- Email: [email protected]
The genomic context of retrocopies increases their chance of functional relevancy in mammals
Journal Pre-proof The genomic context of retrocopies increases their chance of functional relevancy in mammals
JoãoPaulo Machado, Agostinho Antunes PII:
S0888-7543(18)30577-9
DOI:
https://doi.org/10.1016/j.ygeno.2020.01.013
Reference:
YGENO 9451
To appear in:
Genomics
Received date:
12 October 2018
Revised date:
3 January 2020
Accepted date:
21 January 2020
Please cite this article as: J. Machado and A. Antunes, The genomic context of retrocopies increases their chance of functional relevancy in mammals, Genomics (2019), https://doi.org/10.1016/j.ygeno.2020.01.013
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© 2019 Published by Elsevier.
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The genomic context of retrocopies increases their chance of functional relevancy in mammals
João Paulo Machado 1,2 and Agostinho Antunes2,3,# 1
CIMAR/CIIMAR, Interdisciplinary Centre of Marine and Environmental Research,
University of Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de
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Matos, s/n, 4450–208 Porto, Portugal.
Abel Salazar Biomedical Sciences Institute (ICBAS), University of Porto, Porto, Portugal.
3
Department of Biology, Faculty of Sciences, University of Porto, 4169 007 Porto, Portugal.
#
Corresponding author: Agostinho Antunes,
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2
Keywords:
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Full postal address
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Phone: (+351) – 220402742
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Email: [email protected]
Adaptive, Functional relevancy, Genomics, Mammals, Retrocopies
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Abstract Described as “junk” DNA, pseudogenes are dead structures of previously active genes present in genomes. Pseudogenes are categorized into two main classes: processed pseudogenes, formed through retrotransposition, and non-processed pseudogenes, typically originated from gene decay following duplication events. The term “processed pseudogene” has changed to “retrocopy” since they are likely to evolve new functional roles and became a retrogene. Here, we surveyed 38,080 retrocopies from chimpanzee, dog, human, mouse, and rat
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genomes to assess their potential adaptive value. The retrocopies inserted in the same chromosome of the parental gene have higher chances of remain potentially “active” (absence
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of premature stop codons and frameshifts) (~26.1%), while those placed into a different
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chromosome have a twofold decrease chance of continuing potentially “active” (~7.52%). The genomic context of their placement seems associated with their expression. Retrocopies
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placed in intragenic regions and the same sense of the “host” gene have higher chances of being expressed relative to other genomic contexts. The proximity of retrocopies to their
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parental gene is associated with a lower decay rate, and their location likely influence their
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expression. Thus, despite their unclear role, retrocopies are probably involved in adaptive
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processes. Our results evidence natural selection acting in retrocopies.
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1. Introduction In 1977 a structure termed “pseudogene” was reported for the first time in Xenopus laevis oocyte-type 5S RNA [1]. Pseudogenes are present from bacteria to vertebrates [2] and considered as non-functional sequences of genomic DNA that originated from functional genes [3]. Two main processes may be in the origin of pseudogenes from functional copies: (1) retro-transposition leading to the lack of a promoter ("dead on arrival"), and (2) decay of functional genes (frameshifts and/or premature insertion of stop codons), mostly from
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duplicated copies but also occurring in non-duplicated copies [4]. Therefore, pseudogenes are often distinguished into two different broad classes: processed pseudogenes, corresponding to
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those retro-transposed back into a genome via an RNA intermediate; and non-processed
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pseudogenes, which are genomic remains of duplicated genes or residues of dead genes, also known as unitary pseudogenes [5, 6]. Described as non-functional genomic fragments, and
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commonly referred to as “defunct” genes, it is assumed that they evolve under neutrality [7]. However, their presence might be functionally relevant, supported either by transcription data
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or a high degree of conservation [8, 9].
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Retrotransposed genes were routinely labelled as “processed pseudogenes”, but recently the
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term “retrocopies” has been instead adopted to express their functional roles [10]. Therefore, those reverse-transcribed through an RNAs intermediate and inserted in the genome as “new” DNA are now assigned as retrocopies (originated from retroduplication) [11]. The gene duplication is a pivotal mechanism to generate evolutionary novelty [11, 12], along with the retrocopy duplication trough segmental duplication [11]. Although these two mechanisms differ in the type of copies generated, since the tandem duplications usually produce copies that inherit the genetic features (e.g. introns and promoter), while the retrocopies lack the introns and promoters [11]. The retrocopies lack a promoter and consequently their expression is dependent on the recruitment of regulatory elements such as transcriptional mechanisms of host genes that surround their insertion site [11, 13]. Contrary to the segmental duplication, transcribed retrocopies are more likely to evolve “new expression
Journal Pre-proof patterns” and therefore evolve “new functions” [11]. The high transcriptional activity allows a substantial number of functional retrocopies to emerge as retrogenes highlight the role of natural selection in the retrocopy “repository” [12]. The genomic abundance of retrocopies is associated and dependent on the rate of gene duplication/loss [14]. Mammals have a high number of retrocopies, depending on the species and the annotation pipeline [15, 16]. Two recent works suggested the expression in the human genome of 1,286 out of 7,849 retrocopies (~12.5%) [10], and 615 out of 4,927 retrocopies
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(~16.4%) [17]. Contrary to the theoretical expectation that any gene can form a pseudogene, differences have been reported in the relative proportion, from gene to gene [18].
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Housekeeping genes, highly expressed genes in germ-line cells and those participating in
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basic metabolic regulations show multiple corresponding pseudogenes [16, 19-21]. This may be due to high expression levels of these genes, thus increasing their likelihood to accumulate
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mutations [22] or to be retro-transposed into the genome [23]. Moreover, the GC content of
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the genomic regions where the pseudogenes were placed also affect their mutation rate [24]. Early typified as non-functional units, here we provide support that non-random processes
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shape the retrocopies “repository”. Their destination and closer position to the parental gene
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influence frameshifts insertions and stop codons. Furthermore, retrocopies genomic context act as a major determinant for their expression. Here we present evidence supporting the adaptive role of retrocopies in mammalian genomes.
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2. Methods 2.1. Retrocopies retrieval and identification The retrocopies retrieved from pseudogene.org database include: Cannis familaris (dog, build 50), Homo sapiens (human, build 83), Mus musculus (mouse, build 84), Pan troglodytes (chimpanzee, build 50), and Rattus norgevicus (rat, build 74). From this database were retrieved pseudogenes annotated as processed pseudogenes (retrocopies) after excluding those categorized as “ambiguous” and “duplicated”. The data retrieved from pseudogenes.org
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encompass: C. familaris (6,001) (Table S1), H. sapiens (8,074) (Table S2), M. musculus (9,809) (Table S3), P. troglodytes (7,097) (Table S4) and R. norgevicus (7,099) (Table S5).
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These 38,080 pseudogenes, were reduced to 35,277 after discard those associated with
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mitochondrial DNA or inconclusive identity of the parental gene coordinates (chromosome).
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The parental gene coordinates ID were obtained through its corresponding ENSEMBL protein id using the ENSEMBL Biomart. The parental genes were retrieved for the Ensembl releases: C. familiaris (60), H. sapiens (94), M. musculus (95), P. troglodytes (54), and R. norgevicus
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(67). The retrocopies considered “inactive” are those showing signs of loss of protein coding
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ability (premature stop codons and frameshifts), while the other copies still showing protein
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coding ability were considered as “active” [25]. 2.2 Expression of retrocopies
The expression data from human retrocopies were retrieved from RetrogeneDB [17] and RCPedia [10]. In RetrogeneDB, based on ENSEMBL annotation, was retrieved information about expression and the ORF (Open Reading Frame) (188 retrocopies). While from RCPedia, were retrieved all the 7,849 retrocopies, their expression relative to their genomic context (intragenic or intergenic). Either RetrogeneDB or RCPedia use multi-step pipelines [10, 17], while to evaluate retrocopies expression the first use short read libraries in various tissues from selected records of NCBI SRA database [17] the other uses RNA-seq data from six tissues (brain, cerebellum, heart, liver, kidney and testis) [10, 26].
