Dr. Mayank Darji, Dr. Ashok Chaudhary, Dr. Dhara Gor
Pseudogenes, classically regarded as non-functional vestiges of gene evolution, have been redefined as integral components in gene regulation, particularly gene silencing processes. Their contributions encompass diverse mechanisms that intricately modulate cellular transcriptomic dynamics. Pseudogene-generated endogenous siRNAs can participate in RNA interference cascades, instigating targeted gene down regulation. Intriguingly, pseudogenes exhibit context-dependent expression patterns, suggesting nuanced regulatory roles across tissues and developmental stages. Recent findings also propose scaffold functions, facilitating protein complexes implicated in chromatin remodeling and epigenetic modulation. The conventional view of pseudogenes as genomic fossils has yielded to a paradigm in which they intricately contribute to gene silencing networks. This expanding understanding underscores their significance in the intricate tapestry of cellular governance.
Pseudogenes are widespread and plentiful within genomes. In the past, they were labeled as “genomic fossils” and dismissed as “junk DNA” for a considerable period. However, it is now acknowledged that certain pseudogenes fulfill crucial functions in regulating their parent genes. Furthermore, numerous pseudogenes undergo transcription into RNA. These transcribed pseudogenes might also give rise to small interfering RNAs or lead to a reduction in cellular miRNA concentration. Modern molecular biology is undergoing a transformative shift in perspective as it becomes clear that the extensive quantities of so-called “junk” DNA, once considered remnants of evolution, might indeed possess functionality. Recent research provides compelling evidence for the functional role of non-coding RNAs derived from pseudogenes, which were previously disregarded. These pseudogene-expressed non-coding RNAs play a regulatory role in relation to their protein-coding counterparts. A multitude of pseudogenes—numbering in the hundreds—have been observed to undergo transcription into RNA across a diverse array of tissues and tumors. While much attention has been given to pseudogenes expressed in the sense direction, there are indications from certain reports that pseudogenes can also be transcribed as antisense RNAs (asRNAs).
Transcripts originating from pseudogenes have been identified as influencers of ancestral protein-coding gene expression through molecular mechanisms grounded in RNA sequence similarity. These pseudogene-derived transcripts have been shown to control the ancestral gene’s expression through various means: generating endogenous siRNAs, acting as competitive endogenous RNAs (ceRNAs) by sequestering miRNAs, or modifying the stability of the ancestral mRNA. Furthermore, the transcripts of antisense pseudogenes possess the capacity to affect the promoter activity of ancestral genes.
Gene silenced by epigenetic phenomena
Gene silencing via epigenetic mechanisms involving pseudogenes represents a fascinating aspect of molecular biology. Epigenetic regulation refers to modifications that affect gene expression without altering the DNA sequence itself. Pseudogenes, once considered inert “junk” DNA, have emerged as key players in this process. Epigenetic gene silencing mediated by pseudogenes typically involves the creation of small non-coding RNAs or long non-coding RNAs (lncRNAs). These RNA molecules can interact with chromatin-modifying complexes and guide them to specific genomic regions. The result is the modification of histones and DNA methylation patterns, leading to alterations in the chromatin structure and transcriptional activity of nearby genes.
