep herds worldwide. Infections are usually sub-clinical, however the serovar has the potential to cause diarrhea, abortions and chronic proliferative rhinitis. Although occurrence and significance of SASd infections in sheep have been extensively studied, the genetic mechanism underlying this unusual host-adaptation have remained unknown, due to a lack of (a) available high-quality genome sequence(s). Results We utilized Nanopore and Illumina sequencing technologies to generate a de novo assembly of the 4.88-Mbp complete genome sequence of the SASd strain 16-SA00356, isolated from the organs of a deceased sheep in 2016. We annotated and analyzed the genome sequence with the aim to gain a deeper understanding of the genome characteristics associated with its pathogenicity and host adaptation to sheep. Overall, we found a number of interesting genomic features such as several prophage regions, a VirB4/D4 plasmid and novel genomic islands. By comparing the genome of 16-SA00356 to other S. enterica serovars we found that SASd features an increased number of pseudogenes as well as a high level of genomic rearrangements, both known indicators of host-adaptation. Conclusions With this sequence, we provide the first complete and closed genome sequence of a SASd strain. With this study, we provide an important basis for an understanding of the genetic mechanism that underlie pathogenicity and host adaptation of SASd to sheep.

Hepatocellular carcinoma (HCC) is a malignancy with one of the worst prognoses. Long noncoding RNA (lncRNA) are emerging as an important regulator of gene expression and function, leading to the development of cancer. The aim of this study was to determine the relationship between lncRNA and HCC and to further guide clinical therapy. lncRNA in HCC and adjacent tissues were screened, and the correlation between lncRNA‐PDPK2P expression in liver tissues and the pathological characteristics and severity of HCC was assessed. The effects of PDPK2P on HCC proliferation, apoptosis, metastasis, and invasion were also systematically investigated via CCK‐8 assay, flow cytometry, scratch wound healing, and transwell assay, respectively. The relationship between PDPK2P and PDK1 was verified by RNA pull‐down, rescue experiments and western blot. lncRNA‐PDPK2P was highly expressed in HCC tissues with a distinct positive correlation between PDPK2P and PDK1, and the upregulation was clinically associated with a larger tumor embolus, low differentiation, and poor survival. Mechanistically, lncRNA‐PDPK2P interacted with PDK1 and promoted HCC progression through the PDK1/AKT/caspase 3 signaling pathway. lncRNA‐PDPK2P can promote HCC progression, suggesting it may be a clinically valuable biomarker and serve as a molecular target for the diagnosis, prognosis, and therapy of hepatocellular carcinoma.

Abstract Background Increasing evidence suggests a critical role for long noncoding RNAs (LncRNAs) and pseudogenes in cancer. Renal cell carcinoma (RCC), the most common primary renal neoplasm, is highly aggressive and difficult to treat because of its resistance to chemotherapy and radiotherapy. Despite many identified LncRNAs and pseudogenes, few have been clearly elucidated. Methods This study provides new insights into LncRNAs and pseudogenes in the prognosis of RCC. We searched an online database to interrogate alterations and clinical data on cBioPortal. We analysed LncRNA and pseudogene signatures to predict the prognosis of RCC based on a Cox model. We also found potential serum biomarkers of RCC and validated them in 32 RCC patients, as well as healthy controls. Results Alterations were found in 2553 LncRNAs and 8901 pseudogenes and occurred in up to 23% of all cases. Among these, 27 LncRNAs and 45 pseudogenes were closely related to prognosis. We also identified signatures of LncRNAs and pseudogenes that can predict overall survival and recurrence of RCC. We then validated the relative levels of these LncRNAs and pseudogenes in the serum of 32 patients. Six of these, including LINC00520, PIK3CD-AS1, LINC01559, CEACAM22P, MSL3P1 and TREML3P, could be non-invasive biomarkers of RCC. Finally, we selected PIK3CD-AS1 to determine its role in RCC and found that upregulation of PIK3CD-AS1 was closely associated with higher tumour stage and metastasis. Conclusions These signatures of LncRNAs and pseudogenes can predict overall survival and recurrence of RCC. LINC00520, PIK3CD-AS1, LINC01559, CEACAM22P, MSL3P1 and TREML3P could be non-invasive biomarkers of RCC. These data suggest the important roles of LncRNAs and pseudogenes in RCC, and therefore provides us new insights into the prognosis of RCC.