Journal Pre-proof 2.3. Insertion simulation For the retrocopies inserted in the same chromosome of the parental gene, we conducted a simulation study to test if the distance “retrocopy-parental gene” was random or biased. To accomplish this test, we followed the steps 1) collect the position of the parental genes and retrocopies; 2) for each chromosome was shuffled the position of the retrocopies within the list of retrocopies positions. Then measured the distance between the parental gene and the retrocopy. Both analyses were repeated three times to inspect the consistency of the obtained
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data. While the majority of the retrocopies arose through retro-transposition events, some arose from duplication of previously retro-transposed genes, and these events lead to
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duplicated-retrocopies. To avoid possible bias introduced by segmental duplication regions, highly rich in retrocopies [27], the same distance analysis were performed discarding the
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retrocopies inserted in these regions. Data from the retrocopies and segmental duplication
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were retrieved at Segmental Duplication Database [28]. Additionaly to avoid potential bias due to duplications of the pairs retrocopy parental gene, we filtered for retrocopies inserted in
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the same chromosome. The filter was created by BLASTting 5 kbp flanking the retrocopy
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location and minimum alignment of 2.5 kbp and within 10 kbp.
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To test the possibility of a biased difference in the proportions of retrocopies with stop codons or alteration of the ORF relatively to their insertion chromosome, we performed a simulation randomly inserting these retrocopies in the five studied genomes using an “in house” script. To accomplish this the position of the parental genes and retrocopy was read, after this step the retrocopy was randomly sort as “active” or “inactive” up to the observed number of “active” retrocopies in each species. The proportions were then compared using Fisher’s exact test. 2.4. Gene evolution under neutrality Pseudogenes are assumed to evolve under neutrality. If this assumption is correct then the reminiscent sequences of previous functional copies will “accept” all the mutations. The
Journal Pre-proof solely difference rely in the ratio of transitions and transversions. It is expected that the transitions double the number of transversions, and this is an explanatory model for sequences evolving under neutrality [29]. Here, we tested the time and the mutation insertion effect of pseudogenes evolving under the emulated Kimura model [30], with the parameters of the model settled to a tS/tV (kappa value) = 2, which is a value frequently observed in mammalian genomes [29]. In each step, two random mutations were inserted, with doubled chances to insert a transition rather than a transversions to a maximum of 400 steps (800 mutations). For
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each step the sequences were submitted to blast searches (either BLASTn or BLASTx) and the genes were considered lost when the first of the following criteria were achieved: 1) the
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expected value for the gene of interest is below 1e -10 ; or 2) presence of an unrelated gene in the top-hit results. The mutation rate and the predictive time for gene loss were calculated
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under the assumption of 2.41 mutation/site/year (averaged value observed for fossil record
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and sequences) [31].
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3. Results 3.1. Distribution in the Chromosomes The retrocopies follow a random distribution in the chromosomes, given the relation between chromosome length and retrocopies number (Table S6). This trend appears to be generalized in mammals since similar correlations were observed in the five mammals studied (chimpanzee, P. troglodytes; dog, C. familiaris; human, H. sapiens; mouse, M. musculus; and rat, R. norvegicus) (Table S6). Yet the relation is nearly absent when considering the
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chromosome length and the number of retrocopies formed from each chromosome (Table S6). The mouse chromosome Y has been excluded from these analyses given the high number
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of retrocopies allocated (~7 times more than the average of the others chromosomes).
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We detected a non-random position of the parental gene and retrocopies in the human genome
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without signs of disablement (i.e. without stop codons and maintaining the ORF), similar to the other four species described above . We observed evidences of disablement in 92.4% of
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the 29,832 retrocopies inserted in a different chromosome relatively to the parental gene.
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When it is considered the retrocopies inserted in the same chromosome (5,445), the dismantlement decreased to about 73.9%. The disablement ratio of retrocopies placed in the
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same chromosome is significantly lower when compared to those placed in a different chromosome (Fisher’s exact test, p-value <2.2e-16) (Table 1 and Table S7). The proportions in the five species were significantly different (Table 1). In the human genome we have been able to use 7,446 retrocopies and from these 6,564 are inserted in a different chromosome while 882 are inserted in the same chromosome relatively to the parental gene. The insertion relatively to the parental gene seems associated with disablement, since 559 (~8.51%) are a different chromosome and 104 (~11.79%) in the same chromosome. While less evident, this disproportion between retrocopies inserted in the same or different chromosome (disabled) is statistically significant (Fisher exact test, p-value ~ 0.02). For the others four species the same trend was observed with higher proportion of retrocopies inserted in the same chromosome of the parental gene considered as “active” (Table 1 and Fig. S1). Therefore the proportion of
Journal Pre-proof retrocopies with stop codons and/or frameshift re-allocated in a different chromosome is significantly lesser than those inserted in the same chromosome relatively to the parental gene. From the 7,446 retrocopies of the human genome used here, 2,251 are in segmental duplicated regions. After removing those regions, the same trend is observed, since ~8.69% “active” where in a different chromosome relatively to the parental gene and ~10.6% “active” retrocopies are placed in the same chromosome of the parental gene. The difference in the
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proportion (Fisher exact test, p-value ~ 0.018) is observed even when the segmental duplicated regions were discarded.
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Even when excluding based on distance 1Mbp, 0.1Mbp, 0.01Mbp, 0.001Mbp, 0.0001Mbp, to
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take into consideration possible gene conversion effects, the fisher’s exact test show the same
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trend (Table S8). The proportion of retrocopies “active” copies in the same chromosome are significantly higher than those placed in a different chromosome relatively to the parental
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gene.
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For the retrocopies present in the five genomes, we tested a random distribution of those without stop codons and maintaining the ORF (here considered as “active”). Assuming their
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original positions (parent gene and retrocopy) but allowing randomly retrocopies to be considered as “inactive” or “active” up to the limit of those observed as “active”, the results from 100 replicates [32]. In this scenario and allowing 663 retrocopies (for the human replicate) to be without stop codons, only six replicates had significantly higher proportions of retrocopies without stop codons and maintaining the ORF inserted in the same chromosome compared to those inserted in a different chromosome. Simulations were repeated for the other four species also revealed cases where the proportions of retrocopies inserted in the same chromosome relatively to the parental gene were significantly higher: 2 in chimpanzee, 6 in dog, 1 in mouse and 5 in rat. Overall only 4% of the simulations revealed
Journal Pre-proof a significantly higher proportion of retrocopies “active” in the same chromosome, therefore unlike to be a random observation. 3.2. Insertion in the same Chromosome The Mann-Whitney test shows positions significantly closer to the parental gene than a random insertion (p-value < 0.01). This is also observed in simulations using the original positions to constrain the initial placement (p-value < 0.01) (Fig. 1A and Fig. 1B). This trend was observed for each species suggesting the placement of the retrocopies closer to the
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parental gene. Together, these two different simulation scenarios (original positions and completely random [33]) revealed that retrocopies are closer to the parental gene than
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predicted by a random distribution, either including or excluding segmental duplicated
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regions (Fig. 1A and Fig. 1B).
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The presence of segmental duplications (SDs) may alter the results from the measured distance between the parent “gene- retrocopy”, particularly on SD pairs containing both
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retrocopy and parental gene. In the human genome from 882 retrocopies, 277 are within
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segmental duplications [27]. After removing the SDs regions we also detected a significant result for Mann-Whitney-Wilcoxon test, p < 0.01, consequently the retrocopies are closer to
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the parental gene than a completely random distribution (Fig. 1A and Fig. 1B). The disablement of retrocopies and the distance to the parent gene follows an observable distance-based trend. The distance of the pair “retrocopy-parent gene” is significantly lower for the retrocopies considered as “active” (absence of stop codons and maintain of the ORF) when compared with those considered as “inactive”, Mann-Whitney U Test p-value = 0. 18. Indeed, most retrocopies without premature stop codons are closer to the parental gene (Fig. 2). Around 50% of the retrocopies without stop codons and maintaining the ORF are placed within 3Mbp (Million base pairs) relatively to the parental gene position. Considering those with stop codons or missense mutations, ~50% are placed within 3.6 Mbp distance from parent gene to the retrocopy (Fig. 2). Furthermore within the 25 Mbp was observed ~71.0%
Journal Pre-proof of the total number of retrocopies with in-frame stop codons or missense mutations and ~74.1% of all the retrocopies without stop codons and maintaining the ORF. 3.3. Processed Pseudogenes Expression Along with the absence of stop codons, the expressed retrocopies are considered to act as functional elements. On the other hand, symptoms of non-functionality include frame disablement (premature stop codons) and pseudogenes nucleotide sequence decay or incompleteness. Several transcribed pseudogenes are disabled [8], but some disabled
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pseudogenes revealed evidence of being functional relevant [34]. The data from RetrogeneDB shows that from 615 expressed retrocopies in the human genome (Fig. 3A) only 188
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retrocopies maintain the ORF, i.e. nearly 30.5% (Fig. 3B). Data collected from RCPedia
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showed an association between genomic context and the expression, since 26% expressed retrocopies are located in intragenic regions and in the same sense of the “receiver” gene (Fig.
