Zygotic splicing activation (ZSA) is a crucial process of mRNA post-transcriptional regulation during maternal-to-zygotic transition, ensuring normal embryonic development. However, the key factors and mechanisms underlying ZSA regulation remain unclear. We observed that the nuclear speckle (NS), a key splicing region, was newly established at the 2-cell stage in mice and was consistent with the ZSA period. Moreover, NS and TAR DNA-binding protein-43 (TDP43) always exhibited a partially adjacent and mutually exclusive localization relationship. TDP43 performed its function through liquid-liquid phase separation. The condensation-deficient state of TDP43 was the active form involved in NS regulation in 2-cell embryos. Additionally, TDP43 could also interact with both transcribed RNAs associated with NS and directly with NS proteins. Maternal TDP43 deficiency led to the failure of NS assembly in 2-cell embryos, resulting in the inability to skip exons during transcript splicing. In contrast, ectopic expression of TDP43 in zygotes led to abnormal enlargement of the NS, resulting in excessive skipping of transcript exons. Both bidirectional ZSA disorders led to 2-cell arrest during early embryogenesis. ZSA defects caused by TDP43 deficiency also impaired cell totipotency-pluripotency conversion. In this study, we identified an NS upstream regulatory factor, TDP43, which helps maintain the balance of ZSA, providing a new perspective on the post-transcriptional regulation of early embryos.

The elongation and shortening of poly(A) tails are the most common types of post-transcriptional mRNA regulation in eukaryotic cells. PAN2-PAN3 and CCR4-NOT complexes are the main cytoplasmic enzymes that trim the poly(A) tails of mRNAs. However, the in vivo function of PAN2-PAN3 in mammals remains unclear. Here, we found that the germline-specific deletion of Pan2 causes male infertility due to a step-8/9 spermatogenic arrest, while meiotic prophase proceeds normally. Using PAIso-seq2 and mass spectrometry analyses, we define stage-specific remodeling of poly(A) tails that is disrupted in Pan2-null round spermatids (RS). Ribo-lite reveals a global reduction in translation efficiency in Pan2-deficient RS, correlating with proteomic changes and impairing pathways for spermatid differentiation. Endogenous IP-MS identifies PAN2 association with PABPC1 and initiation factors (EIF4E, EIF4A1, EIF5A), whose protein levels decline upon Pan2 loss, indicating compromised stability of key translational machinery. Together, these findings highlight the important physiological functions of PAN2 in spermiogenesis and expand our understanding of the post-transcriptional regulation of mRNAs in specific physiological processes.

Extrachromosomal circular DNA is an emerging regulatory element implicated in genomic stability and gene regulation, yet its role in preimplantation development remains elusive. Here, we report the widespread presence of extrachromosomal circular DNA in preimplantation embryos, characterized by homologous junction sequences and originating from genomic regions enriched for active histone marks and RNA Polymerase II occupancy. Functional perturbations demonstrate that RNA Polymerase II inhibition suppresses extrachromosomal circular DNA production, whereas disruption of the Fanconi anemia pathway elevates it, suggesting that transcription-replication conflicts affect its biogenesis. Notably, extrachromosomal circular DNA levels surge during major zygotic genome activation. Synthetic extrachromosomal circular DNAs carrying putative enhancers for the zygotic genome activation genes Mycn and Egfl7, and the developmental gene Emx1, significantly upregulate the expression of their respective genes upon transfection into fibroblasts and zygotes. Collectively, this study unveils the extrachromosomal circular DNA landscape in preimplantation embryos, elucidates a transcription-replication conflict mechanism underlying its generation, and establishes its regulatory potential during mammalian preimplantation development.

Gene products vary in mRNA transcript and protein isoforms. During the maternal-to-zygotic transition, massive mRNAs transcribed from either the maternal or the zygotic genome were produced, together with a combination of pre-mRNA processing progress and mRNA decay mechanism. Transcripts harbored by embryos differ from the ones contained by oocytes in an alternative exon inclusion/exclusion manner. The lengths and the compositions of polypyrimidine tracts (PPTs) are crucial in splice site definition. Exons downwards longer PPTs were preferentially included, and less pyrimidine-incorporated PPTs tended to be excluded from 2-cell embryos. The U2AF dimers, U2AF1 and U2AF2, are responsible for PPT recognition and 3' splice site determination. In oocytes, U2AF1 formed low density areas (cavities) and was positioned exclusively outside the nuclear speckles. In pre-implantation embryos, U2AF1 keeps in low abundance and disperses in the 2-cell nucleoplasm. Excessive U2AF1 expression leads to developmental arrest at the 2- to 4-cell stage, defective zygotic genome activation (ZGA), and aberrant zygotic splicing events. U2AF1 promotes exon exclusion in a PPT-dependent manner, modulates U2AF dimer's recognition of more-pyrimidine-containing PPTs, defines 3' splice sites, and selectively excludes the downstream exons. The composition of multiple gene variants, including Bclaf1, Ezh2, and several ZGA genes, is balanced by U2AF1 hypoexpression, which further assist in protein isoform maintenance, guarding proper protein positioning, and governing embryonic development.

