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. 2026 Sep 10;17:10674. doi: 10.1038/s41467-026-77541-w

Human testis-specific TTRP regulates circadian clock as an evolutionarily young natural antisense transcript of PER2

Tongyue Chen 1,#, Lirong Guo 1,#, Han Zhang 2,5, Bing Cai 2,3, Yong Gao 2,3, Xinle He 1, Peiliang Wang 1, Xiaohong Ma 1, Yunzhen Li 1, Minyi He 1, Tianlun Lei 1, Huiying Yang 1, Xiaolin Yang 1, Meimei Liao 4, Ziqing Yu 4, Wenliang Zhou 1, Peng Zhang 1, Dan Liang 1,✉, Yanwen Xu 2,3,✉, Jinhu Guo 1,✉
PMCID: PMC13655170  PMID: 42860421

Abstract

The circadian clock orchestrates a variety of physiological and behavioral processes, endowing the ability to adapt to the cycling environment. Here, we report the identification of TTRP, a TDP-43 pseudogene that emerged in higher Hominoidea species ~8.6 Mya. TTRP is a human testis-specific gene that overlaps with the clock gene PER2 and interferes with PER2 transcription. TTRP encodes a protein that interacts with the clock proteins PER2, BMAL1, and CRY2, and promotes PER2 and CRY1 turnover. The expression of gorilla and bonobo TTRP proteins changes the period and amplitude of circadian rhythms in human cells. Knock-in and expression of human TTRP in mouse testes prolong the brood-interval time and increase the ratio of sperm with high motility. These findings highlight the functional importance of TTRP as a regulator of the circadian clock and suggest that the human circadian clock may be undergoing continuous evolution through the recruitment of novel factors.

Subject terms: Molecular evolution, Molecular fluctuations, Circadian rhythms

TTRP is an evolutionarily novel gene in higher Hominoidea that has been recruited to regulate the human circadian clock. As an antisense gene to PER2, TTRP modulates circadian rhythms through both transcriptional interference and its encoded protein.

Introduction

The circadian clock governs a wide spectrum of physiological and behavioral processes1,2. In mammals, circadian systems consist of the master oscillators, which are located in the suprachiasmatic nucleus, and the peripheral oscillators, which are located in other tissues3. At the molecular level, circadian clocks are controlled by a set of clock genes comprising positive and negative elements, which constitute transcription-translational negative feedback loops4. In mammalian circadian systems, positive elements include BMAL1 and CLOCK, and negative elements include PER1-3 and CRY1,22,5–7. In addition, the nuclear receptors REV-ERBα (Nr1d1) and RORα, whose transcription is directly controlled by CLOCK-BMAL1, form additional feedback loops to modulate BMAL1 expression5,8.

Natural antisense transcripts (NATs) are those RNA molecules transcribed from opposite DNA strands that partially or completely overlap with sense RNA, which play diverse roles in regulating gene expression9,10. Some NATs have been implicated in the circadian clock in multiple species, such as Neurospora, silkworm, plants, and mice11–19. In Neurospora crassa, qrf is the NAT of the core clock gene frequency (frq), which regulates the circadian clock through transcriptional interference with frq transcription, fine control of facultative heterochromatin, and DNA methylation15–19. In mammals, it has been reported that mouse Per2 harbors a NAT gene Per2AS, which is expressed rhythmically and antiphasic to Per213,14,20–25. However, the detailed functions and mechanisms of NATs in different circadian systems, including mammals, remain elusive.

Despite the high degree of conservation of circadian regulatory mechanisms across kingdoms, the evolution of the circadian clock remains largely elusive. In this study, we identified a human testicular gene, Testicular TDP-43 Related Protein gene (TTRP), which is also a natural antisense gene of the core circadian clock gene PER2 and participates in the regulation of circadian rhythms. And we demonstrate that TTRP is an evolutionarily young gene involved in the regulation of circadian clock.

Results

Human testicular gene TTRP is a natural antisense transcript of PER2

We aimed to identify testis-specific natural antisense transcripts of core circadian clock genes. The human genome data (genome.ucsc.edu) were browsed, and some potential NATs of several core circadian clock genes were found, including PER2, PER3, and CLOCK, supported by the expressed sequence tags (ESTs) (Supplementary Fig. 1a–c). Among these NATs, the NATs of PER2 (PER2as) showed a testis-specific expression pattern according to the EST expression information (Supplementary Fig. 1d). We conducted RT-PCR in multiple human tissues to validate the expression of these NATs of circadian clock genes, PASD1 served as a testicular marker26, and the results showed that only PER2as was exclusively expressed in testes (Supplementary Fig. 1e). The expression of PER2as was not detected in a panel of fetal tissues (Supplementary Fig. 1f). Moreover, PER2as was expressed in some cells derived from testicular germ tumors, including Tcam-2, mildly in NTera-2, and NEC8; it was also expressed in the chronic myelogenous leukemia K562 cells, but lowly in HeLa, and undetected in HEK293T cells (Supplementary Fig. 1g).

We next conducted 5’ rapid amplification of cDNA ends (RACE) and obtained the full-length sequence of PER2as (Fig. 1a and Supplementary Fig. 2a), which comprises seven exons in total. PER2as is located on chromosome 2 near PER2, and these two genes are transcribed in a tail-to-tail fashion. Specifically, there are overlapping regions in pairs of PER2as exon 4–PER2 exon 23 and PER2as exon 7–PER2 exon 18 (Fig. 1a). A predicted CpG island with a predicted TATA box (TTTAAA) exists upstream of the 5’ end of TTRP (Fig. 1a), which was validated as a promoter through the luciferase reporter assay in HEK293T cells (Fig. 1b). In situ hybridization revealed that PER2as mRNA is predominantly expressed in Leydig cells, spermatocytes and spermatids (Fig. 1c).

Fig. 1. Characterization of the human testis-specific TTRP gene.

