The World of Pseudogenes: New Diagnostic and Therapeutic Targets in Cancers or Still Mystery Molecules?

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.

Species-specific partial gene duplication in Arabidopsis thaliana evolved novel phenotypic effects on morphological traits under strong positive selection

Gene duplication is increasingly recognized as an important mechanism for the origination of new genes, as revealed by comparative genomic analysis. However, how new duplicate genes contribute to phenotypic evolution remains largely unknown, especially in plants. Here, we identified the new gene EXOV, derived from a partial gene duplication of its parental gene EXOVL in Arabidopsis thaliana. EXOV is a species-specific gene that originated within the last 3.5 million years and shows strong signals of positive selection. Unexpectedly, RNA-seq analyses revealed that, despite its young age, EXOV has acquired many novel direct and indirect interactions in which the parental gene does not engage. This observation is consistent with the high, selection-driven substitution rate of its encoded protein, in contrast to the slowly evolving EXOVL, suggesting an important role for EXOV in phenotypic evolution. We observed significant differentiation of morphological changes for all phenotypes assessed in genome-edited and T-DNA insertional single mutants and in double T-DNA insertion mutants in EXOV and EXOVL. We discovered a substantial divergence of phenotypic effects by principal component analyses, suggesting neofunctionalization of the new gene. These results reveal a young gene that plays critical roles in biological processes that underlie morphological evolution in A. thaliana.

PLEKHA8P1 Promotes Tumor Progression and Indicates Poor Prognosis of Liver Cancer

Hepatocellular carcinoma (HCC) records the second-lowest 5-year survival rate despite the avalanche of research into diagnosis and therapy. One of the major obstacles in treatment is chemoresistance to drugs such as 5-fluorouracil (5-FU), making identification and elucidation of chemoresistance regulators highly valuable. As the regulatory landscape grows to encompass non-coding genes such as long non-coding RNAs (lncRNAs), a relatively new class of lncRNA has emerged in the form of pseudogene-derived lncRNAs. Through bioinformatics analyses of the TCGA LIHC dataset, we have systematically identified pseudogenes of prognostic value. Initial experimental validation of selected pseudogene-derived lncRNA (PLEKHA8P1) and its parental gene (PLEKHA8), a well-studied transport protein in Golgi complex recently implicated as an oncogene in both colorectal and liver cancer, indicates that the pseudogene/parental gene pair promotes tumor progression and that their dysregulated expression levels affect 5-FU-induced chemoresistance in human HCC cell line FT3-7. Our study has thus confirmed cancer-related functions of PLEKHA8, and laid the groundwork for identification and validation of oncogenic pseudogene-derived lncRNA that shows potential as a novel therapeutic target in circumventing chemoresistance induced by 5-FU.

Parental bias in expression and interaction of genes in the equine placenta

Most autosomal genes in the placenta show a biallelic expression pattern. However, some genes exhibit allele-specific transcription depending on the parental origin of the chromosomes on which the copy of the gene resides. Parentally expressed genes are involved in the reciprocal interaction between maternal and paternal genes, coordinating the allocation of resources between fetus and mother. One of the main challenges of studying parental-specific allelic expression (allele-specific expression [ASE]) in the placenta is the maternal cellular remnant at the fetomaternal interface. Horses (Equus caballus) have an epitheliochorial placenta in which both the endometrial epithelium and the epithelium of the chorionic villi are juxtaposed with minimal extension into the uterine mucosa, yet there is no information available on the allelic gene expression of equine chorioallantois (CA). In the current study, we present a dataset of 1,336 genes showing ASE in the equine CA (https://pouya-dini.github.io/equine-gene-db/) along with a workflow for analyzing ASE genes. We further identified 254 potentially imprinted genes among the parentally expressed genes in the equine CA and evaluated the expression pattern of these genes throughout gestation. Our gene ontology analysis implies that maternally expressed genes tend to decrease the length of gestation, while paternally expressed genes extend the length of gestation. This study provides fundamental information regarding parental gene expression during equine pregnancy, a species with a negligible amount of maternal cellular remnant in its placenta. This information will provide the basis for a better understanding of the role of parental gene expression in the placenta during gestation.

Imprints of independent allopolyploid formations on patterns of gene expression in two sibling yarrow species (Achillea, Asteraceae)

Background Polyploid species often originate recurrently. While this is well known, there is little information on the extent to which distinct allotetraploid species formed from the same parent species differ in gene expression. The tetraploid yarrow species Achillea alpina and A. wilsoniana arose independently from allopolyploidization between diploid A. acuminata and A. asiatica. The genetics and geography of these origins are clear from previous studies, providing a solid basis for comparing gene expression patterns of sibling allopolyploid species that arose independently. Results We conducted comparative RNA-sequencing analyses on the two Achillea tetraploid species and their diploid progenitors to evaluate: 1) species-specific gene expression and coexpression across the four species; 2) patterns of inheritance of parental gene expression; 3) parental contributions to gene expression in the allotetraploid species, and homeolog expression bias. Diploid A. asiatica showed a higher contribution than diploid A. acuminata to the transcriptomes of both tetraploids and also greater homeolog bias in these transcriptomes, possibly reflecting a maternal effect. Comparing expressed genes in the two allotetraploids, we found expression of ca. 30% genes were species-specific in each, which were most enriched for GO terms pertaining to “defense response”. Despite species-specific and differentially expressed genes between the two allotetraploids, they display similar transcriptome changes in comparison to their diploid progenitors. Conclusion Two independently originated Achillea allotetraploid species exhibited difference in gene expression, some of which must be related to differential adaptation during their post-speciation evolution. On the other hand, they showed similar expression profiles when compared to their progenitors. This similarity might be expected when pairs of merged diploid genomes in tetraploids are similar, as is the case in these two particular allotetraploids.