In mammals, the precise degradation of maternal mRNAs is essential for oocyte maturation and early embryonic development, as it facilitates the "maternal-to-zygotic transition (MZT)" by eliminating maternal transcripts and enabling zygotic genome activation (ZGA). However, the physiological role of the poly(A)-specific nuclease 2 (PAN2), a deadenylase that initiates cascade degradation of long-tailed transcripts, remains unknown. Here, we generated oocyte-specific Pan2 conditional knockout (cKO) mice to investigate its role. We found that Pan2 cKO females exhibit severe female subfertility despite normal oocyte maturation and ovulation, with embryos arresting at the 2-cell stage. PAIso-seq2 and transcriptome sequencing reveal that PAN2 coordinates maternal mRNA deadenylation and decay. Mechanistically, PAN2 recognizes its substrates through a PAN3-PABPC1 bridging complex, and it preferentially targets transcripts whose poly(A) tails lack guanosine (G) but are enriched for uridine (U). PAN2 deficiency causes poly(A) tail dyshomeostasis, leading to global accumulation of maternal mRNAs, impaired zygotic genome activation, and abnormal protein accumulation in 2-cell embryos. Overexpression of these proteins phenocopies developmental defects. Notably, the PAN2-regulated transcriptome is largely non-overlapping with the LC3B-mediated degradation pathway, highlighting the unique and non-redundant role of PAN2 in maternal mRNA clearance. Our study establishes maternal PAN2 as a critical regulator of poly(A) tail homeostasis, ensuring timely maternal mRNA clearance and proper ZGA, highlighting the stage-specific and tail-composition-dependent functions of the deadenylase cascade during the maternal-to-zygotic transition. These findings offer new perspectives on post-transcriptional regulatory mechanisms in early mammalian embryogenesis.

Preimplantation embryogenesis requires precise synchronization of transcriptional activation, mRNA export and translation, and metabolic reprogramming to sustain developmental requirements. Nuclear cap-binding protein 1 (NCBP1), a conserved subunit of the cap-binding complex, has established roles in mRNA processing and export in somatic cells, but its potential functions in preimplantation embryogenesis remain undefined. The spatiotemporal expression dynamics of Ncbp1 were explored on multiple levels. After microinjecting interfering RNA at zygotic stage to knockdown Ncbp1, embryonic developmental competence was evaluated. Co-injection of small interfering RNA and in vitro transcribed Ncbp1 mRNA into the zygote was used to rescue the knockdown phenotype. Further, poly-adenylated RNA-fluorescence in situ hybridization, RNA sequencing, and quantitative proteomics were used to investigate the effects of Ncbp1 knockdown. In addition, oleic acid (OA) supplementation was used to rescue developmental abnormalities. NCBP1 exhibited dynamic spatiotemporal expression coinciding with nuclear-to-cytoplasmic translocation of protein from morula stage. Depletion of Ncbp1 caused morula arrest or fragmentation, accompanied by nuclear poly-adenylated RNA retention and down-regulation of lipid metabolic pathways, notably, stearoyl-CoA desaturase 1 (SCD1), a key enzyme generating monounsaturated OA. Exogenous OA supplementation partially rescued blastocyst formation, implicating NCBP1 in the regulation of SCD1-OA-mediated metabolic homeostasis during morula-to-blastocyst transition. This study illustrates NCBP1 as a mediator that regulates RNA export and lipid homeostasis during early mouse embryo development. Especially NCBP1 regulates the SCD1-OA metabolic pathways, ensuring metabolic flexibility essential for successful morula-to-blastocyst transition, thereby providing new insights into the molecular basis of embryonic developmental competence.

Programmed establishment of the zygote epitranscriptome orchestrates epitranscriptome fidelity and developmental potency acquisition during mammalian preimplantation embryogenesis. N4-acetylcytidine (ac4C) is a conserved RNA modification catalyzed by N-acetyltransferase 10 (NAT10), the currently established mammalian RNA ac4C writer, but its maternal function during the maternal-to-zygotic transition remains incompletely understood. In this study, we found that oocyte-specific Nat10 ablation from primary follicle stage causes female infertility with embryonic developmental arrest predominantly at the 2-cell stage. Notably, the maternal protein NAT10 is essential for ac4C deposition on zygotic genome activation (ZGA) transcripts and maternal mRNAs, and the loss of Nat10 can lead to abnormal accumulation of maternal transcripts and major ZGA inactivation. By interrogating ac4C LACE-seq, RNA-seq, and Ribo-lite data, we identify splicing-factor transcripts as prominent NAT10-associated ac4C targets whose mRNA abundance and ribosome occupancy are reduced after maternal Nat10 deletion. These molecular changes are accompanied by impaired nuclear speckle organization and widespread disruption of detectable alternative-splicing events during zygotic splicing activation. Together, our findings demonstrate that the RNA ac4C writer NAT10 is essential for the post-transcriptional regulation of mouse zygotic splicing activation and provide valuable view for further exploration of the epigenetic mechanism during maternal-to-zygotic transition.