Fig. 1

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a Genomic organization of the human TTRP gene. TTRP contains seven exons, and the coding region is located at exons 1–4 which are indicated in red. The original pseudogene TARDBPP3 and neighboring retrotransposons are labeled. b Top: predicted promoter region and TATA box of TTRP; bottom: luciferase reporter assay showing that the predicted TTRP promoter can drive the expression of luciferase. Data are presented mean ± SD, n = 3. Statistical significance was determined by Student’s t-test. *** p < 0.001. c In situ hybridization results showing the expression of TTRP in the human testes. Representative results are shown (n = 3). Bars indicate 40 μM. d In vitro transcription-translation assay of TTRP. Luciferase (LUC) was also translated and detected as a control (n = 2). e Western blot of TTRP in human testes samples (n = 1). Asterisks denote nonspecific signals (d, e). U2OS cells were transfected to express PER2as (TTRP). f Schematics of the protein motifs of TDP-43 and TTRP. g Subcellular localization of the GFP-TTRP and TTRP-GFP proteins in HEK293T cells. Representative results are shown (n = 3). Bars indicate 20 μM. Source data are provided as a Source Data file.

The potential of TTRP as a protein-encoding transcript was predicted with CPC27, and the predicted value was close to the threshold (Supplementary Fig. 2b). On the basis of the prediction, the exons 1-4 may contain an open reading frame (ORF) (Fig. 1a and Supplementary Fig. 2a). We performed an in vitro transcription-translation assay and revealed that full-length PER2as and the sequence containing exons 1–4 expressed a short protein of 183 aa; whereas the sequence containing exons 5–7 or the sequence bearing mutations at the initiation codons produced no detectable products (Fig. 1d). We next generated multi-colony antisera of PER2as protein, and the Western blotting results validated its expression in the human testes (Fig. 1e).

The results of sequence searches indicated that the protein encoded by PER2as shows similarity between its N’ terminal region (85 aa) and the N’ terminal region of TAR DNA-binding protein-43 (TDP-43)28 (Supplementary Fig. 2c). Thus, this PER2as was named Testicular TDP–43 Related Protein gene (TTRP). TTRP contains no known domain while TDP-43 contains a nuclear localization sequence (NLS) and two RNA recognition motif (RRM) domains (Fig. 1f). Through GFP fusion, we showed that TTRP was predominantly localized in the cytoplasm (Fig. 1g).

There are predicted methylation sites in the promoter area of PER2as (Fig. 2a), and we performed bisulfite sequencing and compared the methylation status of the PER2as promoter and found that the promoter region was significantly less methylated in cells expressing relatively high levels of PER2as, e.g., K562, NEC8, and Tcam-2 (Supplementary Fig. 3a–h). The methylation level in human testes was much lower than that in blood cells (Fig. 2b and Supplementary Fig. 3i–k). Low methylation at its promoter region might explain the specificity of TTRP expression in human testes.

Fig. 2. Evolution analysis of TTRP and its function in circadian clock.

Fig. 2

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a CpG island distribution in the TTRP promoter region. The locations of the primers (BSP-TTRP-F and BSP-TTRP-R) used for amplification of the promoter region are shown. b Statistical results of the methylation in human blood and testes. The location was numbered from the 5’ end of the reverse primer. Data are presented as mean ± SD, n = 10. Statistical significance was determined by Student’s t-test. c Illustration of the evolutionary origin of the TTRP gene in the human genome. d Schematic phylogenetic tree of TTRP evolution in higher primates. e Expression of full-length TTRP resulted in changes in the circadian rhythms of the circadian oscillation of the BMAL1::luciferase reporter in U2OS cells. Right panels: statistical results of the circadian period and amplitude. Data are presented as mean ± SEM, n = 6. f Effects of expression of TTRP on the stability of PER2 mRNA. Data are presented as mean ± SD, n = 3. g qRT-PCR results showed that TTRP and PER2 display antiphase patterns of circadian expression. Data are presented as mean ± SEM, n = 3. Statistical significance was determined by Student’s t-test. h Expression of TTRP with the entire ORF regulates circadian rhythms of circadian oscillation of the BMAL1::luciferase reporter in U2OS cells. TTRP exons 1–4 [TTRP(1–4)] contain the entire ORF; TTRP exons 5–7 [TTRP(5–7)] contain the 3’UTR region; TTRP ORFmut denotes that the initiation codon was mutated in these constructs. Right panels: statistical results of the circadian periods and amplitudes. The values are mean ± SEM, n = 4. i Gorilla TTRP induced changes in the circadian rhythms of luciferase reporter gene expression driven by the hBMAL1 promoter in U2OS cells. Bottom panels: statistics of the period (left) and amplitude (right). Data are presented as mean ± SEM, n = 9. Statistical significance was determined by one-way analysis of variance (ANOVA) in (h), Student’s t-test in (e), (f) and (i). * p < 0.05, ** p < 0.01, and *** p < 0.001. Source data are provided as a Source Data file.

TTRP is an evolutionarily young gene exclusively present in anthropoids

In silico analysis revealed that TTRP overlaps with the TDP-43-derived pseudogene (TARDBPP3, NR026923.1), supported by the existence of several retrotransposable elements, including SINE, LINE, and LTR29 (Figs. 1a and 2c). It has been revealed that some NATs may originate from pseudogenes30. Thus, it is very likely that TTRP originated from TARDBPP3, which was retrotranscribed from TDP-43 mRNA and integrated into the present locus containing a CpG island, which can drive the transcription of this pseudogene. Subsequently, this region generated mutations which were utilized by the pseudogene as novel splicing sites for the first intron in the coding region. Last, this pseudogene evolved to be the TTRP gene through exon reshuffling31 (Fig. 2c and Supplementary Fig. 4).

