Loss to gain: pseudogenes in microorganisms, focusing on eubacteria, and their biological significance.

Pseudogenes are defined as "non-functional" copies of corresponding parent genes. The cognition of pseudogenes continues to be refreshed through accumulating and updating research findings. Previous studies have predominantly focused on mammals, but pseudogenes have received relatively less attention in the field of microbiology. Given the increasing recognition on the importance of pseudogenes, in this review, we focus on several aspects of microorganism pseudogenes, including their classification and characteristics, their generation and fate, their identification, their abundance and distribution, their impact on virulence, their ability to recombine with functional genes, the extent to which some pseudogenes are transcribed and translated, and the relationship between pseudogenes and viruses. By summarizing and organizing the latest research progress, this review will provide a comprehensive perspective and improved understanding on pseudogenes in microorganisms. KEY POINTS: • Concept, classification and characteristics, identification and databases, content, and distribution of microbial pseudogenes are presented. • How pseudogenization contribute to pathogen virulence is highlighted. • Pseudogenes with potential functions in microorganisms are discussed.

The physical and evolutionary energy landscapes of devolved protein sequences corresponding to pseudogenes.

Protein evolution is guided by structural, functional, and dynamical constraints ensuring organismal viability. Pseudogenes are genomic sequences identified in many eukaryotes that lack translational activity due to sequence degradation and thus over time have undergone "devolution." Previously pseudogenized genes sometimes regain their protein-coding function, suggesting they may still encode robust folding energy landscapes despite multiple mutations. We study both the physical folding landscapes of protein sequences corresponding to human pseudogenes using the Associative Memory, Water Mediated, Structure and Energy Model, and the evolutionary energy landscapes obtained using direct coupling analysis (DCA) on their parent protein families. We found that generally mutations that have occurred in pseudogene sequences have disrupted their native global network of stabilizing residue interactions, making it harder for them to fold if they were translated. In some cases, however, energetic frustration has apparently decreased when the functional constraints were removed. We analyzed this unexpected situation for Cyclophilin A, Profilin-1, and Small Ubiquitin-like Modifier 2 Protein. Our analysis reveals that when such mutations in the pseudogene ultimately stabilize folding, at the same time, they likely alter the pseudogenes' former biological activity, as estimated by DCA. We localize most of these stabilizing mutations generally to normally frustrated regions required for binding to other partners.

New investigation of encoding secondary metabolites gene by genome mining of a marine bacterium, Pseudoalteromonas viridis BBR56.

Pseudoalteromonas viridis strain BBR56 was isolated from seawater at Dutungan Island, South Sulawesi, Indonesia. Bacterial DNA was isolated using Promega Genomic DNA TM050. DNA purity and quantity were assessed using NanoDrop spectrophotometers and Qubit fluorometers. The DNA library and sequencing were prepared using Oxford Nanopore Technology GridION MinKNOW 20.06.9 with long read, direct, and comprehensive analysis. High accuracy base calling was assessed with Guppy version 4.0.11. Filtlong and NanoPlot were used for filtering and visualizing the FASTQ data. Flye (2.8.1) was used for de novo assembly analysis. Variant calls and consensus sequences were created using Medaka. The annotation of the genome was elaborated by DFAST. The assembled genome and annotation were tested using Busco and CheckM. Herein, we found that the highest similarity of the BBR56 isolate was 98.37% with the 16 S rRNA gene sequence of P. viridis G-1387. The genome size was 5.5 Mb and included chromosome 1 (4.2 Mbp) and chromosome 2 (1.3 Mbp), which encoded 61 pseudogenes, 4 noncoding RNAs, 113 tRNAs, 31 rRNAs, 4,505 coding DNA sequences, 4 clustered regularly interspaced short palindromic repeats, 4,444 coding genes, and a GC content of 49.5%. The sequence of the whole genome of P. viridis BBR56 was uploaded to GenBank under the accession numbers CP072425-CP072426, biosample number SAMN18435505, and bioproject number PRJNA716373. The sequence read archive (SRR14179986) was successfully obtained from NCBI for BBR56 raw sequencing reads. Digital DNA-DNA hybridization results showed that the genome of BBR56 had the potential to be a new species because no other bacterial genomes were similar to the sample. Biosynthetic gene clusters (BGCs) were assessed using BAGEL4 and the antiSMASH bacterial version. The genome harbored diverse BGCs, including genes that encoded polyketide synthase, nonribosomal peptide synthase, RiPP-like, NRP-metallophore, hydrogen cyanide, betalactone, thioamide-NRP, Lant class I, sactipeptide, and prodigiosin. Thus, BBR56 has considerable potential for further exploration regarding the use of its secondary metabolite products in the human and fisheries sectors.

