The initial dataset, collected from the study by Foulds et al (13), contained 1,823 lncRNA IDs, with sequences for 1,788 lncRNAs successfully identified and retrieved. Following the removal of the duplicate sequences, the final dataset consisted of 1,753 lncRNAs. These were then classified into two subgroups as follows: The first subgroup contained 778 upregulated lncRNAs and the second subgroup contained 975 downregulated lncRNAs (Table SI).

The MEME algorithm was employed to identify potential motifs within the lncRNA sequences of each subgroup. The results of this analysis led to the identification of six motifs: Three motifs in the upregulated lncRNA group and three in the downregulated lncRNA group.

In the upregulated lncRNAs group following NR activation, the first identified motif was GATCCGCCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGG, found in 140 lncRNAs from the total 778 (Fig. S1). The second motif discovered in this group was TGGCCAGGCTGGTCTCGAACTCCTGACCT, present in 127 lncRNAs (Fig. S2), while the third motif was TAATCCCAGCACTTTGGGAGGCCGAGG, which occurs in 115 lncRNAs (Fig. S3).

For the downregulated lncRNA group following NR activation, the first motif discovered was GTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGG, found in 145 lncRNA out of the total of 975 (Fig. S4). The second motif discovered was AGGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGTGAAAC, which is present in 128 lncRNAs (Fig. S5), and the third motif was CCTCAGCCTCCCGAGTAGCTGGGACTACAGGCGC, found in 129 lncRNAs from this group (Fig. S6).

To calculate the probability of each motif appearing randomly at a position within the 3.2 billion base pairs of human DNA, the following steps were taken:

The first motif from the upregulated lncRNAs subgroup consists of 41 nucleotide bases. Since each base (A, C, G, or T) has a ¼ probability of being in a specific position in the sequence, the probability of finding the full sequence of these 41 nucleotide bases is: (¼)⁴¹=2.23517418x10^-25.

Taking into consideration the total number of human DNA base pairs (3.2 billion or 3.2x10⁹), the probability of this sequence occurring randomly at any position in the genome is: 3.2x10⁹x2.23517418x10^-25=7.15255974x10^-16.

Accordingly, for the second motif in the upregulated lncRNA subgroup, consisting of 29 nucleotides, the probability calculation is as follows: (¼)²⁹=1.8626451x10^-18. The probability of finding this specific sequence randomly in human genome is: 3.2x10⁹x1.8626451x10^-18=5.96174432x10^-10. For the third motif in the upregulated lncRNAs subgroup, which consists of 27 nucleotides, the results are as follows: (¼)²⁷=7.8886x10^-17. Thus, the probability of its random occurrence is: 3.2x10⁹x7.8886x10^-17=2.52435x10^-7.

In the downregulated lncRNA subgroup, both first and second motifs consist of 41 nucleotides; thus, the same probability calculations apply: (¼)⁴¹=2.23517418x10^-25. Thus, the probability of their random occurrence is: 3.2x10⁹x2.23517418x10^-25=7.15255974x10^-16. The third motif in the downregulated lncRNA subgroup, consists of 34 nucleotides: (¼)³⁴=1.421085x10^-21. Thus, the probability of its random occurrence is: 3.2x10⁹x1.421085x10^-21=4.5474755x10^-13.

The calculated probabilities are extraordinarily low compared to common biological standards and are consistent with the hypothesis that these motifs are not randomly occurring in the genome (22).

The above-calculated probabilities suggest that the motifs discovered are non-random occurrences in the human genome, even when assuming independent events. However, the nucleotide sequence of DNA is linked to biological functions. The order of the bases is strictly determined by the way nucleotides bind each other in the double helix structure. The complementary pairing of nucleotides restricts the possible sequences that DNA can form, meaning that the probability of these motifs occurring randomly in the genome would likely be even lower than the values calculated above.

