Junchao Shi

Sperm tsRNAs contribute to intergenerational inheritance of an acquired metabolic disorder

Offspring affected by sperm small RNAs Paternal dietary conditions in mammals influence the metabolic phenotypes of offspring. Although prior work suggests the involvement of epigenetic pathways, the mechanisms remains unclear. Two studies now show that altered paternal diet affects the level of small RNAs in mouse sperm. Chen et al. injected sperm transfer RNA (tRNA) fragments from males that had been kept on a high-fat diet into normal oocytes. The progeny displayed metabolic disorders and concomitant alteration of genes in metabolic pathways. Sharma et al. observed the biogenesis and function of small tRNA-derived fragments during sperm maturation. Further understanding of the mechanisms by which progeny are affected by parental exposure may affect human diseases such as diet-induced metabolic disorders. Science, this issue p. 397, p. 391 Fragments of transfer RNA in sperm serve as paternal epigenetic factors linked to diet-induced metabolic problems in their offspring. Increasing evidence indicates that metabolic disorders in offspring can result from the father’s diet, but the mechanism remains unclear. In a paternal mouse model given a high-fat diet (HFD), we showed that a subset of sperm transfer RNA–derived small RNAs (tsRNAs), mainly from 5′ transfer RNA halves and ranging in size from 30 to 34 nucleotides, exhibited changes in expression profiles and RNA modifications. Injection of sperm tsRNA fractions from HFD males into normal zygotes generated metabolic disorders in the F1 offspring and altered gene expression of metabolic pathways in early embryos and islets of F1 offspring, which was unrelated to DNA methylation at CpG-enriched regions. Hence, sperm tsRNAs represent a paternal epigenetic factor that may mediate intergenerational inheritance of diet-induced metabolic disorders.

A novel class of tRNA-derived small RNAs extremely enriched in mature mouse sperm

Author(s): Peng, Hongying; Shi, Junchao; Zhang, Ying; Zhang, He; Liao, Shangying; Li, Wei; Lei, Li; Han, Chunsheng; Ning, Lina; Cao, Yujing; Zhou, Qi; Chen, Qi; Duan, Enkui

Dynamic transcriptional symmetry-breaking in pre-implantation mammalian embryo development revealed by single-cell RNA-seq.

During mammalian pre-implantation embryo development, when the first asymmetry emerges and how it develops to direct distinct cell fates remain longstanding questions. Here, by analyzing single-blastomere transcriptome data from mouse and human pre-implantation embryos, we revealed that the initial blastomere-to-blastomere biases emerge as early as the first embryonic cleavage division, following a binomial distribution pattern. The subsequent zygotic transcriptional activation further elevated overall blastomere-to-blastomere biases during the two- to 16-cell embryo stages. The trends of transcriptional asymmetry fell into two distinct patterns: for some genes, the extent of asymmetry was minimized between blastomeres (monostable pattern), whereas other genes, including those known to be lineage specifiers, showed ever-increasing asymmetry between blastomeres (bistable pattern), supposedly controlled by negative or positive feedbacks. Moreover, our analysis supports a scenario in which opposing lineage specifiers within an early blastomere constantly compete with each other based on their relative ratio, forming an inclined ‘lineage strength’ that pushes the blastomere onto a predisposed, yet flexible, lineage track before morphological distinction. Highlighted article: Partitioning errors in the zygote, modulated by embryonic transcription, give rise to the unequal distribution of lineage specifiers between blastomeres that subsequently drives lineage specification.

Uterine Rbpj is required for embryonic-uterine orientation and decidual remodeling via Notch pathway-independent and -dependent mechanisms.

Coordinated uterine-embryonic axis formation and decidual remodeling are hallmarks of mammalian post-implantation embryo development. Embryonic-uterine orientation is determined at initial implantation and synchronized with decidual development. However, the molecular mechanisms controlling these events remain elusive despite its discovery a long time ago. In the present study, we found that uterine-specific deletion of Rbpj, the nuclear transducer of Notch signaling, resulted in abnormal embryonic-uterine orientation and decidual patterning at post-implantation stages, leading to substantial embryo loss. We further revealed that prior to embryo attachment, Rbpj confers on-time uterine lumen shape transformation via physically interacting with uterine estrogen receptor (ERα) in a Notch pathway-independent manner, which is essential for the initial establishment of embryo orientation in alignment with uterine axis. While at post-implantation stages, Rbpj directly regulates the expression of uterine matrix metalloproteinase in a Notch pathway-dependent manner, which is required for normal post-implantation decidual remodeling. These results demonstrate that uterine Rbpj is essential for normal embryo development via instructing the initial embryonic-uterine orientation and ensuring normal decidual patterning in a stage-specific manner. Our data also substantiate the concept that normal mammalian embryonic-uterine orientation requires proper guidance from developmentally controlled uterine signaling.

