Origination and Function of Plant Pseudogenes

ABSTRACT

Pseudogenes, nonfunctional genomic sequences derived from functional protein-coding genes, form by duplication or retrotransposition, and loss of gene function by disabling mutations. Studies on the evolution and functional aspects of plant pseudogenes are limited, despite their abundance in the plant genome. To date, most researches on pseudogenes focus on mammals. Here, we summarized current knowledge on pseudogenes including the historical and recent progress, analyzes their essential roles in gene regulation in hope of further stimulating researches in plant species for understanding gene regulation and evolution.

Introduction

Pseudogenes have long been defined as non-functional sequences of genomic DNA that are formed by duplication or retrotransposition and loss of gene function by subsequent disabling mutations. Pseudogene assignments are largely dependent on reliable and stable gene annotations within the genome. As such, the fast genomic sequencing technology offers the possibility for comprehensive comparisons of the Ψ complement in various organisms to specific families or classes of pseudogenes. System studies have been performed in human (Homo sapiens),^1,2 and later in worm (Caenorhabidis elegans), and fly (Drosophila melanogaster)³ and plant species.^4–7

Pseudogenes are by nonfunctional sequences and are expected to be evolving neutrally, however, studies in arabidopsis and rice reveal that plant pseudogenes seem to experience much stronger purifying selection than animal pseudogenes.⁵ Further analyses reveals that the selection pressure along the pseudogene is not uniform, suggesting they were functional for a relatively long time.⁵

Although pseudogenes lack functional signals at the protein level, however, it does not preclude the possibility that some pseudogenes may function as RNA genes.^4,5 Our recent large-scale plant pseudogenes study performed in seven angiosperm organisms reveal that the proximal regions of pseudogenes are highly related with regulatory noncoding RNAs (microRNAs and long noncoding RNAs),⁴ suggesting that pseudogenes may have regulatory roles in plant. The aim of this mini-review is to summarize the current knowledge of pseudogenes for understanding pseudogene origin, evolution and regulation in plant species.

Types and origin of pseudogenes

Pseudogenes are disabled copies of protein-coding genes that are abundant in genomes.⁸ Protein-coding genes become pseudogenes if degenerated features are present, such as frameshifts, in-frame stop codons, or disrupting interspersed repeats in the original protein-coding sequence or functional promoters.^6,9,10 This may occur at coding regions or promoter regions.^6,9 Pseudogenes are thought to arise by duplication of protein-coding genes,¹¹ by duplication of existing pseudogenes with gradual accumulation of disabling mutations.⁵ In 1977, the first “pseudogene” was discovered in Xenopus laevis, which is truncated copy of a 5S rRNA gene with a compromised function.¹² Depending on the mechanism of the duplication event that created the pseudogenes, pseudogenes can be classified into three categories: Processed, nonprocessed and unitary pseudogene. Processed pseudogene, also termed retro-pseudogene, arose by reverse transcription of processed mRNAs, followed by integration into the genome.^5,13 Thus, the rate of new processed pseudogene generated can be disturbed by the burst of retrotransposition, and the mRNA expression levels.^1,2 Nonprocessed pseudogenes are derived from segmental duplication,¹⁴ whole genome duplication (WGD),⁴ tandem duplication.¹⁵ An additional type, termed unitary pseudogene (UPG), become disabled that may not have been duplicated before.¹⁶ This type of pseudogene is similar to an unprocessed pseudogene but its paralogous counterpart is not found.¹⁷ The evolution of land plants is characterized by WGD.¹⁸ Immediately after gene duplication, either copy may become genetic redundancy, for no selective pressure should be against any loss-of-function mutation,¹⁹ resulting the possibility that a substantial number of pseudogenes can be generated from this process.²⁰

Pseudogene prediction

Pseudogenes prediction is necessary for genome annotation. However, laborious experimental evidence is difficult to identify pseudogene at genome scale. Therefore, some bioinformatics algorithms for pseudogene prediction have been developed, such as PseudoPipe,¹⁰ PSF (Pseudogene Finder),²¹ Shiu’s pipeline,⁵ PPFinder (Processed Pseudogene Finder),²² and PlantPseudo.⁴ Owing to the high similarity to their parental genes, homology-based strategy has been predominantly used to initiate a search for pseudogenes by these tools.²³ Therefore, three main steps are taken: (1) the search of intergenic regions (masked genic and transposon regions) with sequence similarity to known proteins. (2) detection of destructive mutations. Pseudogene candidates were aligned to their parental genes to examine disabled gene features, like premature stop codons or frameshifts. (3) quality control of the pseudogene candidates, including the match length, identity. To date, PseudoPipe, Shiu’s pipeline, PSF, and PlantPseudo are publicly available automatic pseudogene prediction pipelines, which have great potential to be applied to plant genomes. Among them, PlantPseudo could detect the WGD-derived pseudogenes.⁴ However, UPGs could not be predicated by all the current bioinformatics tools, for they have no homologous genes.