We analyzed TTRP homologous sequences in primates derived from the genome database (genome.ucsc.edu) and found that the insertion of TARDBPP3 near PER2 occurred in P. anubis and M. mulatta ~ 32 Ma ago, the novel splicing sites started to emerge in anthropoids about 26 Ma ago, and the TTRP genes with correct splicing and ORF arose approximately 8.6 Ma ago (Fig. 2d and Supplementary Fig. 4). Therefore, TTRP is an evolutionarily young gene. Some sequence differences of TTRP homologs in great apes are noteworthy: in P. troglodytes TTRP gene the initiation codon is missing, in N. leucogenys the TTRP gene has a six-nucleotide insertion, and the P. abelii TTRP contains a premature nonsense mutation. The evolutionary analysis based on multiple alignments of 17 primate genomes to the human genome revealed that the TTRP locus is under relatively weak selective constraint. Visualization of phastCons17way and phyloP17way scores across the TTRP genomic region showed uniformly low conservation across all five coding exons, with phastCons values remaining low and phyloP scores centered around zero (Supplementary Fig. 5a).

TTRP regulates the circadian clock at transcriptional and translational levels

As TTRP is a NAT of PER2, it is possible that the former is involved in circadian clock through regulating the expression and function of the latter. The human osteosarcoma cell line U2OS engineered to express luciferase under the control of BMAL1 was used to assess the effects of expression of full-length TTRP on circadian oscillation32, and the results revealed that expression of TTRP cDNA covering the open reading frame (ORF) with a SV40 promoter led to a lengthened period and an accelerated damping amplitude (Fig. 2e). As PER2 and TTRP present in opposite orientations, it is possible that these two genes repress each other by facilitating mRNA turnover. In U2OS cells stably transfected with TTRP or a control plasmid and treated with the transcription inhibitor thiolutin33, the decay rates of PER2 mRNA were comparable, which rules out the possible influence on PER2 turnover (Fig. 2f).

NATs may also regulate the expression of sense genes through transcription interference at the transcriptional level or RNA interference at the posttranscriptional level19,34,35. Thus, we measured the levels of PER2 and TTRP transcripts in NEC8 cells via qRT-PCR and found that they are simultaneously transcribed and oscillate in an antiphasic pattern (Fig. 2g), suggesting a mutual inhibitory function. Together, these data suggest that TTRP represses PER2 expression and clock function via transcriptional interference.

We next investigated whether TTRP protein is involved in the circadian clock. To this end, the intact TTRP ORF region (exons 1–4), TTRP ORF with initiation codons mutated (TTRP ORFmut), and TTRP 3’ UTR region (exons 5-7) were stably transfected into the U2OS cells with a luciferase reporter driven by the BMAL1 promoter, respectively. The results revealed that the luciferase rhythms of cells expressing TTRP(5–7) and TTRP ORFmut were comparable to those of the control, respectively; in contrast, expression of the TTRP ORF region [TTRP(1–4)] dramatically repressed the circadian amplitude after the first day, and the period was lengthened due to expression of the TTRP ORF region (Fig. 2h). These data demonstrate the role of TTRP protein in regulating the circadian clock.

To assess functional conservation, we synthesized the TTRP genes of Gorilla gorilla (G. gorilla) (gorilla, gTTRP) and Pan paniscus (P. paniscus) (bonobo, bTTRP), and stably transfected and expressed in U2OS cells. Expression of both gTTRP and bTTRP resulted in significant changes of circadian rhythms, characterised by increased oscillation amplitudes of the luciferase reporter gene expression, and shortened circadian periods which are in contrast to the results of human TTRP expression (Fig. 2e, i and Supplementary Fig. 5b–d). These results suggest that TTRP proteins in these two species also possess the potential to regulate circadian clocks. These results also indicate that TTRP retains a conserved involvement in circadian regulation across primates, but with species-specific functional effects.

Expression of TTRP rewires the circadian rhythmicity of global gene expression

U2OS cells were synchronized with dexamethasone and harvested over two consecutive days with TTRP expression or without TTRP expression (control). The RNA samples were isolated and subjected to RNA sequencing (RNA-seq), which revealed oscillating genes with approximately 24 h periods (JTK_CYCLE, p < 0.01) in TTRP-expressing and control cells (Fig. 3a and Supplementary Fig. 6a–c)36. The expression of TTRP led to extensive changes in the rhythmicity of downstream genes; 238 genes became rhythmic, whereas 177 were arrhythmic (Fig. 3a, b). In addition, the phase distribution of the genes rhythmically expressed in TTRP-expressing cells was more centered than that of the genes rhythmically expressed in control cells (Fig. 3c).

Fig. 3. Expression of TTRP rewires the rhythmic expression of global genes.

Fig. 3

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a Heatmap of genes showing altered circadian rhythms over 48 h between the TTRP-expressing cells compared with control cells. The cells were harvested every 6 h. b Venn diagram displaying the numbers of genes showing rhythmic expression in the cells expressing TTRP and control cells. c Phase distribution of the genes showing circadian rhythms in the cells expressing TTRP and control cells, respectively. d,e GO (d) and KEGG (e) pathway analysis of the genes showing circadian rhythms in the control cells but not in the cells expressing TTRP. f,g qRT–PCR validation of PER2 (f) and CRY1 (g) mRNA levels in TTRP-expressing and control cells. Data are presented as mean ± SD, n = 3. Statistical significance was determined by Student’s t-test (f,g). * p < 0.05, and ** p < 0.01. Source data are provided as a Source Data file.

The expression of TTRP resulted in changes in several pathways involved in circadian rhythms (Fig. 3d, e and Supplementary Fig. 7a, b). The expression of some genes of interest was validated by quantitative reverse transcription PCR (qRT-PCR). For instance, the mRNA levels of PER2 were significantly decreased due to TTRP expression, while CRY1 showed similar rhythmic patterns, although differences at some time points were observed (Fig. 3f, g). The changes in expression or rhythmicity characteristics were also observed in BMAL1, PER1, CRY2, and a set of clock-controlled genes (Supplementary Fig. 7c–f). Moreover, as TTRP is a testicular gene, we also validated the altered rhythmicity of genes associated with cell division and male meiosis were also identified, e.g., Tubulin gamma complex associated protein 6 (TUBGCP6)37 (Supplementary Fig. 7g–i). Together, these data suggest that TTRP protein acts to reprogram circadian gene expression which might further regulate the circadian clock and male reproduction.