Identification and characterization of an ancient class of small RNAs enriched in serum associating with active infection

Author(s): Zhang, Yunfang; Zhang, Ying; Shi, Junchao; Zhang, He; Cao, Zhonghong; Gao, Xuan; Ren, Wanhua; Ning, Yunna; Ning, Lina; Cao, Yujing; Chen, Yongchang; Ji, Weizhi; Chen, Zi-Jiang; Chen, Qi; Duan, Enkui

Dnmt2 mediates intergenerational transmission of paternally acquired metabolic disorders through sperm small non-coding RNAs

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 disorders4–6. 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.Zhang et al. report that tRNA methyltransferase Dnmt2 is required for sperm small-non-coding-RNA-mediated transmission of paternal metabolic disorders to the offspring.

Hormonal regulation of ovarian bursa fluid in mice and involvement of aquaporins.

In rodent species, the ovary and the end of oviduct are encapsulated by a thin membrane called ovarian bursa. The biological functions of ovarian bursa remain unexplored despite its structural arrangement in facilitating oocytes transport into oviduct. In the present study, we observed a rapid fluid accumulation and reabsorption within the ovarian bursa after ovarian stimulation (PMSG-primed hCG injection), suggesting that the ovarian bursa might play an active role in regulating local fluid homeostasis around the timing of ovulation. We hypothesized that the aquaporin proteins, which are specialized channels for water transport, might be involved in this process. By screening the expression of aquaporin family members (Aqp1-9) in the ovarian tissue and isolated ovarian bursa (0, 1, 2 and 5 h after hCG injection), we found that AQP2 and AQP5 mRNA showed dynamic changes after hCG treatment, showing upregulation at 1–2 h followed by gradually decrease at 5 h, which is closely related with the intra-bursa fluid dynamics. Further immunofluorescence examinations of AQP2 and AQP5 in the ovarian bursa revealed that AQP2 is specifically localized in the outer layer (peritoneal side) while AQP5 localized in the inner layer (ovarian side) of the bursa, such cell type specific and spatial-temporal expressions of AQP2 and 5 support our hypothesis that they might be involved in efficient water transport through ovarian bursa under ovulation related hormonal regulation. The physiological significance of aquaporin-mediated water transport in the context of ovarian bursa still awaits further clarification.

BTG4 is a key regulator for maternal mRNA clearance during mouse early embryogenesis

Dear Editor, The maternal to zygotic transition (MZT) is a crucial process in the early development of almost all animals, during which maternal mRNAs are degraded and the zygotic genome is activated (Li et al., 2013). How maternal mRNAs are degraded is one of the long-standing questions in the field of developmental and reproductive biology. Recently, high-throughput sequencing and genetic studies have determined that the elimination of maternal mRNAs is accomplished by two modes: the first mode is dependent on maternally encoded transcripts, while the second mode relies on zygotic transcription (Yartseva and Giraldez, 2015). The first mode is well characterized in Drosophila, in which the maternally encoded RNA-binding proteins SMAUG and PUMILIO are important mediators of maternal mRNA clearance (Semotok et al., 2005; Gerber et al., 2006). The second mode is exemplified by zygotically transcribed miR-430 in zebrafish that directly targets and triggers the deadenylation and subsequent clearance of maternal mRNAs (Giraldez et al., 2006). Despite these encouraging findings in model animals of lower species, the mechanisms governing the selective maternal mRNA clearance are still largely unclear in mammals. Deadenylation is the first and often ratelimiting step accounting for mRNA turnover (Garneau et al., 2007). The CCR4–NOT complex plays predominant roles in mRNA deadenylation, whereby it is involved in the SMAUGand PUMILIO-mediated maternal mRNA clearance in Drosophila (Semotok et al., 2005; Gerber et al., 2006). Previous studies have shown that the anti-proliferative Tob/BTG protein family members are important players in CCR4– NOT-mediated mRNA deadenylation and subsequent degradation (Winkler, 2010). To investigate the potential roles of Tob/ BTG proteins during mouse MZT, we first systematically analyzed the expression patterns of all Tob/BTG members (Tob1, Tob2, and Btg1–4) during this process by using RNA-seq data sets that are publicly available. Strikingly, among all the six members, Btg4, the main functional domain of which is highly conserved among vertebrates, showed an absolutely dominant and specific expression pattern during the MZT (Supplementary Figures S1 and S2A). Next, we examined the spatio-temporal expression pattern of Btg4 by quantitative real-time PCR (qRT-PCR) and western blot. Btg4 was exclusively present in ovaries and testes (Supplementary Figure S2B). In oocytes and preimplantation embryos, Btg4 mRNA was highly expressed in germinal vesicle stage (GV) oocytes and gradually perished during mouse preimplantation development, decreasing by ~90% by the 2cell stage (Figure 1A). At the protein level, however, the translation of Btg4 was largely initiated in metaphase II (MII) oocytes and was peaked at the 1-cell (1C) stage (Figure 1B, Supplementary Figure S2C), depending on the cytoplasmic polyadenylation elements and polyadenylation hexanucleotide AAUAAA sequence in the 3′ UTR (Supplementary Figure S3). To investigate the physiological role of Btg4, we depleted Btg4 using the CRISPR/Cas9 system that targets the first exon, and a targeted frame-shift mutant with a 116-bp deletion (Btg4) was successfully obtained (Supplementary Figure S4A and B). qRT-PCR and western blot showed that Btg4 was efficiently disrupted at both RNA and protein levels (Supplementary Figure S4C and D). Disruption of Btg4 had no effect on mouse viability and the fertility of male mice, but led to the infertility of female mice (Figure 1C). Histological analysis of paraffin sections from wild-type (WT) and Btg4 ovaries and superovulation experiments demonstrated that the oogenesis and fertilization of Btg4 oocytes were grossly normal (Supplementary Figure S5), but the development of embryos from Btg4 female mice was arrested at 1–2-cell stage (Figure 1D), indicating essential roles of Btg4 during the MZT of mouse early embryogenesis. We further performed transcriptome analysis on WT and Btg4 GV oocytes, MII oocytes, and 1C embryos (Supplementary Tables S1–S3). Although the transcriptome profile of Btg4 GV oocytes was comparable to that of WT, >46% (6401/13770) and 20% (2898/ 14058) mRNAs were upregulated in Btg4 MII oocytes and the resulting 1C embryos, respectively (Figure 1E). These findings were consistent with the temporal protein expression pattern and thus the functions of Btg4 during mouse preimplantation development. Moreover, all the 12 genes, generated by overlapping the top 50 significantly upregulated genes in MII oocytes and 1C embryos, could be well validated by qRT-PCR, except for one gene with a very low expression level (Figure 1F, Supplementary Figure S6). To test whether the aberrant upregulation of maternal transcripts is caused by the inefficient deadenylation of maternal mRNAs, poly(A) tail length (PAT) assay (Supplementary Figure S7A) was performed with several representative transcripts that were abundant in GV oocytes but significantly reduced in MII oocytes (Ma et al., 2015). The results showed that these transcripts in Btg4 MII oocytes and the resulting 1C embryos failed to be deadenylated and were thus resistant to degradation (Figure 1G and Supplementary Figure S7B). As the CCR4–NOT complex