Evolution and function

Most pseudogenes are devoid of function, thus are expected to evolve neutrally.⁸ In contrast to animal pseudogenes,²⁴ many plant pseudogenes experienced much stronger purifying selection, with the regions 5′ and 3′ end in the pseudogenes have experienced stronger selection pressure, suggesting that the both regions were functional for a longer period of time after the loss-of-function mutation appeared.^4,5 The distribution of pseudogenes is highly associated with recombination rate of the genome, with higher preservation in regions with low recombination rates.^3,4 Processed pseudogenes have apparently evolved neutrally because of lacking active promoters.¹ Studies of pseudogenes have shown that 2%-5% of mammalian pseudogenes and 2%-32.5% of plant pseudogene are expressed.^4,5,25,26 Recent studies revealed that a substantial of pseudogenes, mostly nonprocessed pseudogenes, are enriched in rare alleles,³ have conserved upstream sequences,³ experiencing ongoing constraint, with active transcription factors (TFs) and RNA polymerase II (Pol II) binding sites in the upstream proximal regions,⁴ hinting their potential regulatory roles. Indeed, many pseudogenes exhibit varying degrees of partial activity, such as, transcribing as high active RNA genes,^4,9,25,27 lncRNAs,²⁸ small interfering RNAs,^29-32 and microRNA decoys.^33,34 Some could even translate into short regulatory peptides that have potential in increasing oxidative stress tolerance in Arabidopsis.³⁵ Therefore, it is becoming apparent that many Ψs can be function as regulatory RNA genes or short regulatory peptides. Number of in-depth case studies reported important roles for pseudogenes in physiology and diseases.³⁶ For example, pseudogenes of the frequently mutated cancer genes PTEN, KRAS, and BRAF function as ceRNAs in vitro.^36,37 Pseudogene PTENP1 could function as a competing endogenous RNA to suppress clear-cell renal cell carcinoma progression.³⁸ All these suggest that pseudogenes could participate in transcription and posttranscriptional regulation of protein-coding genes in the context of biological networks as form of non-coding competitive endogenous RNAs and regulatory peptides.

Pseudogenes in plants

Pseudogenes have been reported mostly in mammals.^3,13,14,39 In contrast, studies in plant remain limited. In rice, a total of 1,439 pseudogenes have been identified.²⁰ Zou et al. identified 28,330 and 4,771 pseudogenes at intergenic regions of rice and Arabidopsis, respectively.⁵ Some plant pseudogenes have been experiencing selective constraints as strong as those experienced by most functional genes.⁵ In Triticum species, pseudogenes were also identified.⁴⁰ Recently, we performed large-scale pseudogenes prediction in seven plant organisms, including Arabidopsis thaliana, Brachypodium distachion, Glycine max [soybean], Medicago truncatula, Oryza sativa [rice], Populus trichocarpa, and Sorghum bicolor and found that a surprisingly large fraction of nonTE regulatory noncoding RNAs (microRNAs and long noncoding RNAs) originate from transcription of pseudogene proximal upstream regions.⁴ Therefore, we proposed that the rapid rewiring of Ψ transcriptional regulatory regions is an essential mechanism driving the origin of novel regulatory modules.⁴ Studies of Arabidopsis populations revealed that the loss-of-function mutations occurred in coding regions play important roles in adaptation and phenotypic diversification.⁷ Experimental data showed that OSIP108 is contained within a pseudogene and is induced in A. thaliana leaves by both the reactive oxygen species-induced PQ and the necrotrophic fungal pathogen Botrytis cinerea.³⁵ Overall, more comprehensive and functional analyses of pseudogenes for plants remain much needed to accelerate research in this area.

Conclusions

We have summary that pseudogenes can be function by being involved in the regulation of gene expression and generating genetic diversity (Figure 1). Plant genomes contain some pseudogenes that under strong constraints because they are import components of the genome. Mechanisms of regulation include the formation of endogenous siRNAs, lncRNAs, miRNAs and providing transcription factors (TFs) and RNA polymerase II (Pol II) binding sites. While additional studies are needed to confirm their regulation in plant genomes.

Figure 1.

Illustration of pseudogenes formation and function. (*) indicates deleterious mutations.

Funding Statement

This work was supported by the the Fundamental Research Funds for the Central Universities [2018ZY32]; Young Elite Scientists Sponsorship Program by CAST [2018QNRC001]; the Project of the National Natural Science Foundation of China [31670333]; the Project of the National Natural Science Foundation of China [31600537]; Guangdong Provincial Key Laboratory of Silviculture, Protection and Utilization [SPU 2018-01].

Acknowledgments

This work was supported by the Fundamental Research Funds for the Central Universities (2018ZY32), Young Elite Scientists Sponsorship Program by CAST (2018QNRC001), Guangdong Provincial Key Laboratory of Silviculture, Protection and Utilization (No. SPU 2018-01), the Project of the National Natural Science Foundation of China (No. 31600537 and 31670333).

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.