Cardiovascular disease is the main disease that affects human life span. In recent years, the disease has been increasingly addressed at the molecular levels, for example, pseudogenes are now known to be involved in the pathogenesis and development of cardiovascular diseases. Pseudogenes are non-coding homologs of protein-coding genes and were once called “junk gene.” Since they are highly homologous to their functional parental genes, it is somewhat difficult to distinguish them. With the development of sequencing technology and bioinformatics, pseudogenes have become readily identifiable. Recent studies indicate that pseudogenes are closely related to cardiovascular diseases. This review provides an overview of pseudogenes and their roles in the pathogenesis of cardiovascular diseases. This new knowledge adds to our understanding of cardiovascular disease at the molecular level and will help develop new biomarkers and therapeutic approaches designed to prevent and treat the disease.

Pseudogenes were once considered as “junk DNA”, due to loss of their functions as a result of the accumulation of mutations, such as frameshift and presence of premature stop-codons and relocation of genes to inactive heterochromatin regions of the genome. Pseudogenes are divided into two large groups, processed and unprocessed, according to their primary structure and origin. Only 10% of all pseudogenes are transcribed into RNAs and participate in the regulation of parental gene expression at both transcriptional and translational levels through senseRNA (sRNA) and antisense RNA (asRNA). In this review, about 150 pseudogenes in the different types of cancers were analyzed. Part of these pseudogenes seem to be useful in molecular diagnostics and can be detected in various types of biological material including tissue as well as biological fluids (liquid biopsy) using different detection methods. The number of pseudogenes, as well as their function in the human genome, is still unknown. However, thanks to the development of various technologies and bioinformatic tools, it was revealed so far that pseudogenes are involved in the development and progression of certain diseases, especially in cancer.

Background Species in the genus Morus (Moraceae) are deciduous woody plants of great economic importance. The classification and phylogenetic relationships of Morus, especially the abundant mulberry resources in China, is still undetermined. Internal transcribed spacer (ITS) regions are among the most widely used molecular markers in phylogenetic analyses of angiosperms. However, according to the previous phylogenetic analyses of ITS sequences, most of the mulberry accessions collected in China were grouped into the largest clade lacking for phylogenetic resolution. Compared with functional ITS sequences, ITS pseudogenes show higher sequence diversity, so they can provide useful phylogenetic information. Methods We sequenced the ITS regions and the chloroplast DNA regions TrnL-TrnF and TrnT-TrnL from 33 mulberry accessions, and performed phylogenetic analyses to explore the evolution of mulberry. Results We found ITS pseudogenes in 11 mulberry accessions. In the phylogenetic tree constructed from ITS sequences, clade B was separated into short-type sequence clades (clades 1 and 2), and a long-type sequence clade (clade 3). Pseudogene sequences were separately clustered into two pseudogroups, designated as pseudogroup 1 and pseudogroup 2. The phylogenetic tree generated from cpDNA sequences also separated clade B into two clades. Conclusions Two species were separated in clade B. The existence of three connection patterns and incongruent distribution patterns between the phylogenetic trees generated from cpDNA and ITS sequences suggested that the ITS pseudogene sequences connect with genetic information from the female progenitor. Hybridization has played important roles in the evolution of mulberry, resulting in low resolution of the phylogenetic analysis based on ITS sequences. An evolutionary pattern illustrating the evolution history of mulberry is proposed. These findings have significance for the conservation of local mulberry resources. Polyploidy, hybridization, and concerted evolution have all played the roles in the evolution of ITS sequences in mulberry. This study will expand our understanding of mulberry evolution.