TTRP binds with clock proteins and regulates circadian negative feedback loop

The expression of TTRP at a near-endogenous level led to decreased levels of PER2 and CRY1 although there was no overt change in the phosphorylation of the circadian clock proteins (Fig. 4a and Supplementary Fig. 8a, b). Expression of TTRP in HEK293T cells expressing luciferase reporter gene driven by PER2 promoter led to decreased level of bioluminescence (Fig. 4b, c), suggesting the involvement of TTRP in the regulation of circadian clock through affecting the transcriptional-translational negative feedback loop4.

Fig. 4. TTRP binds with clock components and regulates circadian rhythm.

Fig. 4

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a Clock protein levels in TTRP-expressing and control cells. The Flag-TTRP plasmid was transfected into and expressed in HEK293T cells, and pcDNA3.1-flag plasmid was transfected as a control. Data are presented as mean ± SEM, n = 4. b Luciferase reporter assay to determine whether expression of TTRP affects the expression of luciferase reporter with a PER2 promoter. Asterisks denote nonspecific bands. c Statistical results from (b). Data are presented as mean ± SEM, n  =  3. d–f Representative Co-IP results for TTRP and clock proteins BMAL1 (d), CRY2 (e), and PER2 (f) in HEK293T cells. Representative of at least two independent experiments is shown. Asterisks denote nonspecific signals. g–i Degradation rates of PER2, BMAL1, CRY2 (g), and CRY1 (h) from Western blot results, and statistical analysis (i). Data are presented as mean ± SEM, n = 3. j ChIP assay results showing the binding of BMAL1 to the promoter regions of the indicated clock genes. Data are presented as mean ± SD, n = 3. Statistical significance was determined by Student’s t-test (a, c, i, j). * p < 0.05, ** p < 0.01, *** p < 0.001. Source data are provided as a Source Data file.

Co-immunoprecipitation (Co-IP) revealed interactions between TTRP and PER2, CRY2, and BMAL1, but not other circadian components, including PER1, CRY1, CLOCK, RORα, and REV-ERBα (Fig. 4d–f and Supplementary Fig. 8c–g). We further analyzed the stability of PER2, CRY1, CRY2, and BMAL1, and found faster degradation of PER2 and CRY1 but not the other tested clock proteins (Fig. 4g–i). The CRY1 mRNA level was comparable in TTRP-expressing cells to that in control cells while the PER2 mRNA was lower in the TTRP-expressing cells (Figs. 3f and 4g, i), suggesting that the lower PER2 protein level in TTRP-expressing cells may be due to decreased transcript amount and accelerated degradation, while the lower level of CRY1 may be solely attributed to the elevated turnover (Fig. 3g and Fig. 4h, i). Moreover, the chromatin immunoprecipitation (ChIP) assay results showed decreased binding of BMAL1 at the promoter regions of PER1/2 and CRY1/2 (Fig. 4j). Together, these data suggest that TTRP binds with clock proteins and controls the degradation and function of clock proteins in the negative feedback loop.

hTTRP modulates the circadian clock and reproduction in mouse testes

We also transfected human TTRP (hTTRP) into the mouse adult fibroblast (MAF) cells, which express a luciferase reporter gene driven by a Per2 promoter. The transduced cells presented altered circadian variables with a prolonged period and a slightly but significantly faster damping rate (Fig. 5a–c). Co-IP experiments revealed that hTTRP was associated with the mouse circadian components mPER2, mCRY2, and mBMAL1, consistent with the results in human cells (Fig. 5d–f). These data suggest that hTTRP may also affect the mouse circadian rhythms by modulating the expression and function of circadian clock components.

Fig. 5. Human TTRP controls circadian clock in mice.

Fig. 5

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a–c Transfection and expression of hTTRP affected circadian rhythms in mouse metastasis-associated fibroblasts (MAF) cells. The rhythms of relative luminescence are shown in (a), and the statistics of the period and amplitude are shown in (b) and (c), respectively. Data are presented as mean ± SEM, n = 6. Statistical significance was determined by Student’s t-test. d–f Representative Co-IP results showing the interaction between human TTRP (hTTRP) and mouse clock proteins, including mBMAL1 (d), mPER2 (e), and mCRY2 (f). Representative of at least two independent experiments is shown. Asterisks denote nonspecific bands (d–f). g Schematic of CRISPR/Cas9-mediated knock-in of the hTTRP gene at the H11 locus. Panel g was created using Microsoft PowerPoint (version 16) and Adobe Illustrator (version 26.0.2). h qRT-PCR results showing the levels of hTTRP in the testes of homozygous (Ki/Ki), heterozygous (Ki/Wt), and wild-type (Wt/Wt) male mice. Data are presented as mean ± SEM, n = 3 mice per group. Statistical significance was determined by One-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons versus the control. *** p < 0.001. Source data are provided as a Source Data file.

As the expression of testes-specific TTRP affected the expression of signaling pathways associated with meiosis (Figs. 1c, 2b and Supplementary Fig. 7g, i), it is worth investigating its potential role in the regulation of male reproduction. As TTRP is present only in anthropoids, observing its effects on testes physiology is very challenging because of ethical and practical issues. Alternatively, we generated the knock-in mice bearing the human TTRP gene (hTTRPKI mice) by CRISPR/Cas9 technique, and the derived mice were crossed with Cre mice with a Dazl promoter that drives the testis-specific expression38,39 (Fig. 5g and Supplementary Fig. 9a, b). The mice were further validated by qRT-PCR of hTTRP levels (Fig. 5h).

The body weights of the Ki/Ki and Ki/Wt mice were significantly greater than the control mice at 17 dpc (days post coitum) during development, while the testes weight did not significantly differ at 17 dpc although the testes weight of Ki/Wt mice was greater than the Wt/Wt control mice (Supplementary Fig. 9c–f). The expression of hTTRP in the mouse testes may affect the testosterone secretion, which accounts for the difference in body weight during development40. Every male Ki/Ki, Ki/Wt or Wt/Wt mouse was mated with two female WT mice, and the number of pups was comparable between the offspring of Ki/Ki, Ki/Wt and Wt/Wt mice (Fig. 6a). There was no significant difference between the quantity of vaginal copulation plugs among the homozygotes, heterozygotes and control mice (Fig. 6b), suggesting that mating behavior was not significantly affected.