Structural Features and Physiological Associations of Human 14-3-3ζ Pseudogenes.

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.

Many purported pseudogenes in bacterial genomes are bona fide genes.

BACKGROUND: Microbial genomes are largely comprised of protein coding sequences, yet some genomes contain many pseudogenes caused by frameshifts or internal stop codons. These pseudogenes are believed to result from gene degradation during evolution but could also be technical artifacts of genome sequencing or assembly. RESULTS: Using a combination of observational and experimental data, we show that many putative pseudogenes are attributable to errors that are incorporated into genomes during assembly. Within 126,564 publicly available genomes, we observed that nearly identical genomes often substantially differed in pseudogene counts. Causal inference implicated assembler, sequencing platform, and coverage as likely causative factors. Reassembly of genomes from raw reads confirmed that each variable affects the number of putative pseudogenes in an assembly. Furthermore, simulated sequencing reads corroborated our observations that the quality and quantity of raw data can significantly impact the number of pseudogenes in an assembler dependent fashion. The number of unexpected pseudogenes due to internal stops was highly correlated (R2 = 0.96) with average nucleotide identity to the ground truth genome, implying relative pseudogene counts can be used as a proxy for overall assembly correctness. Applying our method to assemblies in RefSeq resulted in rejection of 3.6% of assemblies due to significantly elevated pseudogene counts. Reassembly from real reads obtained from high coverage genomes showed considerable variability in spurious pseudogenes beyond that observed with simulated reads, reinforcing the finding that high coverage is necessary to mitigate assembly errors. CONCLUSIONS: Collectively, these results demonstrate that many pseudogenes in microbial genome assemblies are actually genes. Our results suggest that high read coverage is required for correct assembly and indicate an inflated number of pseudogenes due to internal stops is indicative of poor overall assembly quality.

Exosomal lncRNA DUXAP8 affecting CHPF2 in the pathogenesis of intracranial aneurysms.

OBJECTIVE: This study endeavored to explore the relationship between exosome-derived lncRNA Double Homeobox A Pseudogene 8 (DUXAP8) and Chondroitin Polymerizing Factor 2 (CHPF2), and their roles in the pathogenesis of intracranial aneurysm (IA). METHODS: The shared targeted molecules (DUXAP8 and CHPF2) were detected via GSE122897 and GSE75436 datasets. A total of 312 patients with IAs were incorporated into this study. Exosomes were isolated from serum samples, and their identity was confirmed using Western blotting for exosomal markers (CD9, CD63 and ALIX). Inflammatory responses in IA tissues were evaluated using Hematoxylin-Eosin staining. CHPF2 protein concentration and the expression levels of DUXAP8 and CHPF2 mRNA in exosomal samples were assessed using Immunochemistry (IHC), Western Blotting, and qRT-PCR, respectively. Cell-based assays involving Human Umbilical Vein Endothelial Cells (HuvECs), including transfection with exosomal DUXAP8, Western Blotting, qRT-PCR, and Cell Counting Kit-8, were conducted. Receiver Operating Characteristic (ROC) curves were derived using SPSS. RESULTS: DUXAP8 level affects the level of CHPF2. DUXAP8 expression within exosomes was associated with increased CD9, CD63, ALIX and CHPF2 levels during IA development and inflammatory stress. In HuvECs, transfection with exosomes carrying DUXAP8 siRNA resulted in reduced CHPF2 expression, whereas DUXAP8 mimic increased CHPF2 concentrations. The Area Under the ROC Curve (AUC) for exosomal DUXAP8 expression and CHPF2 levels, and aneurysm size was 0.768 (95% CI, 0.613 to 0.924), 0.937 (95% CI, 0.853 to 1.000), and 0.943 (95% CI, 0.860, 1.000), respectively. CONCLUSION: Exosome-derived DUXAP8 promotes IA progression by affecting CHPF2 expression.