Abstract Background Universal stress proteins (USPs) are present in all domains of life. Their expression is upregulated in response to a large variety of stress conditions. The functional diversity found in this protein family, paired with the sequence degeneration of the characteristic ATP-binding motif, suggests a complex evolutionary pattern for the paralogous USP-encoding genes. In this work, we investigated the origin, genomic organization, expression patterns and evolutionary history of the USP gene family in species of the phylum Platyhelminthes. Results Our data showed a cluster organization, a lineage-specific distribution, and the presence of several pseudogenes among the USP gene copies identified. The absence of a well conserved -CCAATCA- motif in the promoter region was positively correlated with low or null levels of gene expression, and with amino acid changes within the ligand binding motifs. Despite evidence of the pseudogenization of various USP genes, we detected an important functional divergence at several residues, mostly located near sites that are critical for ligand interaction. Conclusions Our results provide a broad framework for the evolution of the USP gene family, based on the emergence of new paralogs that face very contrasting fates, including pseudogenization, subfunctionalization or neofunctionalization. This framework aims to explain the sequence and functional diversity of this gene family, providing a foundation for future studies in other taxa in which USPs occur.

The discovery of microRNAs (miRNAs) has fundamentally transformed our understanding of gene regulation. The competing endogenous RNA (ceRNA) hypothesis postulates that messenger RNAs and other RNA transcripts, such as long non-coding RNAs and pseudogenes, can act as natural miRNA sponges. These RNAs influence each other’s expression levels by competing for the same pool of miRNAs through miRNA response elements on their target transcripts, thereby modulating gene expression and protein activity. In recent years, these ceRNA regulatory networks have gained considerable attention in cancer research. Several studies have identified cancer-specific ceRNA networks. Nevertheless, prior bioinformatic analyses have focused on long-non-coding RNA-associated ceRNA networks. Here, we identify an extended ceRNA network (including both long non-coding RNAs and pseudogenes) shared across a group of five hormone-dependent (HD) cancers, i.e., prostate, breast, colon, rectal, and endometrial cancers, using data from The Cancer Genome Atlas (TCGA). We performed a functional enrichment analysis for differentially expressed genes in the shared ceRNA network of HD cancers, followed by a survival analysis to determine their prognostic ability. We identified two long non-coding RNAs, nine genes, and seventy-four miRNAs in the shared ceRNA network across five HD cancers. Among them, two genes and forty-one miRNAs were associated with at least one HD cancer survival. This study is the first to investigate pseudogene-associated ceRNAs across a group of related cancers and highlights the value of this approach to understanding the shared molecular pathogenesis in a group of related diseases.

Abstract Background Genome duplication has played a pivotal role in the evolution of many eukaryotic lineages, including the vertebrates. A relatively recent vertebrate genome duplication is that in Xenopus laevis, which resulted from the hybridization of two closely related species about 17 million years ago. However, little is known about the consequences of this duplication at the level of the genome, the epigenome, and gene expression. Results The X. laevis genome consists of two subgenomes, referred to as L (long chromosomes) and S (short chromosomes), that originated from distinct diploid progenitors. Of the parental subgenomes, S chromosomes have degraded faster than L chromosomes from the point of genome duplication until the present day. Deletions appear to have the largest effect on pseudogene formation and loss of regulatory regions. Deleted regions are enriched for long DNA repeats and the flanking regions have high alignment scores, suggesting that non-allelic homologous recombination has played a significant role in the loss of DNA. To assess innovations in the X. laevis subgenomes we examined p300-bound enhancer peaks that are unique to one subgenome and absent from X. tropicalis. A large majority of new enhancers comprise transposable elements. Finally, to dissect early and late events following interspecific hybridization, we examined the epigenome and the enhancer landscape in X. tropicalis × X. laevis hybrid embryos. Strikingly, young X. tropicalis DNA transposons are derepressed and recruit p300 in hybrid embryos. Conclusions The results show that erosion of X. laevis genes and functional regulatory elements is associated with repeats and non-allelic homologous recombination and furthermore that young repeats have also contributed to the p300-bound regulatory landscape following hybridization and whole-genome duplication.