Potential Diagnostic Power of Blood Circular RNA Expression in Active Pulmonary Tuberculosis

Background Circular RNAs (circRNAs) are a class of novel RNAs with important biological functions, and aberrant expression of circRNAs has been implicated in human diseases. However, the feasibility of using blood circRNAs as disease biomarkers is largely unknown. Methods We explored the potential of using human peripheral blood mononuclear cell (PBMC) circRNAs as marker molecules to diagnose active pulmonary tuberculosis (TB). Findings First, we demonstrated that circRNAs are widely expressed in human PBMCs and that many are abundant enough to be detected. Second, we found that the magnitude of PBMC circRNAs in TB patients was higher than that in the paired healthy controls. Compared with host linear transcripts, the circRNAs within several pathways are disproportionately upregulated in active TB patients, including “Cytokine-cytokine receptor interaction”, “Chemokine signaling pathway”, “Neurotrophin signaling pathway”, and “Bacterial invasion of epithelial cells”. Based on the differentially expressed circRNAs within these pathways, we developed a PBMC circRNA-based molecular signature differentiating active TB patients from healthy controls. We validated the classification power of the PBMC circRNA signature in an independent cohort with the area under the receiver operating characteristic curve (AUC) at 0.946. Interpretation Our results suggest that PBMC circRNAs are potentially reliable marker molecules in TB diagnosis.

Senescence of human skin-derived precursors regulated by Akt-FOXO3-p27KIP1/p15INK4b signaling

Multipotent skin-derived precursors (SKPs) are dermal stem cells with the capacity to reconstitute the dermis and other tissues, such as muscles and the nervous system. Thus, the easily available human SKPs (hSKPs) hold great promises in regenerative medicine. However, long-term expansion is difficult for hSKPs in vitro. We previously demonstrated that hSKPs senesced quickly under routine culture conditions. To identify the underlying mechanisms so as to find an effective way to expand hSKPs, time-dependent microarray analysis of gene expression in hSKPs during in vitro culture was performed. We found that the senescence of hSKPs had a unique gene expression pattern that differs from reported typical senescence. Subsequent investigation ruled out the role of DNA damage and classical p53 and p16INK4a signaling in hSKP senescence. Examination of cyclin-dependent kinase inhibitors revealed the involvement of p15INK4b and p27KIP1. Further exploration about upstream signals indicated the contribution of Akt hypo-activity and FOXO3 to hSKP senescence. Forced activation of Akt and knockdown of FOXO3, p15INK4b and p27KIP1 effectively inhibited hSKP senescence and promoted hSKP proliferation. The unique senescent phenotype of human dermal stem cells and the role of Akt-FOXO3-p27KIP1/p15INK4b signaling in regulating hSKP senescence provide novel insights into the senescence and self-renewal regulation of adult stem cells. The present study also points out a way to propagate hSKPs in vitro so as to fulfill their promises in regenerative medicine.