As previously mentioned, lncRNAs can function as miRNA sponges, regulating the expression of mRNA targets (8,9). Considering this regulatory role, we searched for potential miRNAs complementary to the six identified motifs in miRBase. In the present study, the search revealed that the motif GATCCGCCCGCCTCGGCCTCCCAAAGTGCTGGGATTACAGG was complementary to hsa-miR-1908-3p.

miRNAs typically bind to complementary sequences in mRNAs, mainly targeting the 3' untranslated regions (UTRs) (23), while there is evidence to indicate that they can also target the 5' UTRs and coding regions, thus inhibiting mRNA expression (24). To predict the potential targets of miRNAs, several computational approaches have been developed, such as miRanda (25), mirSVR (26), PicTar (27), TargetScan (28), TargetScanS (29), RNA22(30), PITA (31), RNAhybird (32) and DIANA-microT (33). In addition, there are miRNA target prediction databases such as TarBase (34), PicTar (27) and TargetScan (28).

Thus, to identify mRNAs targeted by hsa-miR-1908-3p, whose expression may be affected by the interaction between lncRNAs and this miRNA, the miRDB database was used. This database, through its miRTarget bioinformatics tool, predicts the mRNA based on high-throughput sequencing experiments. By this analysis, 11 targets were identified, including METTL21A, CHFR, MTA1, CAVIN4, IGFBP3, GRB2, DYNLL2, H2AFX, TNRC6C, C1orf229 and ELOVL2 (Table I). In addition, the TargetScanS (Release 8.0) database (17-19,35) was also searched, and 487 predicted targets were found (Table SII), including the aforementioned 11 targets.

miR-1908 is encoded by the first intron of the FADS1 gene and is highly expressed in mature human adipocytes, where it is likely involved in regulating the preadipocyte differentiation (36). The promoter region of miR-1908 contains two binding sites of NF-κB, suggesting its transcription is regulated by this transcription factor (37). miR-1908 was first discovered in human embryonic stem cells in 2008(38), and its functional role in melanoma metastasis and angiogenesis was explored in 2012(39). More specifically, miR-1908, in conjunction with miR-199a-3p and miR-199a-5p, inhibits apolipoprotein E and DNAJ heat shock protein family 23 member A4, which reduces the interaction of Apo-E with low-density lipoprotein receptor-related protein (LRP)-1 and LRP-8. This interaction promotes melanoma cell invasion and endothelial cell recruitment, resulting in poor prognosis (39). Moreover, miR-1908 dysregulation has been shown to be associated with various types of cancer, where its dysregulation tends to enhance cell proliferation, invasion, migration and angiogenesis, thus reducing the survival rate of affected patients (40,41). Finally, free fatty acids and adipokines, such as resistin and leptin, are considered to downregulate the expression of miR-1908, suggesting a role of this miRNA in regulating obesity-related insulin resistance (42).

For the analysis of motif sequences across the whole human genome using the FIMO tool, the following results were obtained for each of the six motifs. In the upregulated lncRNA subgroup, the first motif appeared 53,524 times, the second motif was detected 51,673 times and the third motif was found 53,936 times. In the downregulated lncRNA subgroup, the first motif (fourth in total) was found 63,126 times, the second motif (fifth in total) was detected 58,442 times and the third motif (sixth in total) was found 57,943 times. Using these results, karyotypes were created for each motif, and the percentage of the occurrences of each motif across all chromosomes was calculated. The karyotypes are presented in Fig. S7, Fig. S8, Fig. S9, Fig. S10, Fig. S11 and Fig. S12.

Subsequently, the present study focused on the 1,000 highest-scoring loci which occurred using the FIMO tool for each motif across the human genome. Using these 1,000 loci, karyotypes for each motif were also created, and the percentage of occurrences of each motif was also calculated for each chromosome (Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5 and Fig. 6).

In addition, the chromosome plots for each motif were created, where the 1,000 loci are represented as red dots beside the corresponding region. These chromosome plots are presented in Fig. S13, Fig. S14, Fig. S15, Fig. S16, Fig. S17 and Fig. S18.

Based on these karyotypes and chromosome plots, it was observed that chromosome 19 exhibited a fairly high percentage of motif occurrence relative to its size. In fact, for all six motifs, the percentage of occurrence in chromosome 19 was higher than that of the longer chromosomes.