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3C). By contrast, only 14% and 16% of those are expressed when placed in intergenic or intragenic positions on the opposite sense, respectively (Fig. 3C). In these two cases, the
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expression is significantly lower when compared to those inserted in intragenic positions in
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the same sense of the gene “host” (Z-Score, p-values < 0.0001).
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3.4. Genes evolving under neutrality Given the detection of retrocopies under adaptation [35] it is relevant to access the time course for a gene to become undetectable through blast searchers (BLASTn or BLASTx) while evolving under complete neutrality. Using the cut-off value 1x10-10 and assuming an evolutionary rate of 2.415x10-9 mutations/site/year [31] we estimated for eight genes (BTF3, CYC1, GADPH, H3F3B, PPIA, RPL7A, SCOP and RPL39) the time needed for the blast searches to be below the empirical cut-off (Fig. S3). Our data suggested nearly 229.46 ± 100.82 million years (Myr) (Table S9) for a gene to be undetectable trough blast searches, but the insertion of the first stop codon would occur earlier, around 26.23 ± 22.95 Myr.
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4. Discussion 4.1. Retrocopies origin and their location The random placement of human retrocopies has been previously described as a “bombardment” along evolution [15], thus hypothesizing retro-transposition as an efficient process to introduce regulatory elements into the genome “in search” of new target genes [36]. Consistently, this “stated war” was detected in the five analyzed mammalian species
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(chimpanzee, dog, human, mouse and rat). This seems a trend for mammals, since larger chromosomes have a higher number of retrocopies. As previously observed in the human
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genome [15], retrocopies allocation is mainly affected by chromosome length [15]. While the results showed a random process on the retrocopies origin given the absence of relation
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between chromosome length and number of “parental” genes retro-copied. Several factors
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were advanced to influence the likelihood of a gene to form a retrocopy, such as expression level and gene length [18], or mRNA stability [37]. Additionally, gene length has been
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mentioned as a major determinant influencing pseudogenes formation, since longer genes
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tend to “produce” more non-processed pseudogenes, whereas retro-pseudogenes are typically formed by short protein-coding genes [38-40]. Together this suggests that the raw material to
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form “new” retrocopies appears to be highly associated with the nature of genes (parental genes) or the mRNA molecules. These properties have been shown to have an adaptive value, such as mRNA stability [41, 42], gene expression [43, 44] and gene length [45]. 4.2. Location as factor for Retrocopies “survival” Despite the retrocopies random insertion, here we reported an association between their placement location and frequency of premature stop codons or frameshifts (i.e. indicative of function loss) [8]. Those placed in the same chromosome showed less evidences of disablement. Indeed, simulated scenarios showed that the association between the retrocopies distribution and the disablement is highly unlikely to be a random event. And even excluding
Journal Pre-proof those within range that is compatible with gene conversion the same trend is detected and excluding possible genes in duplicated regions. Additionally, those retrocopies inserted in the same chromosome of the parental gene tend to be placed closer to the parental gene than a completely random distribution. The survival of the retrocopies requires a de novo promoter or its recruitment from the genomic environment around the placement site [35, 46]. A closer location seem advantageous since some retrocopies adjacent to the parental gene tend to be co-regulated [47]. Later it has been shown
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an absence of co-regulation and, instead, an correlation between expression of retrocopies and parental gene, irrespective to their distance [48]. Thus, after random allocation, we
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hypothesize that their closer location confers advantages and increase their chances to become
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functionally relevant. Another possibility is that a closer location, as observed in the retrocopies allocated in the same chromosome, implies that they are young retrocopies. The
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detection of age-dependent pseudogenes (retrocopies) was previously described as skewed [49], as newly formed retrocopies are identified easier than older retrocopies [50]. Despite the
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different age of the retrocopies as estimated in previous studies [51], the random insertion
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decline the possibility of an age association with absence of signs of disablement. Their
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placement seems random, but selectively disabled depending on the insertion location. This is also supported by the observation of an average lower similarity between the parental gene and the retrocopy inserted in a different chromosome. Alternatively, a closer location increases their chances to became functional relevant by a more favorable expression context. Therefore, being closer to the parental gene, would lead to a decreased number of substitutions, which would increase the retrocopies detection, and consequently enhance the frequency of pseudogenes closer to the parental gene. 4.3. Retrocopies acting as “repositories” of information Based on the analysis of retrocopies in human and mouse genomes, it has been estimated that the formation of “new” retrocopies occurs in a rate of about 1-2% per gene per million years [52]. In the human genome, gene duplications happen at a predicted rate of 0.9% per gene per
Journal Pre-proof million years [52]. Thus, the arousal of “new genes” through retrocopies may be even more relevant than gene duplications. Their maintenance also appears to be of prime relevance since ~40% of the retrocopies are shared between human and mouse [53]. Under a neutral evolutionary pace, it will take ~244Myr for a gene to become “undetectable”, while a hypothetical frameshift might occur much earlier (~26Myr). This implies that pseudogenes, either processed or non-processed, and detected through blast searches older than ~244 Myr were under a period of non-neutral evolution. The expression of retrocopies,
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and their predicted or proofed functionality, is also observed in some non-processed pseudogenes placing them as interesting and important functional units [54].
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The retrocopies persistence lead to an increased probability of pseudogene resurrection [52].
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Therefore, this shows the great relevance of pseudogenes in the generation of evolutionary
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novelties, likely acting as “repositories” of innovation, even in scenarios evolving under neutral evolutionary pace.
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4.4. Genomic landscape as a contributor to retrocopies expression
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The maintenance of the expression patterns might be important for regulatory processes [23,
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55] and probably enables the functional “resurrection” of pseudogenes, frequently described in human and mouse pseudogenes [52]. The lack of an appropriate regulatory environment often lead to the degeneration of the retrocopies [56], and we found that processed pseudogene near the parental gene lead to an increased probability of retention. Concordantly, a parental gene and its pseudogene interdependence were reported for the gene ABCC6 and the ABCC6P1, since their co-expression resulted in ABCC6 decreased expression [57]. Moreover, gene order in eukaryotes follow a non-random location, as those closely placed tend to be co-expressed and co-functional [58]. The allocation site seems closely associated with the chances for the retrocopies, as those placed in intragenic regions in the same sense of the gene “receiver” provide an adequate “expression context”. By contrast, when they are placed in intergenic regions or even in intragenic regions, their
Journal Pre-proof chances to be expressed decrease. After the insertion of a retrocopy, the first step leading it to a retrogene (functional counterpart of the retrocopies) is their expression [47]. Therefore the genomic context of the retrocopy allocation influence their probability of becoming a retrogene. Our results reveal three relevant aspects of retrocopies. 1) Retrocopies inserted in the same chromosome of the parental gene have higher chances to “survive”. 2) Retrocopies inserted in the same chromosome of the parental genes are closer than a completely random process. 3) Placement of retrocopies influences their chances of expression.
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4.5. Retrocopies as functional units
Searching functional elements in genomes is of prime relevance for evolutionary biology. In
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recent years were raised several arguments regarding the functionality of retrocopies. The
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processes that lead to their arousal and ubiquitous presence in the mammalian genomes is therefore of major relevance to understand their function. While retrocopies seem randomly
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placed and dispersed in the genomes, their location apparently determines their chances to acquire a functional relevancy. Those retro-transposed pseudogenes closer to the parental
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gene (same chromosome) increase twice their chances of survival (i.e. lack of perceived stop
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codons and maintain the ORF). In addition, those placed in intragenic regions, in the same
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strand of the ‘receiver’ gene, have higher chances to be expressed. Thus, our results highlight a mechanism of natural selection acting on those retrocopies, revealing a functional relevancy in their maintenance and placing them potentially as functional units. Here, we described several non-random processes occurring in retrocopies, which suggest that retrocopies are subjected to natural selection and their perseverance in genomes is a nonrandom process.