The transforming growth factor-β (TGF-β) family of cytokines performs a multifunctional signaling, which is integrated and coordinated in a signaling network that involves other pathways, such as Wintless, Forkhead box-O (FOXO) and Hedgehog and regulates pivotal functions related to cell fate in all tissues. In the hematopoietic system, TGF-β signaling controls a wide spectrum of biological processes, from immune system homeostasis to the quiescence and self-renewal of hematopoietic stem cells (HSCs). Recently an important role in post-transcription regulation has been attributed to two type of ncRNAs: microRNAs and pseudogenes. Ciona robusta, due to its philogenetic position close to vertebrates, is an excellent model to investigate mechanisms of post-transcriptional regulation evolutionarily highly conserved in immune homeostasis. The combined use of NGS and bioinformatic analyses suggests that in the pharynx, the hematopoietic organ of Ciona robusta, the Tgf-β, Wnt, Hedgehog and FoxO pathways are involved in tissue homeostasis, as they are in human. Furthermore, ceRNA network interactions and 3′UTR elements analyses of Tgf-β, Wnt, Hedgehog and FoxO pathways genes suggest that different miRNAs conserved (cin-let-7d, cin-mir-92c, cin-mir-153), species-specific (cin-mir-4187, cin-mir-4011a, cin-mir-4056, cin-mir-4150, cin-mir-4189, cin-mir-4053, cin-mir-4016, cin-mir-4075), pseudogenes (ENSCING00000011392, ENSCING00000018651, ENSCING00000007698) and mRNA 3′UTR elements are involved in post-transcriptional regulation in an integrated way in C. robusta.

Abstract Background Only a small portion of human long non-coding RNAs (lncRNAs) appear to be conserved outside of mammals, but the events underlying the birth of new lncRNAs in mammals remain largely unknown. One potential source is remnants of protein-coding genes that transitioned into lncRNAs. Results We systematically compare lncRNA and protein-coding loci across vertebrates, and estimate that up to 5% of conserved mammalian lncRNAs are derived from lost protein-coding genes. These lncRNAs have specific characteristics, such as broader expression domains, that set them apart from other lncRNAs. Fourteen lncRNAs have sequence similarity with the loci of the contemporary homologs of the lost protein-coding genes. We propose that selection acting on enhancer sequences is mostly responsible for retention of these regions. As an example of an RNA element from a protein-coding ancestor that was retained in the lncRNA, we describe in detail a short translated ORF in the JPX lncRNA that was derived from an upstream ORF in a protein-coding gene and retains some of its functionality. Conclusions We estimate that ~ 55 annotated conserved human lncRNAs are derived from parts of ancestral protein-coding genes, and loss of coding potential is thus a non-negligible source of new lncRNAs. Some lncRNAs inherited regulatory elements influencing transcription and translation from their protein-coding ancestors and those elements can influence the expression breadth and functionality of these lncRNAs.