According to the literature, human chromosome 19 is recognized for its unusual nature, which was noted even before the initial publication of its DNA sequence (43). One of its unusual features is the considerably high gene density, which exceeds the genome-wide average by >2-fold and includes 20 large, tandemly clustered gene families (43). In addition, chromosome 19 contains a considerable number of segmental duplications, with 6.2% of its sequence lying within intrachromosomal segmental duplications (43). The sequence divergence within these duplications implies that these events occurred between 30 and 40 million years ago (44). These duplications likely played a pivotal role in the evolution of phenotypic characteristics that occurred by genes on chromosome 19 across primates, including humans. Moreover, chromosome 19 presents an unusually high repeat content, comprising 55%, with Alu repeats comprising 26% of the sequence of the chromosome (44). Another notable feature is the GC content (48%) of chromosome 19, the highest of any human chromosome, compared to the GC content of the whole human genome of 41%. This elevated GC content enhances the potential for wide gene regulation via DNA methylation, particularly at CpG islands, as well as in CpG regions located within promoters and enhancers (45).

According to the chromosome plots, the six identified motifs were detected in various regions of chromosome 19. Between those regions, all six motifs aligned with a specific locus, AAVS1, located on chromosome 19q13.42. This site was first mapped in 1991 Kotin et al (46) and Samulski et al (47), who were investigating the integration mechanism of the AAV into the host genome. According to these studies, the AAV preferentially intergrades into the AAVS1 region of human chromosome 19q13.42, a unique phenomenon among eukaryotic DNA viruses (48).

Several features make the AAVS1 region particularly noteworthy. Notably, the locus for myotonic dystrophy (49), as well as a common breakpoint related to chronic B-cell leukemia (BCL-3) (47,48), are located at this region. In addition, AAVS1 undergoes frequent sister chromatid exchanges (50). The alignments of motifs with the AAVS1 region were developed using the Clustal Omega program (51) and visualized with Jalview version 2.11.2.6 program (52) (Fig. S19, Fig. S20, Fig. S21, Fig. S22, Fig. S23 and Fig. S24).

The AAVS1 region is located within the first intron of the phosphatase 1 regulatory subunit 12C (PPP1R12C) gene, which encodes a protein with a poorly defined function. This region exhibits several unique features that contribute to its unique nature. First, it includes a tetrad GACT repeat, which functions as an AAV Rep-binding element, also detected in the AAV2 inverted terminal repeats, facilitating the exclusive integration of the AAV genome in the presence of AAV replication protein. Second, chromosome 19 is GC-rich, with a higher number of CpG islands relative to GpC islands, a characteristic linked to regions of transcriptional activity in vertebrates, which may be relevant to gene expression (53-55). Third, several putative transcription factor-binding sites are present in AAVS1, including CREB, AP-1, AP-2 and Sp1 (48,56). Fourth, topoisomerase I is predicted to cleave at least two sites within AAVS1(48). Fifth, Lamartina et al (56) demonstrated an open chromatin conformation of AAVS1, accompanied by promoter activity on chromosome 19 in cultured cells. All these features position AAVS1 as an approachable region, subject to regulation by both host and viral regulatory elements.

The potential of the AAVS1 site as a target for gene therapy applications has been investigated and well-characterized. This site is known for its ability to integrate exogenous DNA with limited disruption to endogenous genes, providing a preferred site for stable transgene expression (57). A key feature that facilitates the usage of the AAVS1 site in gene therapy is its open chromatin structure, which is accompanied by endogenous insulator elements that protect the integrated DNA sequences from trans-activation or repression (58).