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5. Acknowledgements We are thankful for the comments provided by the Associate Editor and two anonymous reviewers, which helped improving a previous version of this manuscript. The authors acknowledge the Portuguese Fundação para a Ciência e a Tecnologia (FCT) for financial support to JPM (SFRH/BD/65245/2009). This work was further supported by a grant from Iceland, Liechtenstein and Norway through the EEA Financial Mechanism and the Norwegian Financial Mechanism. AA was partially supported by the Strategic Funding
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UID/Multi/04423/2019 through national funds provided by FCT and European Regional Development Fund (ERDF) in the framework of the programme PT2020, and the FCT project
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PTDC/AAG-GLO/6887/2014 (POCI-01-0124-FEDER-016845) and PTDC/CTA-
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AMB/31774/2017 (POCI-01-0145-FEDER/031774/2017).
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Tables Table 1 – Comparison between retrocopies inserted in the same and different chromosome, the proportion of “active” and identity compared with parental gene . The term active is associated with those maintain the ORF and without stop codons, (%) proportion of “active”. SD – Segmental Duplications, w/o – without, ǂ - Total without considering Human only in non-segmental duplicated regions. Different Chromosome
Fische r Te st Exact
“Inactive”
Active (%)
Identity (%)
Fraction (%)
“Active”
“Inactive”
Active (%)
Identity (%)
Fraction (%)
(p-value )
Chimpanzee
150
600
20.00
72.73
92.15
527
5,344
8.98
76.56
94.80
<2.20E-16
Dog
168
487
25.65
73.17
91.02
415
4,774
8.00
75.77
94.88
<2.20E-16
Human
104
778
11.79
72.92
92.97
559
6,005
8.51
77.21
94.64
0.001
M ouse
389
1,591
19.65
80.02
95.50
521
6,716
7.20
75.27
95.50
<2.20E-16
Rat
316
862
26.82
76.76
91.79
259
4,712
5.21
72.25
94.46
<2.20E-16
Human (w/o SD) Total (ǂ)
58
547
10.60
70.63
92.30
390
4,489
8.69
75.73
94.64
0.104
1,127
4,318
26.10
76.58
93.47
2,281
27,551
7.52
75.42
94.89
<2.20E-16
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Spe cie s
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“Active”
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Same Chromosome
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Figures
Fig. 1 - Retrocopies inserted in the same chromosome: distance to parental gene and influence of segmental duplication in human genome. Distance of retrocopies retro-transposed to the same chromosome and the parental gene. The distance observed, a random scenario, with the retrocopies were randomly distributed in each
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chromosome. The observed distance from retrocopies and parental gene after the removal of segmental duplication regions. A) Retrocopies inserted in the same chromosome in the human
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genome. B) After excluding segmental duplicated regions.
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Fig. 2 - Retrocopies inserted in the same chromosome: distance to parental gene and presence of stop codons.
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Comparative empirical cumulative distribution of retrocopies inserted in the same
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chromosome. The orange line represents retrocopies containing stop codons and the blue line
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represents the retrocopies free of stop codons.
Fig. 3 - Retrocopies expression and genomic context. Data retrieved from RetrogeneDB: A) Expressed retrocopies; B) Expressed retrocopies that maintain the ORF, text labels refers to parent gene name’s, the linkers are colored accordingly to the chromosome where the pseudogene is placed. Data retrieved from RCPedia C) Schematic representation of the expressed retrocopies and the genomic context: 1) hypothetical “host” gene with two exons, 2) additional hypothetical gene. The ribbons indicate orientation of the reading frame.
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[1] C. Jacq, J.R. Miller, G.G. Brownlee, A pseudogene structure in 5S DNA of Xenopus laevis, Cell, 12 (1977) 109-120. [2] A.J. Mighell, N.R. Smith, P.A. Robinson, A.F. Markham, Vertebrate pseudogenes, FEBS letters, 468 (2000) 109-114. [3] E.S. Balakirev, F.J. Ayala, Pseudogenes: are they "junk" or functional DNA?, Annual review of genetics, 37 (2003) 123-151. [4] A.N. Khachane, P.M. Harrison, Assessing the genomic evidence for conserved transcribed pseudogenes under selection, BMC genomics, 10 (2009) 435. [5] D. Zheng, A. Frankish, R. Baertsch, P. Kapranov, A. Reymond, S.W. Choo, Y. Lu, F. Denoeud, S.E. Antonarakis, M. Snyder, Y. Ruan, C.L. Wei, T.R. Gingeras, R. Guigo, J. Harrow, M.B. Gerstein, Pseudogenes in the ENCODE regions: consensus annotation, analysis of transcription, and evolution, Genome research, 17 (2007) 839851. [6] Z.D. Zhang, A. Frankish, T. Hunt, J. Harrow, M. Gerstein, Identification and analysis of unitary pseudogenes: historic and contemporary gene losses in humans and other primates, Genome Biol, 11 (2010) R26. [7] R. Martinez-Arias, E. Mateu, J. Bertranpetit, F. Calafell, Profiles of accepted mutation: from neutrality in a pseudogene to disease-causing mutation on its homologous gene, Human genetics, 109 (2001) 7-10. [8] P.M. Harrison, D. Zheng, Z. Zhang, N. Carriero, M. Gerstein, Transcribed processed pseudogenes in the human genome: an intermediate form of expressed retrosequence lacking protein-coding ability, Nucleic acids research, 33 (2005) 23742383. [9] O. Svensson, L. Arvestad, J. Lagergren, Genome-wide survey for biologically functional pseudogenes, PLoS computational biology, 2 (2006) e46. [10] F.C. Navarro, P.A. Galante, RCPedia: a database of retrocopied genes, Bioinformatics, 29 (2013) 1235-1237. [11] H. Kaessmann, N. Vinckenbosch, M. Long, RNA-based gene duplication: mechanistic and evolutionary insights, Nature Reviews Genetics, 10 (2009) 19-31. [12] J. Zhang, Evolution by gene duplication: an update, Trends in ecology & evolution, 18 (2003) 292-298. [13] N. Vinckenbosch, I. Dupanloup, H. Kaessmann, Evolutionary fate of retroposed gene copies in the human genome, Proceedings of the National Academy of Sciences of the United States of America, 103 (2006) 3220-3225. [14] O. Podlaha, J. Zhang, Pseudogenes and their evolution, eLS, (2010). [15] Z. Zhang, P.M. Harrison, Y. Liu, M. Gerstein, Millions of years of evolutio n preserved: a comprehensive catalog of the processed pseudogenes in the human genome, Genome research, 13 (2003) 2541-2558. [16] Z. Zhang, M. Gerstein, Large-scale analysis of pseudogenes in the human genome, Current opinion in genetics & development, 14 (2004) 328-335. [17] M. Kabza, J. Ciomborowska, I. Makalowska, RetrogeneDB - a database of animal retrogenes, Molecular biology and evolution, (2014). [18] W. Li, W. Yang, X.J. Wang, Pseudogenes: pseudo or real functional elements?, Journal of genetics and genomics = Yi chuan xue bao, 40 (2013) 171-177. [19] S. Frederiksen, H. Cao, B. Lomholt, G. Levan, C. Hallenberg, The rat 5S rRNA bona fide gene repeat maps to chromosome 19q12-->qter and the pseudogene repeat maps to 12q12, Cytogenetics and cell genetics, 76 (1997) 101-106.
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[57] A.P. Piehler, M. Hellum, J.J. Wenzel, E. Kaminski, K.B. Haug, P. Kierulf, W.E. Kaminski, The human ABC transporter pseudogene family: Evidence for transcription and gene-pseudogene interference, BMC genomics, 9 (2008) 165. [58] P. Michalak, Coexpression, coregulation, and cofunctionality of neighboring genes in eukaryotic genomes, Genomics, 91 (2008) 243-248.
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Supplemental Material Fig. S1. Processed pseudogenes inserted in the same chromosome: distance to parental gene. Distance of retrocopies retro-transposed to the same chromosome and the parental gene. a) Chimpanzee, b) Dog, c) Mouse and d) Rat. Fig. S2. Blast results from the simulation of 8 genes evolving under neutrality. On each left subfigure are the results from BLASTn and on right for BLASTx for the following genes:
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a) BTF3, b) CYC1, c) H3F3B, d) GADPH, e) PPIA, f) RPL7A, g) RPL39 and h) SCOP. The y-axis is log-scaled. The x-axis (n) represent the natural number of the blast order, and for
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each step (n) corresponds to two mutations. The reference line corresponds to the empirical 1E-10 , value above this value were discarded or partially discarded and not represented in the
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plots.
