Functional roles of conserved lncRNAs and circRNAs in eukaryotes

Long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) have emerged as critical regulators in essentially all biological processes across eukaryotes. They exert their functions through chromatin remodeling, transcriptional regulation, interacting with RNA-binding proteins (RBPs), serving as microRNA sponges, etc. Although non-coding RNAs are typically more species-specific than coding RNAs, a number of well-characterized lncRNA (such as XIST and NEAT1) and circRNA (such as CDR1as and ciRS-7) are evolutionarily conserved. The studies on conserved lncRNA and circRNAs across multiple species could facilitate a comprehensive understanding of their roles and mechanisms, thereby overcoming the limitations of single-species studies. In this review, we provide an overview of conserved lncRNAs and circRNAs, and summarize their conserved roles and mechanisms.


Transcriptional regulation
Numerous lncRNAs exhibit the cis regulatory roles in gene transcription [11,16].5S rRNA is an ancient non-coding RNA expressed in all domains of life, which is transcribed by RNA pol III from the corresponding internal promoter [46,47].Hu and colleagues identified a transcript from 5S rDNA loci named 5S-OT (5S rRNA Overlapped Transcript) with extensive expressions from fission yeast to mammals [48].5S-OT is a highly conserved lncRNA synthesized by RNA pol II and localized to the nucleus in human and mice [48].Antisense oligonucleotides (ASO)-mediated 5S-OT knockdown reduces the nascent 5S rRNA level and human/murine 5S-OT both co-localize to the 5S rDNA gene cluster, indicating the existence of the RNA-mediated pol II-pol III coupling mechanism in gene transcription (Fig. 1A) [48].Additionally, 5S-OT also evolves trans regulatory roles in the anthropoidea suborder of primates due to the insertion of an antisense Alu element.Human 5S-OT interacts with the splicing factor U2AF65 to modulate alternative splicing of hundreds of genes via anti-Alu/Alu pairing [48].Based on this mechanism, the artificial RNA with a polypyrimidine tract (Py) site at the 5′ end and a gene-specific antisense sequence at the 3' end could be used as an effective biotechnological tool to manipulate alternative splicing for several genes [48,49].FENDRR (Fetal-lethal Noncoding Developmental Regulatory RNA) is another conserved lncRNA in human and mice [50,51].The expression of FENDRR is decreased in the fibrotic lungs of patients with idiopathic pulmonary fibrosis and in an induced pulmonary fibrosis mouse model [50].Depletion of FENDRR increases cellular senescence in human lung fibroblasts, while overexpression of human FENDRR reduces lung fibrosis in the pulmonary fibrosis mouse model [50].Further investigation demonstrates that FENDRR decreases pulmonary fibrosis by competing with the binding of IRP1 to iron metabolism genes and then controlling the iron concentrations [50].Additionally, murine Fendrr has also been found to directly form a RNA: dsDNA triplex at promoters of targeted genes associated with lung fibrosis [51].Meanwhile, Fendrr and the Wnt-signaling pathway regulate these genes to participate lung fibrosis in a synergistic manner [51].However, whether human FENDRR is involved in lung fibrosis via the RNA:dsDNA triplex requires further investigation.

Chromatin remodeling
LncRNAs have been reported to exert their functions by chromatin remodeling [11,16,[55][56][57][58].For instance, the lncRNA EPR (Epithelial cell Program Regulator) is prominently expressed in epithelial tissues such as the mammary gland, gastrointestinal tract, lung, and kidney [57,58].In both human and mice, the EPR level is significantly decreased in breast cancer cell lines compared to normal mammary gland cells and EPR overexpression reduces breast cancer cell proliferation [57].Murine Epr recruits Smad3 to the promoter of Cdkn1a to enhance its transcription.Meanwhile, human EPR overexpression also induces Cdkn1a levels in murine cells [57].When examined at the organismal level, the Epr genetic knockout mice (Epr − /− ) demonstrate higher susceptibility to colitis and tumor formation under dextran sodium sulfate induction through tridimensional chromatin remodeling to decrease the expression of genes involved in mucus metabolism [58].
CARINH (Colitis Associated IRF1 antisense Regulator of Intestinal Homeostasis) is another mammal-conserved lncRNA, which is localized close to the 3' end of the coding gene IRF1 in human and mice [59].Both human and murine CARINH are highly expressed in lymphoid and mucosal tissues.Murine Carinh is upregulated in myeloid cells sorted from colitis colons than healthy ones, and human CARINH levels are increased in gut specimens from inflammatory bowel disease (IBD) patients than control biopsy tissues [59].Moreover, CARINH RNA levels are positively correlated with IRF1 levels both in mouse myeloid cells and human monocyte THP-1 cells [59].Mechanistic studies demonstrate that Carinh interacts with p300/CBP at the Irf1 locus to promote Irf1 expression by increasing H3K27ac deposition (Fig. 1B) [59].Further investigation shows that IRF1 also directly binds to the promoter of Carinh and promotes its transcription.All these data indicate that CARINH and IRF1 form a feedforward loop, which is essential for against IBD both in human and mice.