Oocyte-specific isoforms play crucial roles in oocyte maturation, while current understanding of the oocyte transcriptome is mainly focused on gene level. Here, we utilize single-cell full-length isoform sequencing to detect entire transcripts in human and mouse oocytes. Isoform diversity during oocyte maturation is systematically profiled, including 7154 and 4875 putative novel human and mouse transcripts, respectively. More than half of novel isoforms are categorized as novel-not-in-catalog (NNC) and may serve specific functions in oocytes. For example, ARHGAP18 mainly encoded by novel isoforms colocalizes with microtubules, and targeted knockdown of novel isoforms disrupts oocyte maturation. Moreover, approximately 30% of NNC isoforms are derived from transposable elements, and their incorporation within transcripts could enhance isoform stability during oocyte maturation. Altogether, our findings represent a valuable resource showcasing the complexity and diversity of RNA isoforms in oocytes, as well as transposable element co-option for novel isoform generation and isoform stability enhancement.

Amycolatopsis sp. TNS106 produces ristomycin (ristocetin), a type III glycopeptide antibiotic (GPA) featuring extensive glycosyl modifications and potent antimicrobial activity against Gram-positive pathogens. Unlike the well-documented nonribosomal peptide synthetase-assembled peptide scaffold, the timing and specificity of its postassembly tailoring steps remain poorly understood. In this study, we generated a series of A. sp. TNS106 mutants and characterized accumulated derivatives to delineate the postaglycone tailoring steps for ristomycin maturation. In vitro biochemical reactions confirmed the function and timing of the RgtfB and RgtfC glycosyltransferases and MtfA carboxyl methyltransferase. By integrating our findings with prior studies, we propose a comprehensive model for the sequential glycosylation and C-terminal methylation processes governing ristomycin biosynthesis. Notably, we identified multiple ristomycin glycoforms that demonstrate improved antimicrobial activity compared to the parent molecule. Strikingly, removal of the ristosamine on β-hydroxytyrosine 6 (βht6) or the presence of rhamnose on 4-hydroxyphenylglycine 4 (Hpg4) markedly impaired its antibacterial activity, particularly against vancomycin-resistant Enterococcus. The engineered strains constructed here provide a versatile platform for generating novel ristomycin analogs through combinatorial biosynthesis or chemical synthesis, advancing the development of new-to-nature GPAs with improved potency.

The precise translational regulation of maternal messenger RNAs (mRNAs) drives mammalian oocyte maturation. However, the function and mechanism of posttranscriptional chemical modifications, especially the newly identified N4-acetylcytidine (ac4C) modification catalyzed by N-acetyltransferase 10 (NAT10), are unknown. In this study, we developed a low-input ac4C sequencing technology, ac4C LACE-seq, and mapped 8241 ac4C peaks at the whole-transcriptome level using 50 mouse oocytes at the germinal vesicle stage. Oocyte-specific Nat10 knockout wiped out ac4C signals in oocytes and caused severe defects in meiotic maturation and female infertility. Mechanically, Nat10 deletion led to a failure of ac4C deposition on mRNAs encoding key maternal factors, which regulate transcriptome stability and maternal-to-zygotic transition. Nat10-deleted oocytes showed decreased mRNA translation efficiency due to the direct inhibition of ac4C sites on specific transcripts during meiotic maturation. In summary, we developed a low-input, high-sensitivity mRNA ac4C profiling approach and highlighted the important physiological function of ac4C in the precise regulation of oocyte meiotic maturation by enhancing translation efficiency.