Species-specific gene duplication in Arabidopsis thaliana evolved novel phenotypic effects on morphological traits under strong positive selection

Gene duplication is increasingly recognized as an important mechanism for the origination of new genes, as revealed by comparative genomic analysis. However, the ways in which new duplicate genes contribute to phenotypic evolution remain largely unknown, especially in plants, owing to a lack of experimental and phenotypic data. In this study, we identified the new gene Exov, derived from a partial gene region duplication of its parental gene Exov-L, which is a member of an exonuclease family, into a different chromosome in Arabidopsis thaliana. We experimentally investigated the phenotypic effects of Exov and Exov-L in an attempt to understand how the new gene diverged from the parental copy and contributes to phenotypic evolution. Evolutionary analysis demonstrated that Exov is a species-specific gene that originated within the last 3.5 million years and shows strong signals of positive selection. Unexpectedly, RNAseq analyses reveal that the new gene, despite its young age, has acquired a large number of novel direct and indirect interactions in which the parental gene does not engage. This is consistent with a high, selection-driven substitution rate in the protein sequence encoded by Exov in contrast to the slowly evolving Exov-L, suggesting an important role for Exov in phenotypic evolution. We analyzed phenotypic effects of exov and exov-l single T-DNA-insertion mutants; double exov, exov-l T-DNA insertion mutants; and CRISPR/Cas9-mediated exovcrp and exov-lcrp knockouts on seven morphological traits in both the new and parental genes. We detected significant segregation of morphological changes for all seven traits when assessed in terms of single mutants, as well as morphological changes for seven traits associated with segregation of double exov, exov-l mutants. Substantial divergence of phenotypic effects between new and parental genes was revealed by principal component analyses, suggesting neofunctionalization in the new gene. These results reveal a young gene that plays critical roles in biological processes that underlie morphological and developmental evolution in Arabidopsis thaliana.

DiseaseLinc: Disease Enrichment Analysis of Sets of Differentially Expressed LincRNAs

Long intergenic non-coding RNAs (LincRNAs) are long RNAs that do not encode proteins. Functional evidence is lacking for most of them. Their biogenesis is not well-known, but it is thought that many lincRNAs originate from genomic duplication of coding material, resulting in pseudogenes, gene copies that lose their original function and can accumulate mutations. While most pseudogenes eventually stop producing a transcript and become erased by mutations, many of these pseudogene-based lincRNAs keep similarity to the parental gene from which they originated, possibly for functional reasons. For example, they can act as decoys for miRNAs targeting the parental gene. Enrichment analysis of function is a powerful tool to discover the functional effects of a treatment producing differential expression of transcripts. However, in the case of lincRNAs, since their function is not easy to define experimentally, such a tool is lacking. To address this problem, we have developed an enrichment analysis tool that focuses on lincRNAs exploiting their functional association, using as a proxy function that of the parental genes and has a focus on human diseases. The tool is available at: http://cbdm-01.zdv.uni-mainz.de:3838/piyusmor/DiseaseLinc/.

Differential CircRNA Expression Signatures May Serve as Potential Novel Biomarkers in Prostate Cancer

Circular RNAs (circRNAs), a recently discovered non-coding RNA, have a number of functions including the regulation of miRNA expression. They have been detected in a number of malignancies including prostate cancer (PCa). The differential expression pattern of circRNAs associated with PCa and androgen receptor (AR) status was investigated in this study. circRNA profiling was performed using a high throughout microarray assay on a panel of prostate cell lines, which consisted of normal, benign, and malignant cells (n = 9). circRNAs were more commonly significantly up-regulated (p < 0.05) than downregulated in malignant cell lines (n = 3,409) vs. benign cell lines (n = 2,949). In a grouped analysis based on AR status, there were 2,127 down-regulated circRNAs in androgen independent cell lines compared to 2,236 in androgen dependent cell lines, thus identifying a potential circRNA signature reflective of androgen dependency. Through a bioinformatics approach, the parental genes associated with the top 10 differentially expressed circRNAs were identified such as hsa_circ_0064644, whose predicted parental gene target is RBMS3, and hsa_circ_0060539, whose predicted gene target is SDC4. Furthermore, we identified three circRNAs associated with the parental gene Caprin1 (hsa_circ_0021652, hsa_circ_0000288, and hsa_circ_0021647). Other studies have shown the importance of Caprin1 in PCa cell survival and drug resistance. Given the modified circRNA expression signatures identified here, these hypothesis generating results suggest that circRNAs may serve as potential putative diagnostic and predictive markers in PCa. However, further validation studies are required to assess the true potential of these markers in the clinical setting.