Volume 14 Supplement 8
The de novosequence origin of two long non-coding genes from an inter-genic region
- Yulin Dai†1, 2,
- Shengdi Li†1, 2,
- Xiao Dong1, 2,
- Han Sun1, 2, 3,
- Chao Li1, 2,
- Zhi Liu1, 2,
- Beili Ying4,
- Guohui Ding1, 3Email author and
- Yixue Li1, 3Email author
© Dai et al.; licensee BioMed Central Ltd. 2013
Published: 9 December 2013
The gene Polymorphic derived intron-containing, known as Pldi, is a long non-coding RNA (lncRNA) first discovered in mouse. Although parts of its sequence were reported to be conserved in rat and human, it can only be expressed in mouse testis with a mouse-specific transcription start site. The consensus sequence of Pldi is also part of an antisense transcript AK158810 expressed in a wide range of mouse tissues.
We focused on sequence origin of Pldi and Ak158810. We demonstrated that their sequence was originated from an inter-genic region and is only presented in mammalians. Transposable events and chromosome rearrangements were involved in the evolution of ancestral sequence. Moreover, we discovered high conservation in part of this region was correlated with chromosome rearrangements, CpG demethylation and transcriptional factor binding motif. These results demonstrated that multiple factors contributed to the sequence origin of Pldi.
We comprehensively analyzed the sequence origin of Pldi-Ak158810 loci. We provided various factors, including rearrangement, transposable elements, contributed to the formation of the sequence.
KeywordsOverlapping transcripts Sequence Origin of Pldi and Ak158810 loci Conserved Element Substitution rate
Although pervasively transcribed, only 5%-10% of the human genome is covered by mRNA and spliced non-coding RNAs, and the majority of which does not encode proteins . Long non-coding RNAs (lncRNAs) are defined as transcribed non-coding RNA larger than 200 nt in length, which plays an essential role in regulating gene expression, chromatin functions . As lncRNAs act as biological building blocks, it is necessary to understand the process of developing new lncRNA genes .
The emergence of a functional lncRNA gene could be summarized into various evolutionary scenarios, including metamorphosis of a protein coding gene, derived from a genomic region previously devoid of exonic sequence, duplication by retro-transposition, and emergence following tandem duplication or insertion of transposable elements [1, 4]. For most of the scenarios, comprehensive studies have been established on specific lncRNA genes with well-known functions, such as XIST, HOTAIR [5, 6]. However, little was known about developing a new lncRNA gene from a non transcribed genomic region. The mechanism of the de novo origin of a lncRNA gene remains to be clarified.
Previous study on de novo protein has accounted for that those seemingly dispensable sequences in non-genic regions could generate adaptive functional proteins through evolution. The de novo birth and development of a potential protein coding gene is in line with increasing open reading frame (ORF) length and conservation through the natural selection benefited from random translation on genome [3, 7, 8]. Like proteins, the occasional transcription and changing events in non-genic sequences could provide raw material generating de novo lncRNAs . Here, we focused on the sequence origin of a lncRNA in an intergenic region, demonstrating its sequence components and changes within species.
Pldi gene was previously identified and defined as an intergenic originated lncRNA gene, which is overlapped with a putative opposite-strand transcript, AK158810 (Additional File 1). Pldi locates within a 200 kb region that is free of annotated transcripts or expressed sequence tags (ESTs) in rat and humans, which raise the possibility of de novo emergence of the Pldi-AK158810 loci (about 20 kbps-long). Knocking out Pldi would reduce sperm motility and testis weight, indicating that Pldi has the ability in regulating the expression levels of other genes in testis . Numerous functional non-coding RNAs have been demonstrated to regulate gene expressions through an antisense mechanism, playing an important role of gene overlapping in non-coding RNA functions [11–13]. On the contrary, few studies discussed the origin of overlapping non-coding RNAs due to lacking of clear markers, like ORF in protein.
In this study, we conducted a comprehensive analysis on the sequence origin of mouse Pldi-Ak158810 loci. We evaluated various factors that contribute to the origin, and gave adequate evidence to prove the de novo origin of this loci. Moreover, we found that Pldi-Ak158810 established its fixation from a specific overlapping region some time before emergence. We further discussed the potential role of the local element in the evolution and fixation of this orphan lncRNA gene loci.
Materials and methods
Genomes and sequences
The 13 genomes of vertebrates used in this study were downloaded from UCSC genome database http://hgdownload.soe.ucsc.edu/downloads.html. Genome versions of these 13 genomes are in Additional File 2. The sequence of Pldi-Ak158810 loci was picked from mouse (GRCm 38) export data in Ensembl http://www.ensembl.org.