Abstract Background Analyzing close species with diverse developmental modes is instrumental for investigating the evolutionary significance of physiological, anatomical and behavioral features at a molecular level. Many examples of trait loss are known in metazoan populations living in dark environments. Tunicates are the closest living relatives of vertebrates and typically present a lifecycle with distinct motile larval and sessile adult stages. The nervous system of the motile larva contains melanized cells associated with geotactic and light-sensing organs. It has been suggested that these are homologous to vertebrate neural crest-derived melanocytes. Probably due to ecological adaptation to distinct habitats, several species of tunicates in the Molgulidae family have tailless (anural) larvae that fail to develop sensory organ-associated melanocytes. Here we studied the evolution of Tyrosinase family genes, indispensible for melanogenesis, in the anural, unpigmented Molgula occulta and in the tailed, pigmented Molgula oculata by using phylogenetic, developmental and molecular approaches. Results We performed an evolutionary reconstruction of the tunicate Tyrosinase gene family: in particular, we found that M. oculata possesses genes predicted to encode one Tyrosinase (Tyr) and three Tyrosinase-related proteins (Tyrps) while M. occulta has only Tyr and Tyrp.a pseudogenes that are not likely to encode functional proteins. Analysis of Tyr sequences from various M. occulta individuals indicates that different alleles independently acquired frameshifting short indels and/or larger mobile genetic element insertions, resulting in pseudogenization of the Tyr locus. In M. oculata, Tyr is expressed in presumptive pigment cell precursors as in the model tunicate Ciona robusta. Furthermore, a M. oculata Tyr reporter gene construct was active in the pigment cell precursors of C. robusta embryos, hinting at conservation of the regulatory network underlying Tyr expression in tunicates. In contrast, we did not observe any expression of the Tyr pseudogene in M. occulta embryos. Similarly, M. occulta Tyr allele expression was not rescued in pigmented interspecific M. occulta × M. oculata hybrid embryos, suggesting deleterious mutations also to its cis-regulatory sequences. However, in situ hybridization for transcripts from the M. occulta Tyrp.a pseudogene revealed its expression in vestigial pigment cell precursors in this species. Conclusions We reveal a complex evolutionary history of the melanogenesis pathway in tunicates, characterized by distinct gene duplication and loss events. Our expression and molecular data support a tight correlation between pseudogenization of Tyrosinase family members and the absence of pigmentation in the immotile larvae of M. occulta. These results suggest that relaxation of purifying selection has resulted in the loss of sensory organ-associated melanocytes and core genes in the melanogenesis biosynthetic pathway in M. occulta.

Objective/Background: Clinical diagnosis of indeterminate and tuberculoid leprosy is often difficult due to limited and confounding signs and symptoms. In the current study, we evaluated the utility of new multiplex polymerase chain reaction (PCR) using Mycobacterium leprae-specific DNA sequences in the pseudogene regions of ML1545, ML2180, and ML2179 for PCR-based diagnosis of indeterminate leprosy (IND) and leprosy cases across the immunological spectrum. The sensitivity was compared with that of RLEP PCR. Methods: DNA was extracted from paraffin-embedded skin biopsy specimens of 220 leprosy cases, which were divided into IND (41), tuberculoid form (3), borderline tuberculoid (42), midborderline (3), borderline lepromatous (n=59), and lepromatous leprosy (72) cases. PCR positivity of both multiplex and RLEP PCR were compared in all the samples. A decision tree was constructed using the classification and regression trees algorithm to predict the probability of PCR positivity with the new multiplex PCR scheme in various clinical groups of leprosy. Sensitivity of each pseudogene target was determined using real-time PCR assays, and specificity was confirmed by PCR amplification of DNA extracted from three other mycobacterial species and skin biopsies of 44 non-leprosy cases. Results: A multiplex PCR positivity of 75.61% was noted in IND cases when compared to that of 58.54% using RLEP PCR (P < 0.05). Enhanced multiplex PCR positivity was noted across various clinical groups in comparison to RLEP PCR. The decision tree classifier has predicted statistically significant probability for multiplex PCR positivity among RLEP-PCR negative group and clinical groups with a low bacillary load. Conclusion: This new multiplex PCR scheme can support the diagnosis of indeterminate and tuberculoid forms of leprosy with limited clinical manifestations and can be implemented in basic clinical/diagnostic setting that possess conventional PCR facilities.