Four classic "de novo" genes all have plausible homologs and likely evolved from retro-duplicated or pseudogenic sequences.

Despite being previously regarded as extremely unlikely, the idea that entirely novel protein-coding genes can emerge from non-coding sequences has gradually become accepted over the past two decades. Examples of "de novo origination", resulting in lineage-specific "orphan" genes, lacking coding orthologs, are now produced every year. However, many are likely cases of duplicates that are difficult to recognize. Here, I re-examine the claims and show that four very well-known examples of genes alleged to have emerged completely "from scratch"- FLJ33706 in humans, Goddard in fruit flies, BSC4 in baker's yeast and AFGP2 in codfish-may have plausible evolutionary ancestors in pre-existing genes. The first two are likely highly diverged retrogenes coding for regulatory proteins that have been misidentified as orphans. The antifreeze glycoprotein, moreover, may not have evolved from repetitive non-genic sequences but, as in several other related cases, from an apolipoprotein that could have become pseudogenized before later being reactivated. These findings detract from various claims made about de novo gene birth and show there has been a tendency not to invest the necessary effort in searching for homologs outside of a very limited syntenic or phylostratigraphic methodology. A robust approach is used for improving detection that draws upon similarities, not just in terms of statistical sequence analysis, but also relating to biochemistry and function, to obviate notable failures to identify homologs.

Stage II oesophageal carcinoma: peril in disguise associated with cellular reprogramming and oncogenesis regulated by pseudogenes.

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.

Evidence That Aquaporin 11 (AQP11) in the Spiny Dogfish (Squalus acanthias) May Represent a Pseudogene.

Various attempts to amplify an AQP11 cDNA from tissues of the spiny dogfish (Squalus acanthias) were made. Two pairs of deoxy-inosine-containing degenerate primers were designed based on conserved amino acid sequences from an AQP11 alignment. These primers yielded some faint bands from gill cDNA that were sequenced. Blast searches with the sequences showed they were not AQP11. An elasmobranch AQP11 nucleotide sequence alignment was produced to identify conserved regions to make further degenerate primers. One primer pair produced a short 148 bp fragment showing particularly strong amplification in gill and intestine. It was sequenced and represented a piece of the AQP11 gene. However, as the fragment may have resulted from contaminating genomic DNA (in total RNA used to make cDNA), 5' and 3' RACE were performed to amplify the two ends of the putative cDNA. Furthermore, 5' and 3' RACE amplifications depend on the presence of a 5' cap nucleotide and a poly A tail, respectively on the putative AQP11 mRNA. Hence, successful amplification was only possible from cDNA and not genomic DNA. Nested RACE amplifications were performed using gill and intestinal RACE cDNA, but none of the DNA fragments sequenced were AQP11. Consequently, the spiny dogfish AQP11 gene may represent a pseudogene.

Pseudogenomic insights into the evolution of Mycobacterium ulcerans.

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.

SIGNIFICANCE: Prevention and treatment of Buruli ulcer is still a problem but large whole genome datasets on M. ulcerans are readily available. However, genomic studies fail to thoroughly investigate pseudogenes to probe evolutionary changes in the bacteria, and this can be attributed to the lack of bioinformatic tools. This work studied pseudogenes in Mycobacterium ulcerans (MU) to understand its adapted niche and evolutionary differences across African strains. Our results posit an MU niche-adapted model important in understanding transmission. Also, MU pseudogene profiles vary based on lineage and country, suggesting their influence on pseudogenization patterns in the genome. We further identify a reduction in insertion sequences that are used for the detection of the bacteria which may affect the sensitivity of diagnosis.