Sequences of EST Ak158810 were checked to find all the potential open reading frames, by using ORF finder (Open Reading Frame Finder) by default minimum frame size. The ORF finder is accessible in this website server http://www.ncbi.nlm.nih.gov/gorf/gorf.html
Sequence comparison and alignment, phylogeny analysis
We used nucleotide Blast (Basic Local Alignment Search Tool) to detect homology between Pldi-Ak158810 nucleotide sequence and vertebrate genomes, a cutoff for identity was set at 80%. Protein Blast was used to find protein coding genes homologue to the genes flanking with Pldi and Ak15880. ClustalW http://www.clustal.org/download/current/ was used to align protein and nucleotide sequences . MEGA5.1 was used to construct neighbor-joining phylogenic tree . The genomic alignment of 30 vertebrates by MultiZ was downloaded from UCSC [17, 18]. All genomes were mapped to the mouse chromosomes.
Repeats and transposable elements annotation
Repeats and transposable elements were annotated by Repeatmasker program. Sequences of Pldi-Ak158810 were submitted to the Repeatmasker website http://www.repeatmasker.org version 4.0.1, which uses default parameters. The repeat class were transformed and grouped as SINE, LINE, DNA, LTR and others. In the analysis of ancient transposable elements, we did not include simple repeats and low complexity sequences .
Model for substitution rate change
where dtestHD, dtestHR, dtestMR are the genetic distance of the test sequences between human and dog, human and rat, mouse and rat, respectively, tHD, tHR, tMR represent for the divergent time between each pair of species.
where dRefHD, dRefHR, and dRefMR are the genetic distance of the reference exon sequences between human and dog, human and rat, mouse and rat.
where 0 < k1, k2 <1. raverHD, raverHR and raverMR are the average substitution rates at different stages. If raverHR <(>) raverHR , we get r0<(>) r1. Similarly, a reduced r2 will produce a lower raverMR, as the only envolving path affected by r2.
We used ClustalW to realign the conserved elements in Pldi-Ak158810 and exons of surrounding four genes, manually remove sites with low similarity by Bioedit http://www.mbio.ncsu.edu/bioedit/bioedit.html. All the four genes were merged into one single alignment. Then a Maximum Likelihood (ML) tree and distance matrix was estimated by PAML 4.6 baseml for each alignment) .
We collected two sources of methylation data as a comparison, one is from mouse tissue, the other is from human ENCODE data.
Mouse brain methylation data was obtained from forebrain tissue of lab mouse (GSM809309)
Demethylation data from human UCSF brain methylation database viewed with UCSC genome browser was implemented to detect the DNA methylation in the human homologue region of Pldi-Ak158810 loci, which was displaced in Additional file 3.
RNA-seq data is from Encode Cold Spring Harbor Lab (CSHL) RNA-seq, and there are 5 types of tissues included (heart, kidney, ovary, spleen and testis). We viewed this data using UCSC genome browser .
Transcriptional factor binding site data
Human, Mouse, Rat (HMR) Conserved Transcriptional Factor Binding Site (TFBS) was implemented to displace the potential binding sites of these two highly conserved regions .
The co-emergence of Pldi and Ak158810transcription
We studied the emergence time of Pldi and Ak158810. Pldi locates in an inter-genic region free of any human and rat EST signals, indicating that Pldi and its antisense putative gene generating Ak158810 were not transcribed before the divergence of rodents. In mouse lineages, RNA transcript of Pldi has been discovered . To validate the transcript Ak158810, we compared its 2.9 kb sequence with mouse EST database from NCBI. The result confirmed the transcription of Pldi antisense strand, and EST hits matched with splicing of the first and second exons of Ak158810 (Additional File 4). We further analyzed the open reading frames (ORFs) in Ak158810 RNA, The longest ORF is shorter than 110 amino acids. (Two AUG codons with shorter reading frames ~70 amino acids preceded this long ORF) (Additional File 5). It indicates that Ak158810 is not likely to encode proteins. Our results, along with previous knowledge , showed that the Pldi and Ak158810 are two mouse-specific lncRNAs located on anti-sense strand to each other. These evidences suggest that Pldi, and its putative antisense lncRNA, Ak158810, were first transcribed at similar time between the divergence of mouse and rat.
Pldi-Ak158810loci is conserved in mammals and originated from an intergenic region
The origins of exons and introns
The sequence alignment also revealed that were involved in the evolution of Pldi region. We identified two inversions at the loci. First one is the inversion of a ~800 bps fragment, containing the first exon of Ak158810 (Figure 5 & Addition File 1). Another inversion is overlapped with Ak158810 exon 5. The regions, homologue to the two inversed fragments of non-rodent mammalians, are in opposite direction to those of mouse, which reveals both inversions occurred before the divergence of mouse and rat, and after the divergence of primates and rodents. Interestingly, the first inversed region is highly conserved, which is discussed in following section.
Transposable events contribute to the formation of Pldi-Ak158810loci
Transposable elements (TEs) that contributed to the formation of ancestral Pldi-Ak158810 sequences.