mRNA stability
One mechanism of lncRNAs is to regulate mRNA stability [8,60,61].The lncRNA THOR (Testis-associated Highly-conserved Oncogenic long non-coding RNA) is highly conserved in human, mice and zebrafish [60].THOR is extensively expressed in the testis and a variety of human cancers, including non-small-cell lung cancer (NSCLC) and melanoma [60].THOR knockdown results in a significant reduction of cell proliferation in NSCLC and melanoma cell lines.Further murine tumor xenografts derived from THOR knockout and overexpression cells support the proliferation-promoting activity of human THOR [60].Moreover, transgenic THOR knockout in zebrafish leads to fertilization failure and confers resistance to melanoma development, whereas ectopic expression of human THOR accelerates melanoma development in zebrafish [60].Mechanistically, THOR interacts with the RBP IGF2BP1 in both human and zebrafish to increase the stability of IGF2BP1's targets.Overall, THOR is a cancer/testis lncRNA and has undergone favorable evolutionary selection.The lncRNA AC006064.4-201 (human) or Gm49317-201 (mouse) is another conserved lncRNA with a conserved mode of action in mammals.Human AC006064.4-201 is decreased in senescent and degenerated human chondrocytes and protects against Osteoarthritis [61].Additionally, Gm49317-201 knockdown results in an increase in the number of senescent mouse chondrocytes [61].The lncRNA directly interacts with PTBP1 to perturb PTBP1 binding to CDKN1B mRNA, leading to a decrease of CDKN1B mRNA stability and protein level, and this regulatory axis is conserved between human and mice (Fig. 1C) [61].
The lncRNA Cyrano, also known as OIP5-AS1 (OIP5 Antisense RNA 1), is broadly conserved in vertebrates [44,56,70].It is expressed throughout the developing nervous system and is highly expressed in the brain [44,56,70].Kleaveland and colleagues have utilized a series of genetic knockout mice to demonstrate that Cyrano triggers much more effective miR-7 destruction via an extensively miR-7-paired site.By destabilizing miR-7 levels through TDMD, Cyrano prevents inhibition of miR-7-targeted mRNAs and promotes the accumulation of Cdr1as, a circular RNA known to regulate neuronal activity (Fig. 1F) [44].In addition, human H19 is an imprinted lncRNA originating from the H19-IGF2 imprinting cluster of the genome [71,72].In addition to functioning as a spliced full-length transcript, H19 could also be a miR-675 precursor for miR-675 generation.A full predicted H19 genomic sequence is also identified in wallabies, with approximately 51 % similarity to the human H19.Furthermore, the H19 ortholog in therian species demonstrates preservation of both miRNA production and exon-intron structure, indicating functional selection for these characteristics [72].Overall, lncRNAs associate with miRNAs to form complex regulatory networks in physiology and pathology.

X chromosome inactivation
XIST (X-inactive Specific Transcript) and TSIX (Xist antisense RNA) are two well-characterized lncRNAs associated with X chromosome inactivation.They are transcribed from the same gene loci with distinct orientations.XIST is a sense transcript, and TSIX is an antisense transcript [40,79].XIST contains conserved repeats and predominantly resides in the nucleus [40].XIST gradually encapsulates X chromosome starting from the X inactivation center (XIC) followed by instant gene silencing (Fig. 1H) [40].TSIX is conserved in the human XIC and mouse XIC [40].TSIX prevents the accumulation of XIST on the future active female X chromosome through RNA-RNA interaction to maintain the active euchromatin state of the selected chromosome [79].