Transcriptome sequencing has led to the widespread identification of long non-coding RNAs (lncRNAs). Subsequently, these genes have been shown to hold functional importance in human cellular biology, which can be exploited by tumors to drive the hallmarks of cancer. Due to the complex tertiary structure and unknown binding motifs of lncRNAs, there is a growing disparity between the number of lncRNAs identified and those that have been functionally characterized. As such, lncRNAs deregulated in cancer may represent critical components of cancer pathways that could serve as novel therapeutic intervention points. Pseudogenes are non-coding DNA sequences that are defunct relatives of their protein-coding parent genes but retain high sequence similarity. Interestingly, certain lncRNAs expressed from pseudogene loci have been shown to regulate the protein-coding parent genes of these pseudogenes in trans particularly because of this sequence complementarity. We hypothesize that this phenomenon occurs more broadly than previously realized, and that aberrant expression of lncRNAs overlapping pseudogene loci provides an alternative mechanism of cancer gene deregulation. Using RNA-sequencing data from two cohorts of lung adenocarcinoma, each paired with patient-matched non-malignant lung samples, we discovered 104 deregulated pseudogene-derived lncRNAs. Remarkably, many of these deregulated lncRNAs (i) were expressed from the loci of pseudogenes related to known cancer genes, (ii) had expression that significantly correlated with protein-coding parent gene expression, and (iii) had lncRNA protein-coding parent gene expression that was significantly associated with survival. Here, we uncover evidence to suggest the lncRNA-pseudogene-protein-coding gene axis as a prominent mechanism of cancer gene regulation in lung adenocarcinoma, and highlights the clinical utility of exploring the non-coding regions of the cancer transcriptome.

Ferritin, the principal intracellular iron-storage protein localized in the cytoplasm, nucleus, and mitochondria, plays a major role in iron metabolism. The encoding ferritin genes are members of a multigene family that includes some pseudogenes. Even though pseudogenes have been initially considered as relics of ancient genes or junk DNA devoid of function, their role in controlling gene expression in normal and transformed cells has recently been re-evaluated. Numerous studies have revealed that some pseudogenes compete with their parental gene for binding to the microRNAs (miRNAs), while others generate small interference RNAs (siRNAs) to decrease functional gene expression, and still others encode functional mutated proteins. Consequently, pseudogenes can be considered as actual master regulators of numerous biological processes. Here, we provide a detailed classification and description of the structural features of the ferritin pseudogenes known to date and review the recent evidence on their mutual interrelation within the complex regulatory network of the ferritin gene family.

A small phylogenetically conserved sequence of 11,231 bp, termed FAM247, is repeated in human chromosome 22 by segmental duplications. This sequence forms part of diverse genes that span evolutionary time, the protein genes being the earliest as they are present in zebrafish and/or mice genomes, and the long noncoding RNA genes and pseudogenes the most recent as they appear to be present only in the human genome. We propose that the conserved sequence provides a nucleation site for new gene development at evolutionarily conserved chromosomal loci where the FAM247 sequences reside. The FAM247 sequence also carries information in its open reading frames that provides protein exon amino acid sequences; one exon plays an integral role in immune system regulation, specifically, the function of ubiquitin-specific protease (USP18) in the regulation of interferon. An analysis of this multifaceted sequence and the genesis of genes that contain it is presented.

Not all pseudogenes are transcriptionally silent as previously thought. Pseudogene transcripts, although not translated, contribute to the non-coding RNA pool of the cell that regulates the expression of other genes. Pseudogene transcripts can also directly compete with the parent gene transcripts for mRNA stability and other cell factors, modulating their expression levels. Tissue-specific and cancer-specific differential expression of these “functional” pseudogenes has been reported. To ascertain potential pseudogene:gene interactions in HIV-1 infection, we analyzed transcriptomes from infected and uninfected T-cells and found that 21 pseudogenes are differentially expressed in HIV-1 infection. This is interesting because parent genes of one-third of these differentially-expressed pseudogenes are implicated in HIV-1 life cycle, and parent genes of half of these pseudogenes are involved in different viral infections. Our bioinformatics analysis identifies candidate pseudogene:gene interactions that may be of significance in HIV-1 infection. Experimental validation of these interactions would establish that retroviruses exploit this newly-discovered layer of host gene expression regulation for their own benefit.