Fig. 6. Effects of hTTRP on mouse male reproduction.

Fig. 6

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a Statistics of the offspring quantities of the Ki/Ki, Ki/Wt and Wt/Wt male mice. Data are presented as mean ± SEM, Wt/Wt: n = 10 mice; Ki/Wt: n = 7 mice; Ki/Ki: n = 10 mice. b Statistics of the copulatory plugs of the female mice. Each male mouse was accompanied by two females in the cage. Data are presented as mean ± SEM, Wt/Wt: n = 5 mice; Ki/Wt: n = 5 mice; Ki/Ki: n = 4 mice. The statistical significance was calculated by one-way ANOVA. c Statistics of the interbrood days of the offspring of Ki/Ki, Ki/Wt and Wt/Wt male mice. Data are presented as mean ± SEM, Wt/Wt: n = 10 mice; Ki/Wt: n = 11 mice; Ki/Ki: n = 10 mice. Statistical significance was determined by One-way ANOVA followed by Dunnett’s post hoc test for multiple comparisons versus the control (a–c). d Comparison of proportions of the four categories of sperm in Wt/Wt, Ki/Wt, and Ki/Ki mice. “a” stands for sperm showing fast forward motion, “b” for sperm showing slow forward motion, “c” for sperm showing non-forward motion, “d” for sperm showing very slow motion or motionlessness. More than 200 sperm were counted. Data are presented as mean ± SEM, Ki/Ki: n = 5 mice; Ki/Wt: n = 5 mice; Wt/Wt: n = 4 mice. Statistical significance was determined by one-way ANOVA followed by Fisher’s least significant difference (LSD) post hoc test for multiple comparisons (d). e Schematic model of TTRP in the regulation of circadian clock in the human testes. The transcription of TTRP may interfere with the transcription of PER2, which regulates the expression of the latter. Panel e was created using Microsoft PowerPoint (version 16) and Adobe Illustrator (version 26.0.2). In addition, the TTRP protein binds to clock components and regulates the degradation of PER2 and the expression levels of CRY1 and PER2. TTRP may function in male reproduction through pathways that are dependent on and independent of the circadian clock. The trash bins denote degradation, and the dashed lines denote hypothetical regulations. * p < 0.05, ** p < 0.01, *** p < 0.001. Source data are provided as a Source Data file.

The interval time between broods denotes the time duration between the broods of the two female mice for each male mouse, which showed a significant difference between Ki/Ki ( ~ 14 d) and Wt/Wt ( ~ 6 d), and between Ki/Wt ( ~ 13 d) and Wt/Wt (Fig. 6c). According to the WHO manual (2021 edition), the sperm motility can be evaluated on the basis of four categories: rapidly progressive motility, slowly progressive motility, nonprogressive motility, and immotility41. We isolated sperm from epididymis and compared the proportions of the four categories of sperm and found that the proportion of sperm showing rapidly progressive motility was significantly greater in Ki/Ki than in Ki/Wt and in Wt/Wt (Fig. 6d), suggesting that TTRP may promote sperm motility.

Discussion

TAR DNA-binding protein 43 (TDP-43) plays a role in regulating multiple steps of RNA biogenesis and processing, and its mutations are associated with neurodegenerative diseases28. Interestingly, deficiency of TDP-43 in suprachiasmatic nucleus also led to disturbed circadian rhythms42. TTRP is a young testis-specific natural antisense gene of the human core clock gene PER2, which originated through retrotranslocation and evolution from a TDP-43-derived pseudogene TARDBPP3 approximately 8.6 Ma ago. TTRP occurs in anthropoids, and gorilla and bonobo TTRPs could mediate circadian rhythms in human cells (Fig. 2d, e, i and Supplementary Fig. 5b-d).

An antisense gene Per2AS has been previously identified in the mouse Per2 locus13,14, however, there are several different features between it and TTRP. First, TTRP is a descendent of TDP-43 pseudogene in primates, which is not present in mouse genome. Second, Per2AS and TTRP have different gene structures. Mouse Per2AS is transcribed relatively spanning the 5’ part of Per2 while TTRP is located at the 3’ part of PER2 in human14 (Fig. 1a). Moreover, the Per2AS is abundant in the liver while TTRP is a testis-specific gene (Fig. 1c; Supplementary Fig. 1e).

The presence of natural antisense transcripts of clock genes has been reported in a variety of species11–14,16–19,22–25. Many of these antisense genes exhibit antiphasic rhythms with the sense clock genes, which suggests that these NATs and clock gene pairs are expressed in a temporally and spatially overlapping fashion, and they may inhibit each other through transcriptional interference11–25. Among these NATs, Neurospora qrf exhibits an antiphasic expression profile to frq, and the expression of qrf is controlled by light-dependent and light-independent mechanisms, respectively. Qrf represses frq transcription and mediates circadian clock through transcriptional interference, chromatin modification at frq region, and premature termination of transcription15–19. In mice, Per2AS mediates Per2 expression and the amplitude of circadian rhythms14. TTRP also shows an antiphasic rhythm to PER2, and it may regulate circadian rhythms through transcriptional interference (Fig. 2g and Fig. 6e). TTRP also regulates the circadian clock through the interaction of its encoded protein with clock components, including PER2, CRY2, and BMAL1, and regulates the turnover of CRY1 and PER2 (Fig. 4d–f), which modulates the circadian oscillator and subsequently the expression rhythmicity of a subset of genes (Fig. 2e, h and Fig. 3a).

TTRP is expressed in spermatocytes and spermatids in the human testes (Fig. 1c). The expression of TTRP led to alterations in the abundance and circadian rhythms of a set of genes involved in meiosis and cell division (Supplementary Fig. 7g, i). In mice, the expression of hTTRP protein in testes influences certain aspects of reproduction, e.g., interbrood duration and sperm motility (Fig. 6c, d). These results suggest that TTRP may play a fine-tuning role in male reproduction through a circadian clock-dependent pathway, although a circadian clock-independent pathway may also exist (Fig. 6e).