The 75-99 C-Terminal Peptide of URG7 Protein Promotes α-Synuclein Disaggregation.

Up Regulation Gene seven (URG7) is the pseudogene 2 of the transporter ABCC6. The translated URG7 protein is localized with its single transmembrane α-helix in the endoplasmic reticulum (ER) membrane, orienting the N- and C-terminal regions in the lumen and cytoplasm, respectively, and it plays a crucial role in the folding of ER proteins. Previously, the C-terminal region of URG7 (PU, residues 75-99) has been shown to modify the aggregation state of α-synuclein in the lysate of HepG2 cells. PU analogs were synthesized, and their anti-aggregation potential was tested in vitro on α-synuclein obtained using recombinant DNA technology. Circular dichroism (CD), differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and microscopic techniques were used to assess the sample's behavior. The results show that the peptides studied by themselves are prone to clathrate-like structure formation of variable stability. Aggregation of α-synuclein is accompanied by desolvation of its peptide chain and an increase in intermolecular ß-sheets. The PU analogs all interact with α-synuclein aggregates and those possessing the most stable clathrate-like structures have the highest disaggregating effect. These findings suggest that the C-terminal region of URG7 may have a role in interacting and modulating α-synuclein structures and could be used to generate interesting therapeutic candidates as disaggregators of α-synuclein.

UBDP1 pseudogene and UBD network competitively bind miR­6072 to promote glioma progression.

Increasing evidence suggests that pseudogenes play crucial roles in various cancers, yet their functions and regulatory mechanisms in glioma pathogenesis remain enigmatic. In the present study, a novel pseudogene was identified, UBDP1, which is significantly upregulated in glioblastoma and positively correlated with the expression of its parent gene, UBD. Additionally, high levels of these paired genes are linked with a poor prognosis for patients. In the present study, clinical samples were collected followed by various analyses including microarray for long non­coding RNAs, reverse transcription­quantitative PCR, fluorescence in situ hybridization and western blotting. Cell lines were authenticated and cultured then subjected to various assays for proliferation, migration, and invasion to investigate the molecular mechanisms. Bioinformatic tools identified miRNA targets, and luciferase reporter assays validated these interactions. A tumor xenograft model in mice was used for in vivo studies. In vitro and in vivo studies have demonstrated that UBDP1, localized in the cytoplasm, functions as a tumor­promoting factor influencing cell proliferation, migration, invasion and tumor growth. Mechanistic investigations have indicated that UBDP1 exerts its oncogenic effects by decoying miR­6072 from UBD mRNA, thus forming a competitive endogenous RNA network, which results in the enhanced oncogenic activity of UBD. The present findings offered new insights into the role of pseudogenes in glioma progression, suggesting that targeting the UBDP1/miR­6072/UBD network may serve as a potential therapeutic strategy for glioma patients.

Pseudogenes act as a neutral reference for detecting selection in prokaryotic pangenomes.

A long-standing question is to what degree genetic drift and selection drive the divergence in rare accessory gene content between closely related bacteria. Rare genes, including singletons, make up a large proportion of pangenomes (all genes in a set of genomes), but it remains unclear how many such genes are adaptive, deleterious or neutral to their host genome. Estimates of species' effective population sizes (Ne) are positively associated with pangenome size and fluidity, which has independently been interpreted as evidence for both neutral and adaptive pangenome models. We hypothesized that pseudogenes, used as a neutral reference, could be used to distinguish these models. We find that most functional categories are depleted for rare pseudogenes when a genome encodes only a single intact copy of a gene family. In contrast, transposons are enriched in pseudogenes, suggesting they are mostly neutral or deleterious to the host genome. Thus, even if individual rare accessory genes vary in their effects on host fitness, we can confidently reject a model of entirely neutral or deleterious rare genes. We also define the ratio of singleton intact genes to singleton pseudogenes (si/sp) within a pangenome, compare this measure across 668 prokaryotic species and detect a signal consistent with the adaptive value of many rare accessory genes. Taken together, our work demonstrates that comparing with pseudogenes can improve inferences of the evolutionary forces driving pangenome variation.