The insertion of exogenous genes into the human genome is conducted mainly using lentiviral and gamma-retroviral vectors. However, these vectors tend to integrate randomly within the genome (59-61), which can lead to unpredictable interactions between the transgene and the host genome. Such random integration may result in the attenuation or complete silencing of the transgene (62-64) or, more critically, in the dysregulation of host gene expression (64,65), which poses significant risks in gene therapy application. One promising strategy to mitigate these risks involves the use of gene-editing tools that enable site-specific insertion of transgenes into genomic safe harbors (GSHs) (64,66,67). GSHs are genomic locus that support a stable environment for transgene expression without interfering the integrity of endogenous genes (64,68,69). The AAVS1 site is considered a GSH that provides a stable transgene expression (70) due to the presence of flanking insulator regions (58). According to the study by Lombardo et al (66), the GSH AAVS1 can support transgene expression across different human cell types, facilitated by the active and open chromatin structure of the PPP1R12C gene located in this region (66).

The regulatory role of lncRNAs has been well-characterized. Numerous studies have detected that lncRNAs function as crucial regulators of gene expression, participating in various pathways by acting as decoys, guides and scaffolds for molecular interactions. In the present study, it was hypothesized that the regulatory roles of lncRNAs are indisputable, and to investigate this hypothesis, an in-house pipeline was developed to analyze a collection of lncRNAs. This pipeline enabled the identification of conserved motifs within their sequences that may contribute to their regulatory functions. The analysis revealed that one of the motifs may function as a sponge for a specific miRNA, thereby suppressing the function of the miRNA and leading to the upregulation of its mRNA targets. The function of miRNAs in the suppression of the translation of mRNA targets is well known, with implications for numerous pathological conditions, such as neurodegeneration (71-73), cancer (72,74,75) and cardiovascular diseases (74,76,77). Consequently, the interaction between lncRNAs and miRNAs could have significant consequences, either promoting disease prevention and treatment or exacerbating disease progression and severity.

Furthermore, all six motifs identified in the present study were detected across numerous regions of the human genome, with chromosome 19 presenting a markedly high frequency of motif occurrence relative to other chromosomes. Focusing on chromosome 19, it was found that these motifs were aligned with the AAVS1 regulatory region, a genomic locus known for its unique features, including the specific integration of the AAV, the presence of transcription factor binding sites, an AAV Rep-binding element, high GC-content, open chromatin, and two cleavage sites for topoisomerase I. It is noteworthy that the identified motifs demonstrate a pronounced GC content, a feature commonly associated with key regulatory regions, including transcription factor binding sites, polymerase binding sites, and loci involved in chromatin modification and DNA methylation. GC-rich sequences are often enriched at genomic sites that regulate transcription and chromatin remodeling due to their structural properties, such as enhanced stability and the presence of CpG sites that are key for epigenetic regulation (78-81). The existence of these motifs in regions integral to the regulation of the human genome, combined with their GC-rich nature, suggests their role in regulating gene expression through DNA methylation processes. Consequently, these motifs and the lncRNAs containing them, likely function as pivotal regulatory elements in gene expression.

In addition to the potential functions of lncRNAs that contain the identified motifs-based on the presence of these motif sequences in well-characterized regulatory regions of DNA and their interactions with other molecules, including miRNAs, the present study further investigated the documented roles of the lncRNAs containing at least one of these motifs in the literature (82-124) and relevant databases such as NCBI (ncbi.nlm.nih.gov) and GeneCards (125). Despite the incomplete annotation and characterization of these lncRNAs, the majority of the examined lncRNAs are located either within intergenic space, introns of coding genes, or are antisense to coding genes. These lncRNAs are believed to influence the functions of their associated genes, mainly by suppressing gene functions. The lncRNAs examined are involved in various biological processes, such as development, cellular proliferation, immune responses, inflammation, transmembrane transport, gene expression regulation, chromatin and chromosome structure modulation, differentiation, cell cycle control, spermatogenesis, metabolic regulation, and several functions related to the nervous and cardiovascular systems. The results from the present study, as depicted in the diagrams shown in Fig. S25, Fig. S26, Fig. S27, Fig. S28, Fig. S29 and Fig. S30, illustrate the involvement of lncRNAs with each specific motif in these diverse biological pathways.