During evolution, newly emerged genes usually evolve rapidly to possess essential functions through transcriptional interference and posttranslational regulation43. Here, we provide evidence that TTRP is an evolutionarily young gene that occurred only in anthropoids and that it rapidly emerged as a functional gene implicated in the control of the circadian clock and male reproduction. Among the primates, only G. gorilla, and P. paniscus harbor the version of TTRP which can be correctly translated like that in H. sapiens (Supplementary Fig. 4). Functional studies indicate that the human TTRP protein exerts a relatively mild effect on the male reproductive system (Fig. 6c, d and Supplementary Fig. 9e, f).

It is possible that the TTRP protein was only recently integrated into the circadian regulatory network of male germ cells in great apes. As a newly acquired component, the expression of human TTRP lengthens the circadian period, whereas gorilla (gTTRP) and bonobo (bTTRP) TTRPs shorten it (Fig. 2i and Supplementary Fig. 5b–d). Evolutionary analysis of primate genomic sequences revealed low conservation of TTRP genes (Supplementary Fig. 5a), and in several great ape species that carry this gene, nonsense mutations have also been observed (Fig. 2d and Supplementary Fig. 4), suggesting an absence of strong purifying in selection, which is most consistent with near-neutral evolution of the TTRP coding sequence and TTRP is under relatively weak evolutionary constraint. TTRP plays a role in regulating the circadian clock across primates, but with opposite effects on circadian periods (Fig. 2e, i and Supplementary Fig. 5b–d). Such divergent phenotypic outcomes do not contradict functional conservation per se. Instead, they suggest that while TTRP proteins from different primate species are capable of modulating the human circadian clock, the direction and magnitude of their effects have diverged during evolution, potentially reflecting lineage-specific adaptations of circadian regulation to distinct ecological or environmental contexts.

Thus, TTRP appears to be a circadian regulator whose function is conserved in terms of pathway involvement, yet remains evolutionarily flexible in its regulatory output. As TTRP genes continue to evolve, their trajectories may gradually diverge among great ape species, eventually leading to the fixation of species-specific biological functions.

Methods

Plasmids and cells

U2OS cells were cultured under standard conditions (DMEM; 10% FBS, 1% penicillin/streptomycin, all from Invitrogen; 37 °C; 5% CO2). U2OS cells expressing TTRP protein and control cells were used for RNA-seq analysis of the effects of TTRP protein on circadian rhythms at the transcriptomic level. As NEC8 cells simultaneously transcribe both PER2 and TTRP, NEC8 cells were cultured to analyze the transcriptional interference between PER2 and TTRP. HEK293T cells were used for the luciferase reporter assays and the co-immunoprecipitation (Co-IP) to analyze the association between TTRP and clock proteins.

In the subcellular localization study, the TTRP gene fused with GFP was transiently transfected with a CMV promoter. In the study of evolution, the gorilla and bonobo TTRP genes were cloned into the plasmid pBABEDH-SV40-TTRP-puro and stably transfected into U2OS cells harboring luciferase reporter gene driven by a BMAL1 promoter (BMAL1-dLuc); the human TTRP gene was cloned into the plasmid pCDH-CMV-TTRP-EF1-copGFP-T2A-NeoR and stably transfected into mouse MAF cells harboring luciferase reporter gene driven by a Per2 promoter. In Co-IP experiments, TTRP and the circadian clock genes were cloned into pcDNA3.1 plasmid with a CMV promoter and transfected into U2OS cells, respectively. In cell studies, cells transfected with the corresponding vector plasmids served as controls.

Human and animal ethics

All of the experimental procedures involving animals were conducted in accordance with the international, national, and institutional guidelines for the humane treatment of animals, complied with relevant legislation, and were approved by the Administration Committee of the School of Life Sciences, Sun Yat-sen University on Feb 24, 2018. The animals were raised and administered at the animal room in GemPharmatech Co., Ltd. (China).

Human blood and testes biopsy samples were obtained in accordance with the Medical Ethics Committee; all tissue specimens were collected with patients’ informed consent. Human testis specimens from oligozoospermic patients were obtained from cases requiring open testicular biopsy. All the studies involving human tissues have been approved by IEC for Clinical Research and Animal Trials of the First Affiliated Hospital of Sun Yat-sen University (No. [2023]537) on Aug 25, 2023, and have been performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. All these human samples have been destroyed after the analysis.

The kits of Human MTCTM Panel I, Human Fetal MTCTM Panel, and Human MTCTM Panel II containing human cDNAs from multiple tissues were purchased from Takara (Japan). Human Testis Marathon-Ready cDNA was purchased from Clontech (USA).

Cloning of the full length TTRP by 5’ rapid amplification of cDNA ends (RACE)

Touchdown PCR amplification was conducted to clone the full-length sequence of TTRP by 5’ RACE. The cDNA template for RACE was commercially obtained from the Clontech Human Testis Marathon-Ready cDNA kit (Takara, Japan).

Luciferase reporter assay for measuring the activity of TTRP promoter

The UCSC genome browser revealed a CpG island upstream of the transcription start site of TTRP. To assess whether it contains the TTRP promoter, the upstream sequence was subjected to prediction at www-bimas.cit.nih.gov/molbio/proscan, which reported a putative promoter in this region. Next, the upstream 252 nt sequence was fused with the coding sequence of firefly luciferase reporter gene (luc) and cloned into pGL3-basic plasmid. The resultant pGL3-basic-TTRP-p plasmid (2 μg) was transfected into HEK293T cells, the plasmid containing renilla luciferase gene driven by HSV-TK promoter (pRL-TK-renilla luciferase) was simultaneously transfected as control (2 μg). The luciferase signal was measured according to the manufacturer’s instructions (Promega Dual-Glo, Cat.No. E2920) on Multiskan™ FC Microplate Photometer (Thermo Fisher Scientific, USA).