Other mechanisms
LncRNAs could coordinate with key regulators to perform various biological functions [8,11].NEAT1 (Nuclear Enriched Abundant Transcript 1) is a well-known conserved lncRNA that plays an important role in the development of several cancers, including lung cancer, breast cancer, and prostate cancer [42].The accelerated glycolysis (also named Warburg effect) is one of the main metabolic changes observed in cancer [42].Using human cell lines and mouse models, Park et al. found that NEAT1 interacts with PGK1, PGAM1, and ENO1 proteins to facilitate the assembly of the PGK1/PGAM1/ENO1 glycolytic complex, resulting in a higher flux through the penultimate step in glycolysis [42].Human HOXA11AS (Homeobox A11 Antisense) or murine HOXA11os is a conserved lncRNA transcript from the opposite strand of the HOXA11 gene locus in mammals [80].In clinical samples, HOXA11AS is highly expressed in colon biopsies specimens and exhibits a significant reduction in those with ulcerative colitis [80].Similarly, murine HOXA11os is highly expressed in the healthy colon, but downregulated to undetectable levels in colitis [80].Under normal conditions, HOXA11os localizes to mitochondria and interacts with the complex I subunit, NDUFV1, to sustain its function [80].HOXA11os deficiency mice demonstrate complex I deficiency, oxidative phosphorylation dysfunction, and mitochondrial reactive oxygen species production in colonic myeloid cells [80].The conserved lncRNAs with functional roles and molecular mechanisms are summarized in Table 1.
The circRNA CDR1as/ciRS-7 a well-characterized ceRNA, harbors more than 70 conserved binding sites of miR-7 [37,38,43,44].A number of studies have indicated that CDR1as acts as a miRNA sponge for miR-7, derepressing its targets and consequently impacting brain development [37,38].In contrast, other studies have found that the knockout of CDR1as leads to decreased levels of miR-7, suggesting that CDR1as somehow protects miR-7 from degradation [43,44].In addition to numerous conserved miR-7 sites, CDR1as contains a solitary conserved miR-671 binding site to facilitate AGO2-mediated cleavage of the circRNA through almost perfect complementarity to miR-671 (Fig. 2A) [43,44].Interestingly, CDR1as, miR-7 and miR-671 coordinate with the lncRNA Cyrano to form a regulatory network in regulating neuronal activity [44].In addition to regulating neuronal activity, CDR1as has (continued on next page) been proposed to have oncogenic properties because it is highly expressed in tumors and its target, miR-7, is known to be a tumor suppressor [82][83][84].However, Kristensen and colleagues pointed out that, in colon cancer, CDR1as is not expressed in cancer cells but instead is only expressed in stromal cells, while miR-7 is only expressed in cancer cells [85,86].

Modulating RBP functions
CircRNAs always function by interacting with RBPs [2,11,[19][20][21].For instance, circBoule RNAs are generated from a conserved reproductive gene Boule, and are highly conserved and specifically expressed in testes from flies to mammals [87].They directly bind to non-canonical RBPs heat-shock proteins (HSPs; Hsc4 and Hsp60C in flies, and HSPA2 in mammals) through conserved RNA motifs and then facilitate the ubiquitination of these HSPs to decrease their protein levels [87].Genetic circBoule knockout flies and mice both lead to decreased male fertility, especially under heat-stress conditions.In addition, the circ-Boule level is significantly lower in sperm from patients with asthenozoospermia, and is negatively associated with the HSPA2 protein level in normozoospermic sperm (Fig. 2B) [87].All these evidences reveal conserved molecular and physiological functions of circBoule RNAs in fertility protection, stress responses, and evolution.
Recently, Li and colleagues have identified a mammalian conserved circRNA named circLARP1B, which exhibits significantly higher levels in metastatic HCC [45].CircLARP1B consists of exons 2-4 from the LARP1B gene and predominately localizes to the cytoplasm.HCC patients with higher circLARP1B levels had shorter five-year overall survival and disease-free survival rates [45].CircLARP1B facilitates HCC metastasis through remodeling the lipid metabolism in a human HCC cell line and an induced HCC model of genetic knockout mice.Mechanistically, circLARP1B interacts with cytoplasmic HNRNPD via two functional sites and destabilizes LKB1 mRNA via perturbing HNRNPD to modulate AMPK-ACC signaling in human and mice (Fig. 2C) [45].Furthermore, AAV8-mediated hepatocyte-directed knockdown of cir-cLARP1B or Lkb1 in HCC mouse models also demonstrated critical roles of the hepatocytic circLARP1B regulatory pathway in HCC metastasis and lipid metabolism, indicating that circLARP1B might be a potential therapeutic target for HCC [45].

