Demantejlersen3013

Pseudogenes, once considered the "junk remnants of genes," are found to significantly affect the regulatory network of healthy and cancer cells, as well as to be highly specific markers of cancer cell identity. Qualitative and quantitative analysis of pseudogenes has a diagnostic and prognostic value in cancer research via the detection of cell-free pseudogenic DNA circulating throughout the body. Exosomes, nanoparticles with a lipid membrane secreted by almost all types of cells, carry cellular-blueprint molecules, including pseudogenic DNA, as cancer-specific cargo. Therefore, it is vital to develop better laboratory techniques and protocols to identify exosome-associated pseudogenes.Pseudogenes are commonly labeled as "junk DNA" given their perceived nonfunctional status. However, the advent of large-scale genomics projects prompted a revisit of pseudogene biology, highlighting their key functional and regulatory roles in numerous diseases, including cancers. Integrative analyses of cancer data have shown that pseudogenes can be transcribed and even translated, and that pseudogenic DNA, RNA, and proteins can interfere with the activity and function of key protein coding genes, acting as regulators of oncogenes and tumor suppressors. Capitalizing on the available clinical research, we are able to get an insight into the spread and variety of pseudogene biomarker and therapeutic potential. In this chapter, we describe pseudogenes that fulfill their role as diagnostic or prognostic biomarkers, both as unique elements and in collaboration with other genes or pseudogenes. We also report that the majority of prognostic pseudogenes are overexpressed and exert an oncogenic role in colorectal, liver, lung, and gastric cancers. Finally, we highlight a number of pseudogenes that can establish future therapeutic avenues.Although long thought of as "gene relics," pseudogenes have recently gained research and medical interests because of their potential impacts on cellular pathways and of their clinical relevance. Studies have profiled pseudogenes at both DNA and RNA levels in cancers. Differences in pseudogene expression (RNA) or occurrence (DNA) help cancer subtype classification, which in turn can contribute to improving treatment selection in precision medicine. Such differences are also associated with clinical outcomes, such as patient survival.Here we review the existing methods on pseudogene profiling and discuss the application scenarios, as well as their relevant issues and challenges.Aberrant expression of pseudogenes has been observed in many cancer types. Deregulated pseudogenes engage in a multitude of biological processes at the DNA, RNA, and protein levels and eventually facilitate disease progression. To investigate pseudogene functions in cancer, cell lines and cell line transplantation models have been widely used. However, cancer biology is best studied in the context of an intact organism. Here, we present various strategies to investigate pseudogenes in genetically engineered mouse models and discuss advantages and disadvantages of the different approaches.Pseudogenes have been considered as nonfunctional copies of their parental genes for a long time. Indeed, they have been often defined "junk DNA" or "transcriptional noise." However, with the identification of their involvement in several biological processes, the necessity of their study is inevitably growing up. The manipulation of pseudogene expression is complicated by their high homology with parental genes and by the fact that most of them work at the transcriptional level as noncoding RNAs. With the advent of CRISPR/Cas technology, these problems can be overcome. Particularly, as we describe in this chapter, it is possible To perform genome editing, obtaining the complete elimination of the pseudogene genomic sequence (knock-out), preventing pseudogene transcription, introducing specific mutations in the pseudogene sequence, or introducing a specific sequence (knock-in). To positively or negatively manipulate pseudogene transcription. To target pseudogene RNA and negatively regulate its expression. To edit pseudogene DNA and RNA and alter a specific sequence. Moreover, CRISPR/Cas technology can be used as an RNA Binding Protein system for molecular biology techniques (such as RNA immunoprecipitation and pull-down), as well as for transcript tracking and live imaging.NANOG is an embryonic transcription factor, which gets reexpressed in cancer stem or tumor initiating cells. NANOGP8, a retrogene belonging to the NANOG family, is predominantly expressed in cancer cells and shows very high similarity with NANOG both at the nucleotide and at the protein level. selleck chemicals The high similarity makes it extremely challenging to distinguish between these two transcription factors. Here we describe a highly efficient restriction endonuclease-based assay, which is performed on cDNA and allows to distinguish NANOGP8 from NANOG. This assay is critical to understand the specific role of NANOGP8 in cancer stemness, which in turn helps to unravel the therapeutic potential of targeting this undruggable transcription factor through gene therapy, for treatment of various cancers.The technical challenge in proving that a given expressed pseudogene is in fact translated into a functional protein is specificity. To circumvent this challenge, one approach is to use PCR in order to generate a series of clones that allow one to exogenously express the pseudogenic protein of interest, either native or fused to a tag, which can facilitate purification, detection, and complementation in both bacterial and mammalian cells. This approach allows an assessment of whether a putative pseudogenic protein possesses enzymatic activity, to identify its subcellular localization and to test its capacity to complement the parental homolog. An alternative approach is to detect the endogenous protein using targeted proteomics analysis and to assess the full range of endogenous RNA isoforms, in order to consider additional coding and noncoding RNA functionality.Several recent studies support a functional role for pseudogenes, a copy of a parent gene that has lost protein-coding potential, which was for a long time thought to represent only "junk" DNA. Several hundreds of pseudogenes have now been reported as transcribed RNAs in a large variety of tissues and tumor types. Most studies have focused on pseudogenes expressed in sense direction, relative to their protein-coding gene counterpart, but some reports suggest that pseudogenes can be also transcribed as antisense RNAs (asRNAs). Key regulatory genes, such as PTEN and OCT4, have in fact been reported to be under the regulation of pseudogene-expressed asRNAs. Here, we review what is known about pseudogene-expressed asRNAs, we discuss the functional role that these transcripts may have in gene regulation and we summarize the techniques that are available to study them.