We assembled the mitogenome of the Bythograeid crab Segonzacia mesatlantica, using long-range amplification of the mitochondrial genome. The mitogenome is 15,521 base pair long (33.8% A, 21.7% C, 10.5% G, 34% T) with 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNAs, and a 624 bp AT-rich region. The gene arrangement is similar to other Brachyuran species. A whole genome shotgun sequencing approach revealed the presence of mitochondrial pseudogenes in the nuclear genome. This fifth mitogenome for a species of Bythograeidae should help resolve the puzzling question of the evolutionary origin of a family limited to deep-sea hydrothermal vents.

YRNAs are a type of short, noncoding RNAs. A total of four different transcripts can be distinguished, which are YRNA1, YRNA3, YRNA4 and YRNA5. All YRNAs are relatively small, made up of about 100 nucleotides each. YRNAs are characterized by a stem-loop structure and each part of that structure carries a different function. YRNAs are transcribed in the nucleus by RNA polymerase III. Then, the YRNA molecule is bound to the polyuridine tail of the La protein responsible for both its nuclear retention and protection from degradation. They also bind to the Ro60 protein, making the molecule more stable. In turn, YRNA-derived small RNAs (YsRNAs) are a class of YRNAs produced in apoptotic cells as a result of YRNA degradation. This process is performed by caspase-3-dependent pathways that form two groups of YsRNAs, with lengths of either approximately 24 or 31 nucleotides. From all four YRNA transcripts, 75 well-described pseudogenes are generated as a result of the mutation. However, available data indicates the formation of up to 1000 pseudogenes. YRNAs and YRNA-derived small RNAs may play a role in carcinogenesis due to their altered expression in cancers and influence on cell proliferation and inflammation. Nevertheless, our knowledge is still limited, and more research is required. The main aim of this review is to describe the current state of knowledge about YRNAs, their function and contribution to carcinogenesis, as well as their potential role in cancer diagnostics. To confirm the promising potential of YRNAs and YRNA-derived fragments as biomarkers, their significant role in several tumor types was taken into consideration.

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.

Multidrug resistance (MDR) is one of the main limitations of cancer treatment. The overexpression of drug-efflux pumps, such as P-glycoprotein (P-gp), is a major cause of MDR. Importantly, different studies have shown that extracellular vesicles (EVs) participate in the communication between MDR cells and drug-sensitive counterparts, promoting dissemination of the MDR phenotype. In the present work, we aimed to identify RNA species present in MDR cells and in EVs released by those cells, which may be associated with the MDR phenotype. The RNA content from two pairs (leukemia and lung cancer) of MDR (P-gp overexpressing) cells and their drug-sensitive counterparts, as well as from their EVs, was analyzed by deep sequencing. Our results showed distinctive transcripts for MDR cells and their EVs, when compared with their drug-sensitive counterparts. Remarkably, two pseudogenes (a novel pseudogene and RNA 5.8S ribosomal pseudogene 2) were found to be increased in EVs released by MDR cells in both leukemia and lung cancer models. Moreover, six miRs (miR-204-5p, miR-139-5p, miR-29c-5p, miR-551b-3p, miR-29b-2-5p, and miR-204-3p) exhibited altered levels in lung cancer MDR cells and their EVs. This study provides insights into the contribution of EVs to MDR.