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Table S1. Retrocopies in Dog. Data retrieved from pseudogenes.org and the data for the
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parental gene location. #N/D - No data (excluded from the analysis).
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Table S2. Retrocopies in human. Data retrieved from pseudogenes.org, and the data for the parental gene location. #N/D - No data (excluded from the analysis). SDD - Segmental
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duplicated region.
Table S3. Retrocopies in mouse. Data retrieved from pseudogenes.org, and the data for the parental gene location. #N/D - No data (excluded from the analysis).
Table S4. Retrocopies in chimpanzee. Data retrieved from pseudogenes.org, and the data for the parental gene location. #N/D - No data (excluded from the analysis). Table S5. Retrocopies in rat. Data retrieved from pseudogenes.org, and the data for the parental gene location. #N/D - No data (excluded from the analysis).
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Table S8. Retrocopies “active” and “inactive” excluded based on gene conversion range. Table S9. Estimated age of processed pseudogenes misidentification after evolving under
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neutrality.
Journal Pre-proof Authors’ contributions JPM performed the phylogenetic, evolutionary and bioinformatic analyses and drafted the manuscript. AA participated in the design, genetic analyses, drafting and coordination of the
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study. All authors read and approved the final manuscript.
Journal Pre-proof Highlights:
The genomic context of retrocopies increases their chance of functional relevancy in mammals
The retrocopies inserted in the same chromosome of the parental gene have higher chances of
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decrease chance of continuing potentially “active”.
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remain potentially “active”, while those placed into a different chromosome have a twofold
Retrocopies placed in intragenic regions and in the same sense of the “host” gene have higher
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chances of being expressed relative to other genomic contexts.
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The proximity of retrocopies to their parental gene is possibly associated with a lower decay
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rate, and their location likely influence their expression.
Retrocopies are probably involved in adaptive processes.
Figure 1
Figure 2
Figure 3
JoãoPaulo Machado, Agostinho Antunes PII:
S0888-7543(18)30577-9
DOI:
https://doi.org/10.1016/j.ygeno.2020.01.013
Reference:
YGENO 9451
To appear in:
Genomics
Received date:
12 October 2018
Revised date:
3 January 2020
Accepted date:
21 January 2020
Please cite this article as: J. Machado and A. Antunes, The genomic context of retrocopies increases their chance of functional relevancy in mammals, Genomics (2019), https://doi.org/10.1016/j.ygeno.2020.01.013
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier.
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The genomic context of retrocopies increases their chance of functional relevancy in mammals
João Paulo Machado 1,2 and Agostinho Antunes2,3,# 1
CIMAR/CIIMAR, Interdisciplinary Centre of Marine and Environmental Research,
University of Porto, Terminal de Cruzeiros do Porto de Leixões, Av. General Norton de
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Matos, s/n, 4450–208 Porto, Portugal.
Abel Salazar Biomedical Sciences Institute (ICBAS), University of Porto, Porto, Portugal.
3
Department of Biology, Faculty of Sciences, University of Porto, 4169 007 Porto, Portugal.
#
Corresponding author: Agostinho Antunes,
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2
Keywords:
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Full postal address
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Phone: (+351) – 220402742
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Email: [email protected]
Adaptive, Functional relevancy, Genomics, Mammals, Retrocopies
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Abstract Described as “junk” DNA, pseudogenes are dead structures of previously active genes present in genomes. Pseudogenes are categorized into two main classes: processed pseudogenes, formed through retrotransposition, and non-processed pseudogenes, typically originated from gene decay following duplication events. The term “processed pseudogene” has changed to “retrocopy” since they are likely to evolve new functional roles and became a retrogene. Here, we surveyed 38,080 retrocopies from chimpanzee, dog, human, mouse, and rat
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genomes to assess their potential adaptive value. The retrocopies inserted in the same chromosome of the parental gene have higher chances of remain potentially “active” (absence
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of premature stop codons and frameshifts) (~26.1%), while those placed into a different
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chromosome have a twofold decrease chance of continuing potentially “active” (~7.52%). The genomic context of their placement seems associated with their expression. Retrocopies
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placed in intragenic regions and the same sense of the “host” gene have higher chances of being expressed relative to other genomic contexts. The proximity of retrocopies to their
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parental gene is associated with a lower decay rate, and their location likely influence their
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expression. Thus, despite their unclear role, retrocopies are probably involved in adaptive
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processes. Our results evidence natural selection acting in retrocopies.
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1. Introduction In 1977 a structure termed “pseudogene” was reported for the first time in Xenopus laevis oocyte-type 5S RNA [1]. Pseudogenes are present from bacteria to vertebrates [2] and considered as non-functional sequences of genomic DNA that originated from functional genes [3]. Two main processes may be in the origin of pseudogenes from functional copies: (1) retro-transposition leading to the lack of a promoter ("dead on arrival"), and (2) decay of functional genes (frameshifts and/or premature insertion of stop codons), mostly from
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duplicated copies but also occurring in non-duplicated copies [4]. Therefore, pseudogenes are often distinguished into two different broad classes: processed pseudogenes, corresponding to
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those retro-transposed back into a genome via an RNA intermediate; and non-processed
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pseudogenes, which are genomic remains of duplicated genes or residues of dead genes, also known as unitary pseudogenes [5, 6]. Described as non-functional genomic fragments, and
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commonly referred to as “defunct” genes, it is assumed that they evolve under neutrality [7]. However, their presence might be functionally relevant, supported either by transcription data
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or a high degree of conservation [8, 9].
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Retrotransposed genes were routinely labelled as “processed pseudogenes”, but recently the
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term “retrocopies” has been instead adopted to express their functional roles [10]. Therefore, those reverse-transcribed through an RNAs intermediate and inserted in the genome as “new” DNA are now assigned as retrocopies (originated from retroduplication) [11]. The gene duplication is a pivotal mechanism to generate evolutionary novelty [11, 12], along with the retrocopy duplication trough segmental duplication [11]. Although these two mechanisms differ in the type of copies generated, since the tandem duplications usually produce copies that inherit the genetic features (e.g. introns and promoter), while the retrocopies lack the introns and promoters [11]. The retrocopies lack a promoter and consequently their expression is dependent on the recruitment of regulatory elements such as transcriptional mechanisms of host genes that surround their insertion site [11, 13]. Contrary to the segmental duplication, transcribed retrocopies are more likely to evolve “new expression
Journal Pre-proof patterns” and therefore evolve “new functions” [11]. The high transcriptional activity allows a substantial number of functional retrocopies to emerge as retrogenes highlight the role of natural selection in the retrocopy “repository” [12]. The genomic abundance of retrocopies is associated and dependent on the rate of gene duplication/loss [14]. Mammals have a high number of retrocopies, depending on the species and the annotation pipeline [15, 16]. Two recent works suggested the expression in the human genome of 1,286 out of 7,849 retrocopies (~12.5%) [10], and 615 out of 4,927 retrocopies
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(~16.4%) [17]. Contrary to the theoretical expectation that any gene can form a pseudogene, differences have been reported in the relative proportion, from gene to gene [18].
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Housekeeping genes, highly expressed genes in germ-line cells and those participating in
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basic metabolic regulations show multiple corresponding pseudogenes [16, 19-21]. This may be due to high expression levels of these genes, thus increasing their likelihood to accumulate
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mutations [22] or to be retro-transposed into the genome [23]. Moreover, the GC content of
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the genomic regions where the pseudogenes were placed also affect their mutation rate [24]. Early typified as non-functional units, here we provide support that non-random processes
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shape the retrocopies “repository”. Their destination and closer position to the parental gene
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influence frameshifts insertions and stop codons. Furthermore, retrocopies genomic context act as a major determinant for their expression. Here we present evidence supporting the adaptive role of retrocopies in mammalian genomes.