The emergence of NRs dates back >1.5 billion years, originating in early eukaryotic organisms as ligand-dependent transcription factors. These receptors were responsive to environmental cues such as hormones, light, and metabolic shifts. The earliest NRs likely played a critical role in regulating gene expression, helping primitive eukaryotes adapt to dynamic external conditions, thus aiding their survival and proliferation. Of note, unicellular organisms such as Euglena gracilis, a photosynthetic protist, contain an ancestral version of the NR, providing evidence that these mechanisms were present well before the evolution of multicellular organisms. This discovery enhances the understanding of the origins of NRs and their evolutionary significance (126).

As evolution progressed, NRs diversified into a complex protein family with distinct functions. Different subfamilies evolved to regulate a wide variety of biological processes across animals, plants, fungi and even bacteria. In modern organisms, NRs govern critical processes such as metabolism, immune response, reproduction, and development. The diversity of these receptors is reflected in the large number of subfamilies, including steroid hormone receptors, thyroid hormone receptors, peroxisome proliferator-activated receptors and orphan NRs. Despite their varied roles, these receptors retain a conserved ligand-binding domain that allows them to interact with specific signaling molecules to regulate gene expression (126).

The present study discovered six novel sequence motifs within the lncRNA sequences under investigation. These lncRNAs exhibited altered expression levels upon the activation of NRs, either increasing or decreasing in abundance. Notably, several of these lncRNAs, which contain at least one of the identified motifs, are regulated by multiple NRs. This suggests that these motifs may be associated with NR function but do not show specificity to any single receptor, underscoring the complexity of the regulatory network between NRs and ncRNAs. This finding highlights the importance of further research to explore their broader biological implications.

To investigate the evolutionary conservation of these motifs, the FIMO tool was applied to examine their presence in various organisms. The present study analyzed species such as Mus musculus, Squamata (lizard), Danio rerio (zebrafish), Drosophila melanogaster, Caenorhabditis elegans, Escherichia coli (K12 MG1655), Saccharomyces cerevisiae and Bacillus subtilis (PY79). Additionally, the present study explored the presence of these motifs in the genome of Euglena gracilis using the ‘Matcher’ tool on the Galaxy platform. A phylogenetic tree was subsequently constructed using the iTOL tool (Interactive Tree Of Life), which enabled the visualization and annotation of the evolutionary associations among the organisms and the conservation of the motifs across species (127). The resulting phylogenetic tree (Fig. 7) clearly illustrates the evolutionary conservation of these motifs, which were identified repeatedly in the studied organisms (Table II).

The genetic similarities between humans and the other organisms examined in the present study exhibit significant variation. For instance, humans and Mus musculus (mouse) share ~85% genetic similarity, reflecting a divergence of ~70 million years ago, making the mouse an essential model for human genetic research, particularly in disease mechanisms and therapeutic development (128). Squamata, a diverse group of reptiles, includes species that serve as critical models for studying thermoregulation, behavior and reproductive strategies. Research on lizards and snakes has provided important insights into immunity, skin regeneration, and other physiological processes crucial for survival in varied environments (129). The genetic similarity between humans and different Squamata species varies considerably, with lizards sharing ~70% genetic similarity with humans, diverging ~250 million years ago. The six motifs identified in the present study were also present in Squamata, suggesting their ancient evolutionary origins. Danio rerio (zebrafish), with a genetic similarity to humans of ~70%, diverged ~400 million years ago and is an critical model organism for developmental biology and gene function studies (130). Drosophila melanogaster (fruit fly), sharing ~60% of its genes with humans and diverging ~600 million years ago, is extensively used in genetic regulation, neurobiology, and cellular research (131). Caenorhabditis elegans (nematode), with ~40% genetic similarity to humans, has been instrumental in research on aging, developmental biology and cellular processes (132). Escherichia coli K12 MG1655, although lacking direct gene homologs in humans, plays a crucial role in understanding fundamental gene regulation and bacterial genetics (133). Saccharomyces cerevisiae (yeast) shares ~23% of its genes with humans and diverged from humans >1 billion years ago, rendering it a key organism for cell biology, genomics and metabolism research (134). Bacillus subtilis PY79, a soil bacterium, has markedly contributed to the understanding of bacterial gene regulation and stress responses (135). Finally, Euglena gracilis, a single-celled eukaryote, is considered to share ancient NR mechanisms with humans, offering insights into the early evolution of eukaryotic gene regulation (136).