Species with the TE
mouse, rat, human, dog
mouse, rat, human, dog
mouse, rat, human, dog
mouse, rat, human
mouse, human, dog
Gene composition originated from TE
Origin from TE
A, B, C, D
G, H, I, I, K
An inverse element is highly conserved and obtains a reduced substitution rate after rearrangement
From genomic sequence in mammals, we noticed the Pldi-Ak158810 loci was interrupted by chromosome rearrangement in a period of time before its emergence in mouse lineage. Interestingly, one of the rearranged fragments associated with Ak158810 exon 1 is highly conserved among species. From this point of view, we estimated the substitution rate of this highly conserved region among species to test whether inversion contribute to fixation of local region. To better learn the evolution of this loci, we examined the change in substitution rate during the fixation of 4 species, mouse, rat, human and dog. Taking the exon sequences of flanking genes (pcbd1, slc29a3, sgpl1, unc5b) as a reference, we constructed a simple model to test the rate change at two time points: the point of chromosome rearrangement and the emergence of Pldi and Ak158810 in mouse lineage (Figure 5). We extracted sequences of two conserved elements (CE1, conserved element 1 in the inversion; CE2, conserved element 2 near Ak158810 exon 3), which could be detected by Blastn in distant organisms.
A simplified model to test the change of substitution rate at two time point: occurrence of the inversion and emergence of Pldi and Ak158810 gene.
Exons of surrounding genes
Conserved element 1 (inversed region)
Exons of surrounding genes
Conserved element 2
Various factors contribute to the formation of Pldi-Ak158810sequence
A new lncRNA gene could emerge through different scenarios, such as metamorphosis from a protein-coding gene, interrupted by tandem repeat and transposable elements, and de novo origin from an intergenic region. Our analysis further confirmed the inter-genic origin of Pldi-Ak158810 sequence without any clues of long genomic duplication in a recent past. Tracing back in history, both transposable events and chromosome rearrangements were found in the region. In conclusion, the formation of the Pldi-Ak158810 loci, which became a pair of lncRNA genes in mouse lineage, was affected by multiple factors.
Fixation of partial Pldi-Ak158810sequence before gene birth
A previous study indicates that the conservation of non-coding RNA is only slightly higher than that of inter-genic region . In Pldi region, reduced polymorphism has been detected in specific mouse lineage, which suggests the present of purifying selection. Nevertheless, we found in our data that partial Pldi-Ak158810 sequence is conserved in all mammalians. It raises the possibility that purifying selection may be acquired in partial Pldi-Ak158810 region much earlier than the gene birth.
According to these observations, we suggested in species other than mouse, partial region of Pldi-Ak158810 loci could not be simply recognized as "non-functional" before the birth of Pldi.
Birth order of AK158810 and Pldi
It has been known that two neighboring genes may form a transcriptional unit , which is correlated with expression. As for this case, we assumed the earlier developed lncRNA might influence the birth of the other one by expression level. We attempted to detect the birth order of AK158810 and Pldi, According to previous studies, the birth order of Ak158810 and Pldi may not quite clear for the following reasons: first, testis where Pldi was born has been considered as an important organ for the emergence of a novel gene [4, 29]. According to RNA-seq data (CSHL) and previous study, Pldi is a testis-specific lncRNA, while Ak158810 is likely to have a wide expression range, such as heart, spleen and kidney (Additional File 8). That indicates that Ak158810 seems to be a not that young gene as Pldi [4, 30]; Second, considering northern blot experiment, Pldi exists in more species or lineages in mouse testis [10, 31], inferring that it is more likely to be older than Ak158810. The conflict result, together with the phenomenon, that the expressions of both lncRNAs are limited in mouse, demonstrated that AK158810 and Pldi were newly transcribed lncRNAs in a similar age after the divergence of mouse and rat.
In this study, we comprehensively analyzed the sequence origin of a lncRNA antisense gene pair, Pldi-Ak158810. We found out various factors, including rearrangement, transposable elements, contributed to the formation of the sequence. We also figured out partial sequence of the entire loci is highly conserved in mammalians before the birth and provided evidences and correlated factors for the early fixation of conserved elements.
Lists of abbreviation
- Pldi :
Polymorphic derived intron-containing
long non-coding RNA
expressed sequence tags
Conserved Element 1
Conserved Element 2
Short interspersed nuclear elements
Long interspersed nuclear elements
DNA repeat elements
Long terminal repeat elements, which include retroposons
Transcriptional factor binding site
Cold Spring Harbor Lab
Human, mouse, rat.
We thank Prof. Lei Liu, Dr. Guangyong Zheng, Dr. Zhen Wang, Jie Wang, and Tiancheng Liu, for their helpful comments and suggestions. This work was supported by the National Key Basic Research Program of China, known as 973 Program (grant number: 2011CB910204, grant number: 2010CB529206, grant number: 2010CB912702), the Research Program of The Chinese Academy of Sciences (grant number: KSCX2-EW-R-04, grant number: KSCX2-YW-R-190, grant number: 2011KIP204) and National Natural Science Foundation of China (grant number: 31070752).
Funding for the publication fee comes from National Natural Science Foundation of China (grant number: 31070752).
This article has been published as part of BMC Genomics Volume 14 Supplement 8, 2013: Selected articles from the International Conference on Intelligent Biology and Medicine (ICIBM 2013): Genomics. The full contents of the supplement are available online at http://www.biomedcentral.com/bmcgenomics/supplements/14/S8.
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