Zhang, Liwen, Ph.D.

RNA modifications constitute a dynamic regulatory code governing RNA structure, fate, and translation. Modified nucleosides, long dismissed as metabolic waste, are now recognized as stable biomarkers and active signaling messengers involved in metabolic and reproductive regulation. Advances in methods for detecting modified nucleosides could accelerate translation toward clinical diagnostics and monitoring.

The periconception period is critical for embryo development, pregnancy outcomes, and offspring health. During this stage, oviductal and uterine fluids facilitate embryo-maternal interactions and support early embryonic development. Using PANDORA-seq, we identify a diverse repertoire of small non-coding RNAs in female mouse oviduct fluid and uterine fluid during preimplantation, with tRNA-derived small RNAs and rRNA-derived small RNAs being predominant. Maternal high-fat diet during preimplantation period significantly alters tsRNA and rsRNA expression in oviduct fluid and uterine fluid compared to normal diet, disrupting blastocyst metabolic gene expression. While implantation remained unaffected, these alterations impair mid-gestation embryonic and placental growth, resulting in reduced birth weight and length, as well as metabolic disorders in offspring. Furthermore, transfecting embryos with uterine fluid-derived sncRNAs altered by maternal high-fat diet mimics the in vivo effects. These findings suggest that tsRNAs and rsRNAs in reproductive fluids may reflect maternal metabolic status and transmit dietary information to the early embryo, which might influence pregnancy outcomes and offspring health.

Polycystic Ovary Syndrome (PCOS) is the most prevalent ovulatory and endocrine disorder affecting reproductive-aged women, yet the absence of a specific, rapid molecular diagnostic marker results in diagnostic delays and inaccuracies. Given the critical role of RNA modifications in disease pathology, this study utilized a high-throughput RNA modification profiling platform to investigate 15 types of peripheral blood RNA modification patterns in individuals with ovulatory disorders, including PCOS and Primary Ovarian Insufficiency (POI), and control subjects. Our results revealed that distinct modification profiles correspond to specific disease states, with significant shifts in RNA modification inter-correlations observed across conditions. Additionally, specific RNA modifications were associated with clinical features, such as serum levels of testosterone and the follicle number per ovary (FNPO). To optimize diagnostic precision, we evaluated various machine learning models, identifying that combining m6A and m7G modifications in a light gradient boosting machine model achieves the highest accuracy in distinguishing PCOS, outperforming traditional diagnostic markers. This highlights the potential of RNA modification profiling as a novel, high-accuracy diagnostic tool for PCOS in clinical settings.

Small noncoding RNAs (sncRNAs) are a diverse group of RNAs including small interfering RNAs (siRNAs), microRNAs (miRNAs), PIWI-interacting RNAs (piRNAs) and RNAs derived from structured RNAs like transfer RNAs (tRNAs), ribosomal RNAs (rRNAs), and others. These sncRNAs have varied termini and RNA modifications, which can interfere with adaptor ligation and reverse transcription during cDNA library construction, hindering detection of many types of sncRNAs by standard small RNA sequencing methods. To address this limitation, PANDORA-seq introduces a refined methodology. The procedure includes sequential enzymatic treatments of size-selected RNAs with T4PNK and AlkB, which effectively circumvent the challenges presented by the ligation-blocking termini and reverse transcription-blocking RNA modifications, followed by tailored small RNA library construction protocols and deep sequencing. The obtained datasets are analyzed with the SPORTS pipeline, which can comprehensively analyze various types of sncRNAs beyond the traditionally studied classes, to include those derived from various parental RNAs (e.g., from tRNA and rRNA), as well as output the locations on the parental RNA from which these sncRNAs are derived. The entire protocol takes  7 days, depending on the sample size and sequencing turnaround time. PANDORA-seq provides a transformative tool to further our understanding of the expanding small RNA universe and to explore the uncharted functions of sncRNAs.