The presence of the discovered motifs in species that diverged long before humans, including organisms with distinct genomes, such as Euglena gracilis and Saccharomyces cerevisiae, combined with the lack of specificity in these motifs, suggests they may represent ancestral binding sites for the precursors of NRs. The conservation of these motifs across a broad evolutionary spectrum emphasizes their potential role in regulating gene expression via NR binding. Given that these motifs do not exhibit specificity for any particular NR, it was hypothesized that they may have functioned as general binding sites for ancestral NR precursors (137). Moreover, these motifs play a crucial role in the evolution of NR function by enabling NRs to interact with a broader range of binding sites, facilitating gene regulation in response to environmental and internal cues (126,138).

The widespread presence of these motifs across different evolutionary lineages points to their ancient origins, likely predating the highly specific NRs found in modern organisms. In early eukaryotes, these motifs could have functioned as genomic binding sites for precursor forms of NRs, which were less specific in their binding preferences compared to the more specialized NRs currently observed (139). This generalization would have been essential in the early stages of NR evolution, allowing for flexible and robust gene regulation. These early, versatile genomic binding sites could have allowed primordial NR-like proteins to modulate gene expression effectively, even in the absence of the highly refined receptor-genome interactions seen in contemporary systems.

The conservation of these motifs across such diverse species also suggests that they may have functioned as backup mechanisms, ensuring the continued regulation of essential genes when canonical NR binding sites, such as the glucocorticoid response element for GR, were unavailable or dysfunctional (139). In this sense, these motifs may have served as a safety net, allowing the NR system to maintain gene expression and cellular functions even in the absence of optimal binding site availability. This backup role would have been particularly important in the early evolutionary stages, when the molecular machinery for precise gene regulation was still evolving (140).

As NRs became more specialized through evolution, the ancestral motifs likely persisted due to their role in maintaining regulatory flexibility. In modern organisms, these motifs may still contribute to the robustness of gene regulation, particularly in cases where the canonical NR-binding sites are compromised or unable to function. Their ability to interact with multiple NRs enhances the adaptability of the organism to different signaling environments, providing a more versatile and resilient regulatory system (140).

The preservation of these motifs across such a broad evolutionary spectrum underscores their importance in the evolutionary trajectory of gene regulation. These motifs may represent a foundational aspect of NR functionality, offering a mechanism that allowed early organisms to adapt to changing environments by ensuring continued gene regulation, even when specific receptor-binding sites were not fully evolved. The fact that these motifs can still be found in modern organisms suggests that they continue to play a role in gene regulation, particularly in situations where canonical NR interactions are suboptimal or unavailable (126).

This broader, more flexible role in gene regulation may have allowed organisms to thrive in variable environments and adapt to new challenges. The conservation of motifs across both ancient and modern species highlights their functional importance and their potential as key elements in maintaining the adaptability of the NR signaling system throughout evolutionary history.

To date, numerous studies have examined and described the potential function of lncRNAs in the regulation of gene expression and their abilities to act as biomarkers or as pharmaceutical targets, or even therapeutic molecules (141-144). In the present study, six conserved motifs were detected for the first time in the sequences of the lncRNAs studied which are suggested to participate in the regulatory function of lncRNAs that contain them. However, to fully understand the role of these motifs, and the lncRNAs containing them, in gene regulation, future research is required to explore their functional significance in greater detail. Experimental studies could confirm whether these motifs act as alternative binding sites for NRs or other transcription factors. Additionally, further investigations into the mechanisms through which these motifs contribute to the regulation of gene expression in both ancient and modern species are warranted to provide valuable insight into the evolution of NR-mediated gene regulation and its ongoing role in cellular adaptability.