Targeted long-read sequencing for comprehensive detection of CYP21A2 mutations in patients with 21-hydroxylase deficiency.

BACKGROUND: 21-Hydroxylase deficiency (21-OHD) is caused by pathogenic CYP21A2 variations. CYP21A2 is arranged in tandem with its highly homologous pseudogene CYP21A1P; therefore, it is prone to mismatch and rearrangement, producing different types of complex variations. There were few reports on using only one method to detect different CYP21A2 variants simultaneously. AIMS: Targeted long-read sequencing method was used to detect all types of CYP21A2 variants in a series of patients with 21-OHD. METHODS: A total of 59 patients with 21-OHD were enrolled from Peking Union Medical College Hospital. Long-range locus-specific PCR and long-read sequencing (LRS) were performed to detect the pathogenic variants in CYP21A2. RESULTS: Copy-number variants of CYP21A2 were found in 25.4% of patients, including 5.1% with 3 copies of CYP21A2, 16.9% with 1 copy of CYP21A2, and 3.4% with 0 copy of CYP21A2. The remaining 74.6% of patients had 2 copies of CYP21A2. Pathogenic variants were identified in all 121 alleles of 59 patients. Specifically, single-nucleotide variants and small insertions/deletions (< 50 bp) were detected in 79 alleles, of which conversed from CYP21A1P were detected in 63 alleles, and rare variants were found in the other 16 alleles. Large gene conversions (> 50 bp) from pseudogene were detected in 10 alleles, and different chimeric genes (CYP21A1P/CYP21A2 or TNXA/TNXB) formed by large deletions were detected in 32 alleles. Of all variants, p.I173N was the most common variant (19.0%). CONCLUSIONS: Our study demonstrated that targeted long-read sequencing is a comprehensive method for detecting CYP21A2 variations, which is helpful for genetic diagnosis in 21-OHD patients.

PTEN regulates expression of its pseudogene in glioblastoma cells in DNA methylation-dependent manner.

Glioblastoma (GBM) is the most aggressive and frequent type of primary brain cancer in adult patients. One of the key molecular features associated with GBM pathogenesis is the dysfunction of PTEN oncosuppressor. In addition to PTEN gene, humans and several primates possess processed PTEN pseudogene (PTENP1) that gives rise to long non-coding RNA lncPTENP1-S. Regulation and functions of PTEN and PTENP1 are highly interconnected, however, the exact molecular mechanism of how these two genes affect each other remains unclear. Here, we analyzed the methylation level of the CpG islands (CpGIs) in the promoter regions of PTEN and PTENP1 in patient-derived GBM neurospheres. We found that increased PTEN methylation corelates with decreased PTEN mRNA level. Unexpectedly, we showed the opposite trend for PTENP1. Using targeted methylation and demethylation of PTENP1 CpGI, we demonstrated that DNA methylation increases lncPTENP1-S expression in the presence of wild type PTEN protein but decreases lncPTENP1-S expression if PTEN protein is absent. Further experiments revealed that PTEN protein binds to PTENP1 promoter region and inhibits lncPTENP1-S expression if its CpGI is demethylated. Interestingly, we did not detect any effect of lncPTENP1-S on the level of PTEN mRNA, indicating that in GBM cells PTENP1 is a downstream target of PTEN rather than its upstream regulator. Finally, we studied the functions of lncPTENP1-S and demonstrated that it plays a pro-oncogenic role in GBM cells by upregulating the expression of cancer stem cell markers and decreasing cell adhesion.

PMS2 or PMS2CL? Characterization of variants detected in the 3' of the PMS2 gene.