Figure 1. Oct4 gene expression regulation by pseudogene Oct4p4
Anti-sense pseudogene transcripts can impact on the promoter activity of ancestral genes. Scarola et al. (2015) used mESCs (mouse embryonic stem cells) to identify a set of novel transcripts derived from putative Oct4 pseudogenes (Oct4P1, Oct4P2, Oct4P3 Oct4P4 and Oct4P5). Tight regulation of subcellular localization and expression during mESC differentiation anticipate a defined role for a subset of these transcripts in mESC biology. Here, their studies focused on a nuclear-restricted non-coding RNA (ncRNA) that is transcribed in sense orientation from the processed, X-linked Oct4P4. Dramatic upregulation of sense Oct4P4 transcription during mESC differentiation gives rise to a long, non-coding RNA that forms a complex with the repressive H3K9-specific HMTase(Histone methyl transferase) SUV39H1. This complex translocates to the promoter of the ancestral Oct4 protein-coding gene, located on chromosome 17, to impose H3K9 tri-methylation leading to a consecutive recruitment of HP1a and silencing of the ancestral Oct4 gene (Fig. 1). Interfering with the murine Oct4P4–SUV39H1 silencing complex in differentiated cells leads to the re-activation of mESC self-renewal transcription factor expression and telomerase-dependent telomere maintenance mechanism (Fig.1)
Gene silencing by siRNA
Pseudogenes populate the mammalian genome as remnants of artefactual incorporation of coding messenger RNAs into transposon pathways. Tam et al. (2015) show that a subset of pseudogenes generates endogenous small interfering RNAs (endo-siRNAs) in mouse oocytes. These endo-siRNAs are often processed from double-stranded RNAs formed by hybridization of
Figure 2. Hdac1 gene expression regulation by pseudogene by siRNA
spliced transcripts from protein-coding genes to antisense transcripts from homologous pseudogenes. They showed that mammalian oocytes, protein-coding mRNAs interact with pseudogene transcripts to form dsRNAs that are processed into endo-siRNAs. Examination of Dicer knockouts indicates a function for endo-siRNAs in gene regulation. The specific case of HDAC1 may point to a RNA-induced silencing complex (RISC)-based mechanism. Few uniquely mapping siRNAs are generated from the Hdac1 gene itself, suggesting that it is not used prominently as a Dicer substrate. Instead, most uniquely mapping sense and antisense siRNAs can be assigned to a series of Hdac1 pseudogenes. On the basis of its increased expression in Dicer-null oocytes, they propose that pseudogene-derived, antisense siRNAs direct RISC to cleave Hdac1 mRNAs.
Given the vast repertoire of vertebrate pseudogenes, we anticipate the existence of a series of pseudogene-derived lncRNAs that use mechanisms analogous to Oct4P4 to control the expression of ancestral genes on the epigenetic level. Understanding pseudogene lncRNA-dependent mechanism of epigenetic gene regulation and a precise categorization of pseudogene-derived lncRNAs based on subcellular localization, gene expression regulation and deposition at gene promoters will provide important insights into the global role of pseudogene derived lncRNAs in regulating higher order chromatin structure. On the flip side, the absence of a pseudogene transcript might find remedy through the introduction of an external expression mechanism. This could be achieved by employing a viral vector in a traditional gene therapy strategy. A prime illustration lies in the scenario of PTENP1 depletion within melanoma. In this context, introducing an excess of PTENP1 transgene could potentially reinstate the functionality of a tumor-suppressor and decelerate the progression of the tumor.
For some genes, the loss of coding potential and pseudogenization leads to gene death, and ultimately removal from the genome. For others, inactive genes are resurrected as noncoding RNA genes and neo-functionalized as gene-regulatory elements. Pseudogene-derived transcripts can regulate gene expression by generating small RNAs, regulating mRNA stability via direct binding or sequestration of trans-acting RNA decay proteins, sponging of endogenous miRNAs and promoter recruitment of epigenetic remodeling complexes. The extent to which pseudogenes are important in gene regulation and the pathogenesis of disease has so far been underappreciated for a number of reasons. First, only a relatively small number of functional pseudogenes have been extensively characterized. Second, functional pseudogenes exert their effects on gene expression via a plethora of mechanisms that are not always easily inferred by analysis of the primary nucleic acid sequence. Third, working with pseudogenes presents technical difficulties due to the close homology between the pseudo-and parental-mRNA sequences. Finally, the lineage specificity of pseudogenes and particularly the lack of conservation between humans and mice may prove to be an obstacle in the development of pseudogene-targeted therapeutics. Nevertheless, pseudogene-transcribed lncRNAs are emerging as both important regulators of gene expression and as promising novel targets for pharmacological intervention.