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2. Methods 2.1. Retrocopies retrieval and identification The retrocopies retrieved from pseudogene.org database include: Cannis familaris (dog, build 50), Homo sapiens (human, build 83), Mus musculus (mouse, build 84), Pan troglodytes (chimpanzee, build 50), and Rattus norgevicus (rat, build 74). From this database were retrieved pseudogenes annotated as processed pseudogenes (retrocopies) after excluding those categorized as “ambiguous” and “duplicated”. The data retrieved from pseudogenes.org
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encompass: C. familaris (6,001) (Table S1), H. sapiens (8,074) (Table S2), M. musculus (9,809) (Table S3), P. troglodytes (7,097) (Table S4) and R. norgevicus (7,099) (Table S5).
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These 38,080 pseudogenes, were reduced to 35,277 after discard those associated with
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mitochondrial DNA or inconclusive identity of the parental gene coordinates (chromosome).
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The parental gene coordinates ID were obtained through its corresponding ENSEMBL protein id using the ENSEMBL Biomart. The parental genes were retrieved for the Ensembl releases: C. familiaris (60), H. sapiens (94), M. musculus (95), P. troglodytes (54), and R. norgevicus
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(67). The retrocopies considered “inactive” are those showing signs of loss of protein coding
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ability (premature stop codons and frameshifts), while the other copies still showing protein
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coding ability were considered as “active” [25]. 2.2 Expression of retrocopies
The expression data from human retrocopies were retrieved from RetrogeneDB [17] and RCPedia [10]. In RetrogeneDB, based on ENSEMBL annotation, was retrieved information about expression and the ORF (Open Reading Frame) (188 retrocopies). While from RCPedia, were retrieved all the 7,849 retrocopies, their expression relative to their genomic context (intragenic or intergenic). Either RetrogeneDB or RCPedia use multi-step pipelines [10, 17], while to evaluate retrocopies expression the first use short read libraries in various tissues from selected records of NCBI SRA database [17] the other uses RNA-seq data from six tissues (brain, cerebellum, heart, liver, kidney and testis) [10, 26].
Journal Pre-proof 2.3. Insertion simulation For the retrocopies inserted in the same chromosome of the parental gene, we conducted a simulation study to test if the distance “retrocopy-parental gene” was random or biased. To accomplish this test, we followed the steps 1) collect the position of the parental genes and retrocopies; 2) for each chromosome was shuffled the position of the retrocopies within the list of retrocopies positions. Then measured the distance between the parental gene and the retrocopy. Both analyses were repeated three times to inspect the consistency of the obtained
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data. While the majority of the retrocopies arose through retro-transposition events, some arose from duplication of previously retro-transposed genes, and these events lead to
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duplicated-retrocopies. To avoid possible bias introduced by segmental duplication regions, highly rich in retrocopies [27], the same distance analysis were performed discarding the
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retrocopies inserted in these regions. Data from the retrocopies and segmental duplication
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were retrieved at Segmental Duplication Database [28]. Additionaly to avoid potential bias due to duplications of the pairs retrocopy parental gene, we filtered for retrocopies inserted in
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the same chromosome. The filter was created by BLASTting 5 kbp flanking the retrocopy
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location and minimum alignment of 2.5 kbp and within 10 kbp.
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To test the possibility of a biased difference in the proportions of retrocopies with stop codons or alteration of the ORF relatively to their insertion chromosome, we performed a simulation randomly inserting these retrocopies in the five studied genomes using an “in house” script. To accomplish this the position of the parental genes and retrocopy was read, after this step the retrocopy was randomly sort as “active” or “inactive” up to the observed number of “active” retrocopies in each species. The proportions were then compared using Fisher’s exact test. 2.4. Gene evolution under neutrality Pseudogenes are assumed to evolve under neutrality. If this assumption is correct then the reminiscent sequences of previous functional copies will “accept” all the mutations. The
Journal Pre-proof solely difference rely in the ratio of transitions and transversions. It is expected that the transitions double the number of transversions, and this is an explanatory model for sequences evolving under neutrality [29]. Here, we tested the time and the mutation insertion effect of pseudogenes evolving under the emulated Kimura model [30], with the parameters of the model settled to a tS/tV (kappa value) = 2, which is a value frequently observed in mammalian genomes [29]. In each step, two random mutations were inserted, with doubled chances to insert a transition rather than a transversions to a maximum of 400 steps (800 mutations). For
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each step the sequences were submitted to blast searches (either BLASTn or BLASTx) and the genes were considered lost when the first of the following criteria were achieved: 1) the
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expected value for the gene of interest is below 1e -10 ; or 2) presence of an unrelated gene in the top-hit results. The mutation rate and the predictive time for gene loss were calculated
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under the assumption of 2.41 mutation/site/year (averaged value observed for fossil record
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and sequences) [31].
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3. Results 3.1. Distribution in the Chromosomes The retrocopies follow a random distribution in the chromosomes, given the relation between chromosome length and retrocopies number (Table S6). This trend appears to be generalized in mammals since similar correlations were observed in the five mammals studied (chimpanzee, P. troglodytes; dog, C. familiaris; human, H. sapiens; mouse, M. musculus; and rat, R. norvegicus) (Table S6). Yet the relation is nearly absent when considering the
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chromosome length and the number of retrocopies formed from each chromosome (Table S6). The mouse chromosome Y has been excluded from these analyses given the high number
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of retrocopies allocated (~7 times more than the average of the others chromosomes).
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We detected a non-random position of the parental gene and retrocopies in the human genome
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without signs of disablement (i.e. without stop codons and maintaining the ORF), similar to the other four species described above . We observed evidences of disablement in 92.4% of
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the 29,832 retrocopies inserted in a different chromosome relatively to the parental gene.
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When it is considered the retrocopies inserted in the same chromosome (5,445), the dismantlement decreased to about 73.9%. The disablement ratio of retrocopies placed in the
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same chromosome is significantly lower when compared to those placed in a different chromosome (Fisher’s exact test, p-value <2.2e-16) (Table 1 and Table S7). The proportions in the five species were significantly different (Table 1). In the human genome we have been able to use 7,446 retrocopies and from these 6,564 are inserted in a different chromosome while 882 are inserted in the same chromosome relatively to the parental gene. The insertion relatively to the parental gene seems associated with disablement, since 559 (~8.51%) are a different chromosome and 104 (~11.79%) in the same chromosome. While less evident, this disproportion between retrocopies inserted in the same or different chromosome (disabled) is statistically significant (Fisher exact test, p-value ~ 0.02). For the others four species the same trend was observed with higher proportion of retrocopies inserted in the same chromosome of the parental gene considered as “active” (Table 1 and Fig. S1). Therefore the proportion of
Journal Pre-proof retrocopies with stop codons and/or frameshift re-allocated in a different chromosome is significantly lesser than those inserted in the same chromosome relatively to the parental gene. From the 7,446 retrocopies of the human genome used here, 2,251 are in segmental duplicated regions. After removing those regions, the same trend is observed, since ~8.69% “active” where in a different chromosome relatively to the parental gene and ~10.6% “active” retrocopies are placed in the same chromosome of the parental gene. The difference in the
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proportion (Fisher exact test, p-value ~ 0.018) is observed even when the segmental duplicated regions were discarded.
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Even when excluding based on distance 1Mbp, 0.1Mbp, 0.01Mbp, 0.001Mbp, 0.0001Mbp, to
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take into consideration possible gene conversion effects, the fisher’s exact test show the same
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trend (Table S8). The proportion of retrocopies “active” copies in the same chromosome are significantly higher than those placed in a different chromosome relatively to the parental
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gene.
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For the retrocopies present in the five genomes, we tested a random distribution of those without stop codons and maintaining the ORF (here considered as “active”). Assuming their
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original positions (parent gene and retrocopy) but allowing randomly retrocopies to be considered as “inactive” or “active” up to the limit of those observed as “active”, the results from 100 replicates [32]. In this scenario and allowing 663 retrocopies (for the human replicate) to be without stop codons, only six replicates had significantly higher proportions of retrocopies without stop codons and maintaining the ORF inserted in the same chromosome compared to those inserted in a different chromosome. Simulations were repeated for the other four species also revealed cases where the proportions of retrocopies inserted in the same chromosome relatively to the parental gene were significantly higher: 2 in chimpanzee, 6 in dog, 1 in mouse and 5 in rat. Overall only 4% of the simulations revealed
Journal Pre-proof a significantly higher proportion of retrocopies “active” in the same chromosome, therefore unlike to be a random observation. 3.2. Insertion in the same Chromosome The Mann-Whitney test shows positions significantly closer to the parental gene than a random insertion (p-value < 0.01). This is also observed in simulations using the original positions to constrain the initial placement (p-value < 0.01) (Fig. 1A and Fig. 1B). This trend was observed for each species suggesting the placement of the retrocopies closer to the
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parental gene. Together, these two different simulation scenarios (original positions and completely random [33]) revealed that retrocopies are closer to the parental gene than
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predicted by a random distribution, either including or excluding segmental duplicated
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regions (Fig. 1A and Fig. 1B).