Although high-throughput RNA sequencing (RNA-seq) has greatly advanced small non-coding RNA (sncRNA) discovery, the currently widely used complementary DNA library construction protocol generates biased sequencing results. This is partially due to RNA modifications that interfere with adapter ligation and reverse transcription processes, which prevent the detection of sncRNAs bearing these modifications. Here, we present PANDORA-seq (panoramic RNA display by overcoming RNA modification aborted sequencing), employing a combinatorial enzymatic treatment to remove key RNA modifications that block adapter ligation and reverse transcription. PANDORA-seq identified abundant modified sncRNAs-mostly transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs)-that were previously undetected, exhibiting tissue-specific expression across mouse brain, liver, spleen and sperm, as well as cell-specific expression across embryonic stem cells (ESCs) and HeLa cells. Using PANDORA-seq, we revealed unprecedented landscapes of microRNA, tsRNA and rsRNA dynamics during the generation of induced pluripotent stem cells. Importantly, tsRNAs and rsRNAs that are downregulated during somatic cell reprogramming impact cellular translation in ESCs, suggesting a role in lineage differentiation.

The development of life beyond planet Earth is a long-standing quest of the human race, but whether normal mammalian embryonic development can occur in space is still unclear. Here, we show unequivocally that preimplantation mouse embryos can develop in space, but the rate of blastocyst formation and blastocyst quality are compromised. Additionally, the cells in the embryo contain severe DNA damage, while the genome of the blastocysts developed in space is globally hypomethylated with a unique set of differentially methylated regions. The developmental defects, DNA damage and epigenetic abnormalities can be largely mimicked by the treatment with ground-based low-dose radiation. However, the exposure to simulated microgravity alone does not cause major disruptions of embryonic development, indicating that radiation is the main cause for the developmental defects. This work advances the understanding of embryonic development in space and reveals long-term extreme low-dose radiation as a hazardous factor for mammalian reproduction.

The discovery of RNAs (for example, messenger RNAs, non-coding RNAs) in sperm has opened the possibility that sperm may function by delivering additional paternal information aside from solely providing the DNA1. Increasing evidence now suggests that sperm small non-coding RNAs (sncRNAs) can mediate intergenerational transmission of paternally acquired phenotypes, including mental stress2,3 and metabolic disorders4,5,6. How sperm sncRNAs encode paternal information remains unclear, but the mechanism may involve RNA modifications. Here we show that deletion of a mouse tRNA methyltransferase, DNMT2, abolished sperm sncRNA-mediated transmission of high-fat-diet-induced metabolic disorders to offspring. Dnmt2 deletion prevented the elevation of RNA modifications (m5C, m2G) in sperm 30-40 nt RNA fractions that are induced by a high-fat diet. Also, Dnmt2 deletion altered the sperm small RNA expression profile, including levels of tRNA-derived small RNAs and rRNA-derived small RNAs, which might be essential in composing a sperm RNA ’coding signature’ that is needed for paternal epigenetic memory. Finally, we show that Dnmt2-mediated m5C contributes to the secondary structure and biological properties of sncRNAs, implicating sperm RNA modifications as an additional layer of paternal hereditary information.

Caffeine consumption has been widely used as a central nervous system stimulant. Epidemiological studies, however, have suggested that maternal caffeine exposure during pregnancy is associated with increased abnormalities, including decreased fertility, delayed conception, early spontaneous abortions, and low birth weight. The mechanisms underlying the negative outcomes of caffeine consumption, particularly during early pregnancy, remain unclear. In present study, we found that pregnant mice treated with moderate (5 mg/kg) or high (30 mg/kg) dosage of caffeine (intraperitoneally or orally) during preimplantation resulted in retention of early embryos in the oviduct, defective embryonic development, and impaired embryo implantation. Transferring normal blastocysts into the uteri of caffeine-treated pseudopregnant females also showed abnormal embryo implantation, thus indicating impaired uterine receptivity by caffeine administration. The remaining embryos that managed to implant after caffeine treatment also showed increased embryo resorption rate and abnormal development at mid-term stage, and decreased weight at birth. In addition to a dose-dependent effect, significant variations between individual mice under the same caffeine dosage were also observed, suggesting different sensitivities to caffeine, similar to that observed in human populations. Collectively, our data revealed that caffeine exposure during early pregnancy impaired oviductal embryo transport, embryonic development, and uterine receptivity, which are responsible for abnormal implantation and pregnancy loss. The study raises the concern of caffeine consumption during early stages of pregnancy.