PMS2 germline pathogenic variants are one of the major causes for Lynch syndrome and constitutional mismatch repair deficiencies. Variant identification in the 3' region of this gene is complicated by the presence of the pseudogene PMS2CL which shares a high sequence homology with PMS2. Consequently, short-fragment screening strategies (NGS, Sanger) may fail to discriminate variant's gene localization. Using a comprehensive analysis strategy, we assessed 42 NGS-detected variants in 76 patients and found 32 localized on PMS2 while 6 on PMS2CL. Interestingly, four variants were detected in either of them in different patients. Clinical phenotype was well correlated to genotype, making it very helpful in variant assessment. Our findings emphasize the necessity of more specific complementary analyses to confirm the gene origin of each variant detected in different individuals in order to avoid variant misinterpretation. In addition, we characterized two PMS2 genomic alterations involving Alu-mediated tandem duplication and gene conversion. Those mechanisms seemed to be particularly favored in PMS2 which contribute to frequent genomic rearrangements in the 3' region of the gene.

Pathological roles of miRNAs and pseudogene-derived lncRNAs in human cancers, and their comparison as prognosis/diagnosis biomarkers.

This review examines and compares the diagnostic and prognostic capabilities of miRNAs and lncRNAs derived from pseudogenes in cancer patients. Additionally, it delves into their roles in cancer pathogenesis. Both miRNAs and pseudogene-derived lncRNAs have undergone thorough investigation as remarkably sensitive and specific cancer biomarkers, offering significant potential for cancer detection and monitoring. . Extensive research is essential to gain a complete understanding of the precise roles these non-coding RNAs play in cancer, allowing the development of novel targeted therapies and biomarkers for improved cancer detection and treatment approaches.

Construction and analysis of pseudogene-related ceRNA network in breast cancer.

Breast cancer (BC) is one of the leading causes of cancer-related deaths in women. The present study explored the potential role of pseudogenes in BC via construction and analysis of a competing endogenous RNA (ceRNA) network through a three-step process. First, we screened differentially expressed genes in nine BC datasets. Then the gene-pseudogenes pairs (nine hub genes) were selected according to the functional enrichment and correlation analysis. Second, the candidate hub genes and interacting miRNAs were used to construct the ceRNA network. Further analysis of the ceRNA network revealed a crucial ceRNA module with two genes-pseudogene pairs and two miRNAs. The in-depth analysis identified the GBP1/hsa-miR-30d-5p/GBP1P1 axis as a potential tumorigenic axis in BC patients. In the third step, the GBP1/hsa-miR-30d-5p/GBP1P1 axis expression level was assessed in 40 tumor/normal BC patients and MCF-7 cell lines. The expression of GBP1 and GBP1P1 was significantly higher in the tumor compared to the normal tissue. However, the expression of hsa-miR-30d-5p was lower in tumor samples. Then, we introduced the GBP1P1 pseudogene into the MCF-7 cell line to evaluate its effect on GBP1 and hsa-miR-30d-5p expression. As expected, the GBP1 level increased while the hsa-miR-30d-5p level decreased in the GBP1P1-overexprsssing cell line. In addition, the oncogenic properties of MCF-7 (cell viability, clonogenicity, and migration) were improved after GBP1P1 overexpression. In conclusion, we report a ceRNA network that may provide new insight into the role of pseudogenes in BC development.

Ambiguous genes due to aligners and their impact on RNA-seq data analysis.

The main scope of the study is ambiguous genes, i.e. genes whose expression is difficult to estimate from the data produced by next-generation sequencing technologies. We focused on the RNA sequencing (RNA-Seq) type of experiment performed on the Illumina platform. It is crucial to identify such genes and understand the cause of their difficulty, as these genes may be involved in some diseases. By giving misleading results, they could contribute to a misunderstanding of the cause of certain diseases, which could lead to inappropriate treatment. We thought that the ambiguous genes would be difficult to map because of their complex structure. So we looked at RNA-seq analysis using different mappers to find genes that would have different measurements from the aligners. We were able to identify such genes using a generalized linear model with two factors: mappers and groups introduced by the experiment. A large proportion of ambiguous genes are pseudogenes. High sequence similarity of pseudogenes to functional genes may indicate problems in alignment procedures. In addition, predictive analysis verified the performance of difficult genes in classification. The effectiveness of classifying samples into specific groups was compared, including the expression of difficult and not difficult genes as covariates. In almost all cases considered, ambiguous genes have less predictive power.