Monitoring of the circadian dynamics of luciferase reporter

The U2OS cell (Bmal1-dLucU2OS) harbors a cassette dynamically expressing luciferase under the control of the BMAL1 promoter32, which allows to real-time record the circadian oscillation of luciferase signals with the LumiCycle luminometer (ActiMetrics, USA), after synchronizing the cells with 2-hr treatment of 100 nM dexamethasone. Bioluminescence was mounted over 10 min intervals with the LumiCycle and the data were analyzed with ClockLab software by following its instruction32. The immortalized mouse adult fibroblast (MAF) cells containing luciferase reporter gene driven by Per2 promoter were treated and the luciferase signals were monitored in the same way. The MAF cells derived from tail harbor a cassette dynamically expressing luciferase under the control of a Per2 promoter44, which was used to monitor the effects of human TTRP protein on the oscillatory rhythms of mouse cells. The synchronization and observation were following the same protocol above.

Bisulfite PCR analysis and sequencing

The procedure for bisulfite sequencing PCR (BSP) was used to detect methylation of the TTRP promoter region. The genomic DNA samples were isolated from cell lines, testis and blood, respectively, and 200 ng of genomic DNA of each sample was treated using the EZ DNA Methylation-Gold™ Kit (Zymo Research, USA) according to the manufacturer’s instructions45. For bisulfite sequencing, the DNA treated by the EZ DNA Methylation-Gold™ Kit was used for PCR with the primers of BSP-TTRP (forward, 5’- ACCTCTCTCGCCTTCGCTTTC-3’; reverse, 5’-TTCCACTAGGCCGCTGCTG-3’). PCR products were cloned into pEASY-T5-Zero Cloning Vector (TransGen Biotech, China), and 10 clones were sequenced for each sample. The sequencing results were analyzed with QUMA46.

GFP constructs and fluorescent microscopy

The region of TTRP ORF was cloned into pEGFP-N1 and pEGFP-C1, respectively, either at the C’ terminus (pTTRP-EGFP) or the N’ terminus (pEGFP-TTRP). The obtained constructs were transiently transfected into HEK293T cells grew on coverslips in the flasks. After 48h of transfection, the slides were taken out, and the GFP signals were visualized under fluorescent microscopy.

Protein analysis and antibody preparation

TTRP protein was expressed in E. coli, extracted from inclusion bodies, and purified with a column stuffed with Ni2+ -NTA-Superflow agarose. The resultant TTRP protein was used to immunize a rabbit. The serum containing antibodies against TTRP was extracted and validated in TTRP-expressing cells and control cells by Western blot assays.

In vitro transcription and translation

The TNT® T7 Quick Coupled Transcription/Translation System kit (Promega, USA) was used for in vitro translation. The fragments of full-length TTRP, 1-4 exons of TTRP [TTRP(1-4)], 5-7 exons of TTRP [TTRP(5-7)], and TTRP with mutation of the putative ORFs (TTRP-ORFmut) were cloned and the coupled in vitro transcription/translation were conducted by following the manufacturer’s protocol. Afterward, 10 μl of translated samples was denatured and separated by electrophoresis and detected by the kit TranscendTM Chemiluminescent Non-Radioactive Translation Detection System (Promega, USA). Transcription and translation of luciferase (luc) were carried out simultaneously as a control.

RNA in situ hybridization

Human biopsy testes samples or mouse testes were frozen in liquid nitrogen, returned to room temperature, fixed with 4% paraformaldehyde, dehydrated, embedded with paraffin, and sliced. PCR products were purified using E.Z.N.A.®Gel Extraction Kit (Omega, USA) as in vitro transcription templates. Sense and antisense RNA probes were labeled with Digoxigenin by in vitro transcription approach using DIG RNA Labeling Kit (SP6/T7) (Roche, USA). Hybridization and washes were performed at 55 °C, hybridization with the antisense sequence at its 5’ end was used as control. DIG-labeled sense and anti-sense probes were detected using Anti-Digoxigenin-AP and NBT/BCIP phosphate reagents (Roche, USA).

Construction of mice testis-specifically expressing human TTRP and analysis of its role in male reproduction

To determine the potential role of human TTRP (hTTRP) in regulating male reproduction in mice, the H11-Dazl-LSL-hTTRP-polyA cassette was constructed and microinjected into the zygotes of C57BL/6 J mice and Cas9 mRNA, sgRNA and donor were co-injected. sgRNA directs Cas9 endonuclease cleavage at the mouse H11 locus and creates DSB (double-strand break). Such breaks will be repaired, and result in Dazl-hTTRP-polyA fragment inserted into the mouse H11 locus by homologous recombination47. The pups will be genotyped by PCR, followed by sequence analysis. The obtained F0 mice B6JNju-H11em1Cin(Dazl-hTTRP) Nju were crossed with B6;129S7-Amhr2tm3(cre)BhrMmncNju tool mice (Fig. 5g and Supplementary Fig. 9a, b). The hTTRPKI mice were generated by GemPharmatech Co., Ltd. (China). Mice were maintained under controlled environmental conditions with a 12-h light/12-h dark cycle, ambient temperature of 20–26 °C, and relative humidity of 30–70%, with ad libitum access to food and water.

To analyze the effects of hTTRP on mouse reproduction, each male mouse from the same brood at eight to nine weeks of age was raised together with two females. The quantities of offspring and the interval time between productions in each cross-cage were recorded and analyzed. In addition, during this period, the vaginal copulation plug of female mice was checked and counted every morning.

Male homozygote hTTRPKI (Ki/Ki), heterozygote (Ki/Wt) and wild type mice aged about 50 d, were sacrificed and epididymis were dissected, cleaned, and treated with 1 mL BWW culture medium. Thereafter, the testicle was incubated at 37 °C in 5%CO2, 95% O2 for 10 mins. During the incubation, most of the mobile sperm were released into the culture medium. An aliquot of 10µL sample was loaded into a prewarmed disposable standard two-chambered slide (Microptic S.L., Spain), cleaned with ddH2O. The sperm motility was assessed with the Motility/Concentration module of the Sperm Class Analyzer (SCA®) (Microptic S.L., Barcelona, Spain, version 5.2). The capturing parameters included: optics, Ph- (negative phase contrast); automatic analysis; analytical properties, particle area, 10–300. Five fields of view were captured randomly with the SCA® system for sperm samples from each testicle, and all of the captured sperm tracks were visually validated to eliminate incorrect records. The results were classified into 4 levels according to the World Health Organization (W.H.O) standard: fast forward motion (level a), slow forward motion (level b), non-forward motion (level c), and very slow or motionless motion (level d)41.