Transcriptional regulation
Multiple circRNAs could regulate gene transcription [19][20][21].The circRNA cia-cGAS is highly expressed in long-term hematopoietic stem cells (LT-HSCs) among human, mice, and rats [88].The cia-cGAS-deficient mice exhibit decreased numbers of dormant LT-HSCs [88].Under basal conditions, cia-cGAS interacts with DNA sensor cGAS to block its synthase activity in the nucleus, thereby safeguarding dormant LT-HSCs from cGAS-mediated depletion [88].In the absence of cia-cGAS, cGAS binds to its own genomic DNA and catalyzes the synthesis of cGAMP products, which are released into the cytoplasm and then trigger the production of type I interferons, leading to LT-HSCs exhaustion [88].CircIPO11 is another conserved circRNA in human and mice with significant expression in both liver CSCs and HCC tumor tissues [89].Downregulation of human circIPO11 inhibits the self-renewal of liver CSCs and suppresses tumor propagation in vitro and in vivo.Mechanistic study reveals that circIPO11 recruits TOP1, relaxing supercoiled DNA during gene replication and transcription, to the GLI1 promoter, thereby initiating GLI1 transcription and activating Hedgehog signaling for self-renewal maintenance of liver CSCs (Fig. 2D) [89].

Other circRNAs
CircSLC45A4 is a highly conserved circRNA in human neuroblastoma cells and mouse embryonic cortex [90].CircSLC45A4 knockdown in human SH-SY5Y cells causes spontaneous differentiation.Depletion of circSlc45a4 significantly reduces the basal progenitor pool in the developing mouse cortex [90].CircARHGAP5-2 is conservatively expressed in human and mouse adipocytes, which maintains the global adipocyte gene program, and facilitates lipid accumulation [91].Although circSLC45A4 and circARHGAP5-2 are two functionally conserved circRNAs, the mechanisms of their function require further investigation.

Conclusions and perspectives
Current active research in lncRNAs and circRNAs has brought us various interesting findings implying that lncRNAs and circRNAs are of great importance in human diseases [30,[92][93][94][95]. Several of them are evolutionarily conserved and have essential roles in physiology and pathology [1,8,30,31].We have systematically described a series of conserved lncRNAs and circRNAs, and elucidated their underlying molecular mechanisms (Table 1 and Figs. 1 and 2).
Compared to mRNAs and circRNAs, lncRNAs are less conserved across species; thus, conserved lncRNAs might be more dispensable due to their survival under the selection pressure of evolution [1,8,30].For instance, the lncRNA Epr knockout mice have demonstrated a higher susceptibility to colitis and tumor formation when induced with dextran sodium sulfate [58].HOXA11os knockout mice have defective mitochondria in colonic myeloid cells, leading to hypersusceptibility to colitis [80].Transgenic THOR knockout in zebrafish results in fertilization failure and provides resistance to melanoma development [60].Additionally, despite the seemingly normal appearance of Cyrano − /− mice, knockdown of conserved Cyrano in zebrafish causes a neurodevelopmental phenotype [44,70].The organismal phenotype of conserved lncRNAs highlights their importance across species.
It has been demonstrated that ceRNA interactions occur when miRNA and target levels approach equimolar [96].Given that miRNAs are typically present in high copy numbers within cells, if a lncRNA or circRNA is functioning as a miRNA sponge, it should contain multiple binding sites for the target miRNA, such as CDR1as [37,38,43,44].Nevertheless, the majority of lncRNAs and circRNAs that are believed to act as miRNA sponges contain only a single or a few target miRNA binding sites and are expressed exclusively in specific tissues [96].If lncRNAs or circRNAs are capable of inducing TDMD, as exemplified by Cyrano's action on miR-7, the necessity for numerous binding sites and high abundance could be negated [44,97].However, it is noteworthy that a relatively small number of lncRNAs or circRNAs are fully complementary to the entire sequence of the target miRNA [98].Consequently, most physiological alterations in the expression of individual circRNAs or lncRNAs will not impede the activity of miRNAs.
Thus far, a lot of in vitro and in vivo methods have been developed to validate ceRNA hypotheses, such as bioinformatics analyses, luciferase reporter assays, and correlation analysis of circRNA-miRNA-miRNA target genes [96,98].However, some limitations of these research methods have led to questions about the credibility of the conclusions.If a circRNA has a negative correlation with a miRNA and a positive correlation with its target mRNA, this circRNA is always considered to act as a miRNA sponge.Nevertheless, it has recently been demonstrated that this correlation may result from the co-expression or mutual exclusion of subpopulations [85,86].Bulk tissues are typically composed of a heterogeneous mixture of cell types.For example, colon and prostate tumor tissues often contain uneven proportions of muscle cells, which are known to be rich in circRNAs [99].Consequently, the discrepancy in muscle cell content between different samples may give rise to an artifactual bias in circRNA expression [85,86,98,99].Similarly, lncRNA expression is also cell-specific, which means that the analysis of lncRNA/circRNA expression and function using large tissue samples, such as tumor and non-tumor adjacent specimens, may result in erroneous conclusions.
Given that the majority of circRNAs overlap with mRNAs and the functions of most circRNAs are unknown, there is actually some controversy surrounding the question of whether circRNAs are conserved [39].Here, we have reviewed a number of circRNAs that are expressed in more than one species and have analogous biological functions.One of them, the CDR1as, which has no well-expressed linear equivalent, has conserved functions across multiple species with analogous mechanisms [37,38,43,44].These studies about conserved circRNAs have highlighted the potential importance of circRNAs.
Almost all reported conserved circRNAs with conserved roles in multiple species are exonic circRNAs (EcircRNAs).However, up-to-date, no conserved circRNAs have been reported to produce conserved polypeptides, and we are looking forward to future reports on translationally competent conserved circRNAs.In addition to EcircRNAs, Exon-Intron circRNAs (EIciRNAs), circular intronic RNAs (ciRNAs), and mitochondrial-encoded circRNAs (mecciRNAs) are another three wellrecognized subclasses of circRNAs [2,21].EIciRNAs in the nucleus are reported to promote gene transcription in cis via interacting with U1 small nuclear ribonucleoprotein [2,35].CircRNF217, an EIciRNA in the cytoplasm, was reported to play trans roles as a miR-130-3p sponge to facilitate antibacterial responses in teleost fish [100].Interestingly, circCAMSAP1 is conserved between human and mice, but only human circCAMSAP1 shows intron retention [101], indicating the complex regulatory mechanism in intron retention of EIciRNAs.Although ciRNAs are widely expressed across species [2,102,103], conserved ciRNAs are rarely reported, largely owing to the lower conservation of intronic sequences compared to exonic sequences [102,103].MecciRNAs are extensively expressed in human, mouse and zebrafish [22,104].Even though the mitochondrial DNA sequence performs high similarity between human and mice, there are no conserved mecciRNAs with conserved functions reported [105].
In conclusion, the summarized functions and mechanisms of conserved lncRNAs and circRNAs somehow provide new insights into the RNA world from an evolutionary perspective.

Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 1 .
Fig. 1.Alternative mechanisms of conserved lncRNAs.(A) The cis regulatory role of 5S-OT in 5S rRNA transcription.5S-OT is transcribed by pol II and 5S rRNA is transcribed by pol III.(B) The lncRNA CARINH recruits p300/CBP to increase the deposition of H3K27ac at the IRF1 promoter, leading to the upregulated transcription of the IRF1 gene.(C) The lncRNA AC006064.4-201disturbs PTBP1 from binding to CDKN1B mRNA, resulting in the destabilization of CDKN1B mRNA and the downregulation of CDKN1B protein to inhibit chondrocyte senescence.(D) DIO3OS interacts with NONO to block NONO-mediated ZEB1 mRNA nuclear export, thereby inhibiting HCC stemness.(E) SNHG16 acts as a miR-146a sponge to relieve the repressive effect on its target TRAF6, ultimately regulating the NETosis and autophagy.(F) Cyrano promotes mature miR-7 degradation through target RNA-directed miRNA degradation (TDMD) to de-repress the miR-7 targeted mRNAs.(G) The putative lncRNA HOXB-AS3 encoded polypeptide binds to HNRNPA1 and regulates the HNRNPA1-mediated splicing-regulation of PKM to inhibit colon cancer (CRC) growth.(H) XIST encapsulates the X chromosome to contribute to X chromosome inactivation.

Fig. 2 .
Fig. 2. Molecular mechanisms of conserved circRNAs.(A) CDR1as functions as a miR-7 sponge (Left).MiR-671 associates with Ago 2 to mediate CDR1as degradation.(B) Conserved circBoule-HSP interaction and functional mechanisms form flies to mammals.CircBoule RNAs interact with HSPs to facilitate their ubiquitination.Genetic circBoule knockout flies and mice both demonstrate decreased male fertility, especially under heat-stress conditions.In addition, the circBoule level is significantly lower in sperm from patients with asthenozoospermia.(C) CircLARP1B binds to HNRNPD protein via two conserved motifs and inhibits HNRNPD binding to the 3′ UTR of LKB1 mRNA.CircLARP1B destabilizes LKB1 mRNA and downregulates LKB1 protein to promote hepatocellular carcinoma (HCC) metastasis and lipid accumulation.(D) CircIPO11 increases GLI1 transcription by recruiting TOP1 to the GLI1 promoter.