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The presence of segmental duplications (SDs) may alter the results from the measured distance between the parent “gene- retrocopy”, particularly on SD pairs containing both
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retrocopy and parental gene. In the human genome from 882 retrocopies, 277 are within
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segmental duplications [27]. After removing the SDs regions we also detected a significant result for Mann-Whitney-Wilcoxon test, p < 0.01, consequently the retrocopies are closer to
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the parental gene than a completely random distribution (Fig. 1A and Fig. 1B). The disablement of retrocopies and the distance to the parent gene follows an observable distance-based trend. The distance of the pair “retrocopy-parent gene” is significantly lower for the retrocopies considered as “active” (absence of stop codons and maintain of the ORF) when compared with those considered as “inactive”, Mann-Whitney U Test p-value = 0. 18. Indeed, most retrocopies without premature stop codons are closer to the parental gene (Fig. 2). Around 50% of the retrocopies without stop codons and maintaining the ORF are placed within 3Mbp (Million base pairs) relatively to the parental gene position. Considering those with stop codons or missense mutations, ~50% are placed within 3.6 Mbp distance from parent gene to the retrocopy (Fig. 2). Furthermore within the 25 Mbp was observed ~71.0%
Journal Pre-proof of the total number of retrocopies with in-frame stop codons or missense mutations and ~74.1% of all the retrocopies without stop codons and maintaining the ORF. 3.3. Processed Pseudogenes Expression Along with the absence of stop codons, the expressed retrocopies are considered to act as functional elements. On the other hand, symptoms of non-functionality include frame disablement (premature stop codons) and pseudogenes nucleotide sequence decay or incompleteness. Several transcribed pseudogenes are disabled [8], but some disabled
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pseudogenes revealed evidence of being functional relevant [34]. The data from RetrogeneDB shows that from 615 expressed retrocopies in the human genome (Fig. 3A) only 188
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retrocopies maintain the ORF, i.e. nearly 30.5% (Fig. 3B). Data collected from RCPedia
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showed an association between genomic context and the expression, since 26% expressed retrocopies are located in intragenic regions and in the same sense of the “receiver” gene (Fig.
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3C). By contrast, only 14% and 16% of those are expressed when placed in intergenic or intragenic positions on the opposite sense, respectively (Fig. 3C). In these two cases, the
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expression is significantly lower when compared to those inserted in intragenic positions in
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the same sense of the gene “host” (Z-Score, p-values < 0.0001).
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3.4. Genes evolving under neutrality Given the detection of retrocopies under adaptation [35] it is relevant to access the time course for a gene to become undetectable through blast searchers (BLASTn or BLASTx) while evolving under complete neutrality. Using the cut-off value 1x10-10 and assuming an evolutionary rate of 2.415x10-9 mutations/site/year [31] we estimated for eight genes (BTF3, CYC1, GADPH, H3F3B, PPIA, RPL7A, SCOP and RPL39) the time needed for the blast searches to be below the empirical cut-off (Fig. S3). Our data suggested nearly 229.46 ± 100.82 million years (Myr) (Table S9) for a gene to be undetectable trough blast searches, but the insertion of the first stop codon would occur earlier, around 26.23 ± 22.95 Myr.
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4. Discussion 4.1. Retrocopies origin and their location The random placement of human retrocopies has been previously described as a “bombardment” along evolution [15], thus hypothesizing retro-transposition as an efficient process to introduce regulatory elements into the genome “in search” of new target genes [36]. Consistently, this “stated war” was detected in the five analyzed mammalian species
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(chimpanzee, dog, human, mouse and rat). This seems a trend for mammals, since larger chromosomes have a higher number of retrocopies. As previously observed in the human
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genome [15], retrocopies allocation is mainly affected by chromosome length [15]. While the results showed a random process on the retrocopies origin given the absence of relation
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between chromosome length and number of “parental” genes retro-copied. Several factors
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were advanced to influence the likelihood of a gene to form a retrocopy, such as expression level and gene length [18], or mRNA stability [37]. Additionally, gene length has been
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mentioned as a major determinant influencing pseudogenes formation, since longer genes
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tend to “produce” more non-processed pseudogenes, whereas retro-pseudogenes are typically formed by short protein-coding genes [38-40]. Together this suggests that the raw material to
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form “new” retrocopies appears to be highly associated with the nature of genes (parental genes) or the mRNA molecules. These properties have been shown to have an adaptive value, such as mRNA stability [41, 42], gene expression [43, 44] and gene length [45]. 4.2. Location as factor for Retrocopies “survival” Despite the retrocopies random insertion, here we reported an association between their placement location and frequency of premature stop codons or frameshifts (i.e. indicative of function loss) [8]. Those placed in the same chromosome showed less evidences of disablement. Indeed, simulated scenarios showed that the association between the retrocopies distribution and the disablement is highly unlikely to be a random event. And even excluding
Journal Pre-proof those within range that is compatible with gene conversion the same trend is detected and excluding possible genes in duplicated regions. Additionally, those retrocopies inserted in the same chromosome of the parental gene tend to be placed closer to the parental gene than a completely random distribution. The survival of the retrocopies requires a de novo promoter or its recruitment from the genomic environment around the placement site [35, 46]. A closer location seem advantageous since some retrocopies adjacent to the parental gene tend to be co-regulated [47]. Later it has been shown
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an absence of co-regulation and, instead, an correlation between expression of retrocopies and parental gene, irrespective to their distance [48]. Thus, after random allocation, we
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hypothesize that their closer location confers advantages and increase their chances to become
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functionally relevant. Another possibility is that a closer location, as observed in the retrocopies allocated in the same chromosome, implies that they are young retrocopies. The
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detection of age-dependent pseudogenes (retrocopies) was previously described as skewed [49], as newly formed retrocopies are identified easier than older retrocopies [50]. Despite the
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different age of the retrocopies as estimated in previous studies [51], the random insertion
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decline the possibility of an age association with absence of signs of disablement. Their
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placement seems random, but selectively disabled depending on the insertion location. This is also supported by the observation of an average lower similarity between the parental gene and the retrocopy inserted in a different chromosome. Alternatively, a closer location increases their chances to became functional relevant by a more favorable expression context. Therefore, being closer to the parental gene, would lead to a decreased number of substitutions, which would increase the retrocopies detection, and consequently enhance the frequency of pseudogenes closer to the parental gene. 4.3. Retrocopies acting as “repositories” of information Based on the analysis of retrocopies in human and mouse genomes, it has been estimated that the formation of “new” retrocopies occurs in a rate of about 1-2% per gene per million years [52]. In the human genome, gene duplications happen at a predicted rate of 0.9% per gene per
Journal Pre-proof million years [52]. Thus, the arousal of “new genes” through retrocopies may be even more relevant than gene duplications. Their maintenance also appears to be of prime relevance since ~40% of the retrocopies are shared between human and mouse [53]. Under a neutral evolutionary pace, it will take ~244Myr for a gene to become “undetectable”, while a hypothetical frameshift might occur much earlier (~26Myr). This implies that pseudogenes, either processed or non-processed, and detected through blast searches older than ~244 Myr were under a period of non-neutral evolution. The expression of retrocopies,
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and their predicted or proofed functionality, is also observed in some non-processed pseudogenes placing them as interesting and important functional units [54].
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The retrocopies persistence lead to an increased probability of pseudogene resurrection [52].
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Therefore, this shows the great relevance of pseudogenes in the generation of evolutionary
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novelties, likely acting as “repositories” of innovation, even in scenarios evolving under neutral evolutionary pace.