RNA-seq analysis and qRT-PCR

The total RNAs from U2OS cells expressing TTRP or not were extracted by using TRIzol® Reagent (Invitrogen, USA) and subjected to RNA sequencing, which was performed on a NovaSeq 6000 sequencer (Illumina Inc., USA) by Forevergen Co., Ltd. (China). The read length was PE150bp, and the qualified reads were obtained after removing raw reads with adapters or low-quality bases, and then aligned to the human genome (NCBI37/hg9) by SOAP with default parameters48. RSeQC was used to calculate the gene expression level represented as reads per kilobase per million mapped reads (RPKM)49. The JTK_CYCLE software was used to analyze and screen rhythm genes based on the RPKM values, and threshold for ADJ. P ≤ 0.05 and PER ≥ 20 h and ≤ 24 h50. To identify pathways with significant enrichment, pathway enrichment analysis was performed based on the KEGG database and the result of the analysis model with circadian repetition. KEGG pathways fulfilling the criterion of a hypergeometric p < 0.05 & log2(FoldChange) > 1 or < -1 were defined as significantly enriched in DEGs and were evaluated using DAVID (https://david.ncifcrf.gov/tools.jsp). The RNA-seq datasets were deposited at the GEO database (accession no. GSE116976).

Total RNA was isolated and reverse transcribed using Evo M-MLV RT Kit with gDNA Clean for qPCR II (Accurate Biotechnology, China). The obtained cDNA samples were amplified with the SYBR Green Master Mix (Takara, Japan) using a LightCycler 480 (Roche, Germany).

Evolutionary analysis

The primate TTRP homologous genomic sequences were obtained and analyzed at https://genome.ucsc.edu/database (accessed on Sep 02, 2025). The genome sequence of P. anubis is incomplete, and even PER2 failed to be found; therefore, it was precluded from further analysis. Alignment of coding sequences of the TTRP homologs was performed with MEGA 7.0.951. PhastCons17way and phyloP17way scores across the TTRP genomic regions based on 17 primate genomes were displayed according to the data at genome.ucsc.edu (accessed on Jan 12, 2026).

Statistics

For statistical analysis, GraphPad Prism (V8) and Microsoft Excel were used. The results are presented as the mean ± SEM or mean ± SD as indicated. The significance of the qRT-PCR results was determined by Student’s t-test, JTK-CYCLE, and one-way or two-way analysis of variance (ANOVA) as indicated. * represents p-value of the statistical tests being less than a significant level of 0.05 (p < 0.05); ** p < 0.01, and *** p < 0.001.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Supplementary information

Supplementary Information (3.6MB, pdf)
Reporting summary (2.3MB, pdf)
Transparent Peer Review file (248.1KB, pdf)

Source data

Source data (6.2MB, zip)

Acknowledgements

We thank H. Li (Sun Yat-sen University), Y. Xu (Suzhou University, China), E.E. Zhang (National Institute of Biological Sciences, China), and Y. Liu (Fudan University, China) for support or helpful comments.

Author contributions

J.G. conceived the project and designed the experiments. T.C., L.G., H.Z., B.C., Y.G., X.H., P.W., X.M., Y.L., M.H., H.Y., X.Y., performed the experiments. T.C., L.G., H.Z., P.Z., D.L., Y.X., and J.G. analyzed the data. T.L., M.L., Z.Y., and W.Z. supported the experiments. J.G supervised the project. T.C., L.G., P.Z., D.L., Y.X., and J.G. wrote and revised the manuscript. Y.G., P.Z., Y.X., and J.G. acquired the funding. All authors read and approved the manuscript.

Peer review

Peer review information

Nature Communications thanks Yi Liu, and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. A peer review file is available.

Funding

This work was funded by the National Basic Research Program of China grants (2022YFA1604500 and 2012CB947600 to J.G.); the National Natural Science Foundation of China grants (82471884 to Y.X., 32370477 to P.Z., 31871188, 32171162, and 32000824 to J.G.); the Space Medical Experiment Program (HYZHXMN01003 to J.G.); the Ningbo Talent Project (215-432094250 to J.G.); and the Natural Science Foundation of Guangdong Province (2023A1515010240 to Y.G.).

Data availability

All data are available in the main text or as Supplementary Information. All material generated in this study (including but not limited to oligonucleotides, plasmids, purified proteins, antibodies, cell lines, and mice) are available on request. The sequence of TTRP gene generated in this study has been deposited in the GenBank database under accession code PQ565831. The RNA sequencing datasets generated in this study are available at the NCBI Gene Expression Omnibus (GEO) (GSE116976). Source data are provided with this paper.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

These authors contributed equally: Tongyue Chen, Lirong Guo.

Contributor Information

Dan Liang, Email: liangd8@mail.sysu.edu.cn.

Yanwen Xu, Email: xuyanwen@mail.sysu.edu.cn.

Jinhu Guo, Email: guojinhu@mail.sysu.edu.cn.

Supplementary information

The online version contains supplementary material available at https://doi.org/10.1038/s41467-026-77541-w.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Information (3.6MB, pdf)
Reporting summary (2.3MB, pdf)
Transparent Peer Review file (248.1KB, pdf)
Source data (6.2MB, zip)

Data Availability Statement

All data are available in the main text or as Supplementary Information. All material generated in this study (including but not limited to oligonucleotides, plasmids, purified proteins, antibodies, cell lines, and mice) are available on request. The sequence of TTRP gene generated in this study has been deposited in the GenBank database under accession code PQ565831. The RNA sequencing datasets generated in this study are available at the NCBI Gene Expression Omnibus (GEO) (GSE116976). Source data are provided with this paper.

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