Vertebrates can perceive at least five different taste qualities, each of which is thought to have a specific role in the evolution of different species. The avoidance of potentially poisonous foods, which are generally bitter or sour tasting, and the search for more nutritious ones, those with high-fat and high-sugar content, are two of the most well-known examples. The study of taste genes encoding receptors that recognize ligands triggering taste sensations has helped to reconstruct several evolutionary adaptations to dietary changes. In addition, an increasing number of studies have focused on pseudogenes, genomic DNA sequences that have traditionally been considered defunct relatives of functional genes mostly because of the presence of deleterious mutations interrupting their open reading frames. The study of taste receptor pseudogenes has helped to shed light on how the evolutionary history of taste in vertebrates has been the result of a succession of gene gain and loss processes. This dynamic role in evolution has been explained by the “less-is-more” hypothesis, suggesting gene loss as a mechanism of evolutionary change in response to a dietary shift. This mini-review aims at depicting the major lineage-specific loss of function of taste receptor genes in vertebrates, stressing their evolutionary importance and recapitulating signatures of natural selection and their correlations with food habits.

Most of RNA transcripts, instead of serving templates for protein synthesis, perform different functions such as control of embryogenesis, differentiation, imprinting, X-chromosome inactivation, immune response, and stress reactions. A large portion of these RNAs are denoted as long non-coding RNAs (lncRNAs) which function as signaling molecules, navigating systems and platforms for ribonucleic complexes assembly. They also participate in the organization of specific cellular domains. Moreover, they are able to bind regulatory proteins and micro-RNAs, and serve as precursors for small RNAs. It was shown that lncRNAs participate in transcription regulation; they are also involved in alternative splicing, RNA editing, traffic, translation and degradation of RNA. The three-dimensional structure of lncRNAs plays a crucial role in processes of chromatin remodeling and transcription regulation. In this review we discuss various aspects of lncRNAs functioning.

Although bacterial genomes have been traditionally viewed as being very compact, with relatively low amounts of repetitive and non-coding DNA, this view has dramatically changed in recent years. The increase of available complete bacterial genomes has revealed that many species present abundant repetitive DNA (i.e., insertion sequences, prophages or paralogous genes) and that many of these sequences are not functional but can have evolutionary consequences as concerns the adaptation to specialized host-related ecological niches. Comparative genomics analyses with close relatives that live in non-specialized environments reveal the nature and fate of this bacterial junk DNA. In addition, the number of insertion sequences and pseudogenes, as well as the size of the intergenic regions, can be used as markers of the evolutionary stage of a genome.

Pseudogenes are abundant in the human genome and had long been thought of purely as nonfunctional gene fossils. Recent observations point to a role for pseudogenes in regulating genes transcriptionally and post-transcriptionally in human cells. To computationally interrogate the network space of integrated pseudogene and long non-coding RNA regulation in the human transcriptome, we developed and implemented an algorithm to identify all long non-coding RNA (lncRNA) transcripts that overlap the genomic spans, and specifically the exons, of any human pseudogenes in either sense or antisense orientation. As inputs to our algorithm, we imported three public repositories of pseudogenes: GENCODE v17 (processed and unprocessed, Ensembl 72); Retroposed Pseudogenes V5 (processed only) and Yale Pseudo60 (processed and unprocessed, Ensembl 60); two public lncRNA catalogs: Broad Institute, GENCODE v17; NCBI annotated piRNAs; and NHGRI clinical variants. The data sets were retrieved from the UCSC Genome Database using the UCSC Table Browser. We identified 2277 loci containing exon-to-exon overlaps between pseudogenes, both processed and unprocessed, and long non-coding RNA genes. Of these loci we identified 1167 with Genbank EST and full-length cDNA support providing direct evidence of transcription on one or both strands with exon-to-exon overlaps. The analysis converged on 313 pseudogene-lncRNA exon-to-exon overlaps that were bidirectionally supported by both full-length cDNAs and ESTs. In the process of identifying transcribed pseudogenes, we generated a comprehensive, positionally non-redundant encyclopedia of human pseudogenes, drawing upon multiple, and formerly disparate public pseudogene repositories. Collectively, these observations suggest that pseudogenes are pervasively transcribed on both strands and are common drivers of gene regulation.