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4.4. Genomic landscape as a contributor to retrocopies expression
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The maintenance of the expression patterns might be important for regulatory processes [23,
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55] and probably enables the functional “resurrection” of pseudogenes, frequently described in human and mouse pseudogenes [52]. The lack of an appropriate regulatory environment often lead to the degeneration of the retrocopies [56], and we found that processed pseudogene near the parental gene lead to an increased probability of retention. Concordantly, a parental gene and its pseudogene interdependence were reported for the gene ABCC6 and the ABCC6P1, since their co-expression resulted in ABCC6 decreased expression [57]. Moreover, gene order in eukaryotes follow a non-random location, as those closely placed tend to be co-expressed and co-functional [58]. The allocation site seems closely associated with the chances for the retrocopies, as those placed in intragenic regions in the same sense of the gene “receiver” provide an adequate “expression context”. By contrast, when they are placed in intergenic regions or even in intragenic regions, their
Journal Pre-proof chances to be expressed decrease. After the insertion of a retrocopy, the first step leading it to a retrogene (functional counterpart of the retrocopies) is their expression [47]. Therefore the genomic context of the retrocopy allocation influence their probability of becoming a retrogene. Our results reveal three relevant aspects of retrocopies. 1) Retrocopies inserted in the same chromosome of the parental gene have higher chances to “survive”. 2) Retrocopies inserted in the same chromosome of the parental genes are closer than a completely random process. 3) Placement of retrocopies influences their chances of expression.
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4.5. Retrocopies as functional units
Searching functional elements in genomes is of prime relevance for evolutionary biology. In
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recent years were raised several arguments regarding the functionality of retrocopies. The
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processes that lead to their arousal and ubiquitous presence in the mammalian genomes is therefore of major relevance to understand their function. While retrocopies seem randomly
Pr
placed and dispersed in the genomes, their location apparently determines their chances to acquire a functional relevancy. Those retro-transposed pseudogenes closer to the parental
al
gene (same chromosome) increase twice their chances of survival (i.e. lack of perceived stop
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codons and maintain the ORF). In addition, those placed in intragenic regions, in the same
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strand of the ‘receiver’ gene, have higher chances to be expressed. Thus, our results highlight a mechanism of natural selection acting on those retrocopies, revealing a functional relevancy in their maintenance and placing them potentially as functional units. Here, we described several non-random processes occurring in retrocopies, which suggest that retrocopies are subjected to natural selection and their perseverance in genomes is a nonrandom process.
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5. Acknowledgements We are thankful for the comments provided by the Associate Editor and two anonymous reviewers, which helped improving a previous version of this manuscript. The authors acknowledge the Portuguese Fundação para a Ciência e a Tecnologia (FCT) for financial support to JPM (SFRH/BD/65245/2009). This work was further supported by a grant from Iceland, Liechtenstein and Norway through the EEA Financial Mechanism and the Norwegian Financial Mechanism. AA was partially supported by the Strategic Funding
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UID/Multi/04423/2019 through national funds provided by FCT and European Regional Development Fund (ERDF) in the framework of the programme PT2020, and the FCT project
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PTDC/AAG-GLO/6887/2014 (POCI-01-0124-FEDER-016845) and PTDC/CTA-
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rn
al
Pr
e-
AMB/31774/2017 (POCI-01-0145-FEDER/031774/2017).
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Tables Table 1 – Comparison between retrocopies inserted in the same and different chromosome, the proportion of “active” and identity compared with parental gene . The term active is associated with those maintain the ORF and without stop codons, (%) proportion of “active”. SD – Segmental Duplications, w/o – without, ǂ - Total without considering Human only in non-segmental duplicated regions. Different Chromosome
Fische r Te st Exact
“Inactive”
Active (%)
Identity (%)
Fraction (%)
“Active”
“Inactive”
Active (%)
Identity (%)
Fraction (%)
(p-value )
Chimpanzee
150
600
20.00
72.73
92.15
527
5,344
8.98
76.56
94.80
<2.20E-16
Dog
168
487
25.65
73.17
91.02
415
4,774
8.00
75.77
94.88
<2.20E-16
Human
104
778
11.79
72.92
92.97
559
6,005
8.51
77.21
94.64
0.001
M ouse
389
1,591
19.65
80.02
95.50
521
6,716
7.20
75.27
95.50
<2.20E-16
Rat
316
862
26.82
76.76
91.79
259
4,712
5.21
72.25
94.46
<2.20E-16
Human (w/o SD) Total (ǂ)
58
547
10.60
70.63
92.30
390
4,489
8.69
75.73
94.64
0.104
1,127
4,318
26.10
76.58
93.47
2,281
27,551
7.52
75.42
94.89
<2.20E-16
Pr
al
rn Jo u
Spe cie s
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“Active”
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Same Chromosome
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Figures
Fig. 1 - Retrocopies inserted in the same chromosome: distance to parental gene and influence of segmental duplication in human genome. Distance of retrocopies retro-transposed to the same chromosome and the parental gene. The distance observed, a random scenario, with the retrocopies were randomly distributed in each
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chromosome. The observed distance from retrocopies and parental gene after the removal of segmental duplication regions. A) Retrocopies inserted in the same chromosome in the human
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pr
genome. B) After excluding segmental duplicated regions.
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Fig. 2 - Retrocopies inserted in the same chromosome: distance to parental gene and presence of stop codons.
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Comparative empirical cumulative distribution of retrocopies inserted in the same
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chromosome. The orange line represents retrocopies containing stop codons and the blue line
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represents the retrocopies free of stop codons.
Fig. 3 - Retrocopies expression and genomic context. Data retrieved from RetrogeneDB: A) Expressed retrocopies; B) Expressed retrocopies that maintain the ORF, text labels refers to parent gene name’s, the linkers are colored accordingly to the chromosome where the pseudogene is placed. Data retrieved from RCPedia C) Schematic representation of the expressed retrocopies and the genomic context: 1) hypothetical “host” gene with two exons, 2) additional hypothetical gene. The ribbons indicate orientation of the reading frame.
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Supplemental Material Fig. S1. Processed pseudogenes inserted in the same chromosome: distance to parental gene. Distance of retrocopies retro-transposed to the same chromosome and the parental gene. a) Chimpanzee, b) Dog, c) Mouse and d) Rat. Fig. S2. Blast results from the simulation of 8 genes evolving under neutrality. On each left subfigure are the results from BLASTn and on right for BLASTx for the following genes:
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a) BTF3, b) CYC1, c) H3F3B, d) GADPH, e) PPIA, f) RPL7A, g) RPL39 and h) SCOP. The y-axis is log-scaled. The x-axis (n) represent the natural number of the blast order, and for
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each step (n) corresponds to two mutations. The reference line corresponds to the empirical 1E-10 , value above this value were discarded or partially discarded and not represented in the
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plots.
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Table S1. Retrocopies in Dog. Data retrieved from pseudogenes.org and the data for the
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parental gene location. #N/D - No data (excluded from the analysis).
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Table S2. Retrocopies in human. Data retrieved from pseudogenes.org, and the data for the parental gene location. #N/D - No data (excluded from the analysis). SDD - Segmental
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duplicated region.
Table S3. Retrocopies in mouse. Data retrieved from pseudogenes.org, and the data for the parental gene location. #N/D - No data (excluded from the analysis).
Table S4. Retrocopies in chimpanzee. Data retrieved from pseudogenes.org, and the data for the parental gene location. #N/D - No data (excluded from the analysis). Table S5. Retrocopies in rat. Data retrieved from pseudogenes.org, and the data for the parental gene location. #N/D - No data (excluded from the analysis).
Journal Pre-proof Table S6. Individual correlation between the retrocopies location and parental gene origin. (*) significant correlation.
Table S8. Retrocopies “active” and “inactive” excluded based on gene conversion range. Table S9. Estimated age of processed pseudogenes misidentification after evolving under
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Journal Pre-proof Authors’ contributions JPM performed the phylogenetic, evolutionary and bioinformatic analyses and drafted the manuscript. AA participated in the design, genetic analyses, drafting and coordination of the
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study. All authors read and approved the final manuscript.
Journal Pre-proof Highlights:
The genomic context of retrocopies increases their chance of functional relevancy in mammals
The retrocopies inserted in the same chromosome of the parental gene have higher chances of
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decrease chance of continuing potentially “active”.
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remain potentially “active”, while those placed into a different chromosome have a twofold
Retrocopies placed in intragenic regions and in the same sense of the “host” gene have higher
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chances of being expressed relative to other genomic contexts.
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The proximity of retrocopies to their parental gene is possibly associated with a lower decay
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rate, and their location likely influence their expression.
Retrocopies are probably involved in adaptive processes.
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