We previously reported that RNF148 was involved in the ubiquitination-mediated degradation of CHAC2. However, its molecular mechanism was not determined. In this study, we investigated the role and mechanism of RNF148 in the progression of colorectal cancer (CRC), especially in the process of ubiquitination-mediated degradation of CHAC2. Our results revealed that RNF148 was upregulated in most CRC tissues, and its expression significantly correlated with the 3-year overall survival rate and most clinicopathological parameters of CRC patients. Furthermore, RNF148 served as an independent prognostic biomarker of CRC and promoted CRC cell proliferation and migration while inhibiting cell apoptosis and sensitivity to 5-FU. Mechanistically, RNF148 used its protease-associated domain to bind to the CHAC domain of CHAC2 and target it for degradation. In addition, we identified two phosphorylation and three ubiquitination residues of CHAC2 and identified Y118 and K102 as the critical phosphorylation and ubiquitination residues, respectively. We also identified CHAC2’s and RNF148’s interacting proteins and discovered their potential interaction network. In conclusion, our current study unveiled the role of RNF148 in CRC and the mechanism of RNF148 in the ubiquitination-mediated degradation of CHAC2, which shed light on providing potential prognostic biomarkers and molecular targets for CRC patients.

Mammalian oocyte maturation is driven by the strict translational regulation of maternal mRNAs stored in the cytoplasm. However, the function and mechanism of post-transcriptional chemical modifications, especially the newly identified N4-acetylcytidine (ac4C) modification catalyzed by N-acetyltransferase 10 (NAT10), are unknown. In this study, we developed a low-input ac4C sequencing technology, ac4C LACE-seq, and mapped 8241 ac4C peaks at the whole-transcriptome level using 50 mouse oocytes at the germinal vesicle stage. We profiled the mRNA landscapes of NAT10-interactions and ac4C modifications. The NAT10-interacted and ac4C-modified transcripts are associated with high translation efficiency in oocytes. Oocyte-specific Nat10 knockout wiped out ac4C signals in oocytes and caused severe defects in meiotic maturation and female infertility. ac4C LACE-seq results indicated that Nat10 deletion led to a failure of ac4C deposition on mRNAs encoding key maternal factors, such as MSY2, ZAR1, BTG4, and cyclin B1, which regulate transcriptome stability and maternal-to-zygotic transition. Nat10 -deleted oocytes showed decreased mRNA translation efficiency during meiotic maturation, partially due to the direct inhibition of ac4C sites on specific transcripts. In summary, we developed a low-input, high-sensitivity mRNA ac4C profiling approach and highlighted the important physiological function of ac4C in the precise regulation of oocyte meiotic maturation by enhancing translation efficiency.### Competing Interest StatementThe authors have declared no competing interest.

Cancer occurrence and development are closely related to increased lipid production and glucose consumption. Lipids are the basic component of the cell membrane and play a significant role in cancer cell processes such as cell-to-cell recognition, signal transduction, and energy supply, which are vital for cancer cell rapid proliferation, invasion, and metastasis. Sterol regulatory element-binding transcription factor 1 (SREBP1) is a key transcription factor regulating the expression of genes related to cholesterol biosynthesis, lipid homeostasis, and fatty acid synthesis. In addition, SREBP1 and its upstream or downstream target genes are implicated in various metabolic diseases, particularly cancer. However, no review of SREBP1 in cancer biology has yet been published. Herein, we summarized the roles and mechanisms of SREBP1 biological processes in cancer cells, including SREBP1 modification, lipid metabolism and reprogramming, glucose and mitochondrial metabolism, immunity, and tumor microenvironment, epithelial-mesenchymal transition, cell cycle, apoptosis, and ferroptosis. Additionally, we discussed the potential role of SREBP1 in cancer prognosis, drug response such as drug sensitivity to chemotherapy and radiotherapy, and the potential drugs targeting SREBP1 and its corresponding pathway, elucidating the potential clinical application based on SREBP1 and its corresponding signal pathway.

Proper ovarian follicle development,which is required for the maintenance of female fertility,is critical for the production of mature oocytes[1,2].Meanwhile,the correct establishment of the epitranscriptome in oocytes is essential for precise gene repression and the acquisition of developmental competence[1-5].

Massive numbers of modified bases in mRNAs sculpt the epitranscriptome and play vital roles in RNA metabolism. The only known acetylated RNA modification, N-4-acetylcytidine (ac4C), is highly conserved across cell types and among species. Although the GCN5-related acetyltransferase 10 (NAT10) functions as an ac4C writer, the mechanism underlying the acetylation process is largely unknown. In this study, the NAT10/PCBP/TDP43 complex mediated mRNA ac4C formation in mammalian cells is identified. RNA-binding proteins (RBPs) are identified, affiliated with two different families, poly(rC)-binding protein 1/2 (PCBP1/2) and TAR DNA binding protein 43 (TDP43), as NAT10 adaptors for mRNA tethering and substrate selection. Knockdown of the adaptors resulted in decreased mRNA acetylation abundance in HEK293T cells and ablated cytidine-rich ac4C motifs. The adaptors also affect the ac4C sites by recruiting NAT10 to their binding sequences. The presence of the NAT10/PCBP/TDP43 complex in mouse testes highlights its potential physiological functions in vivo. These findings reveal the composition of the mRNA ac4C writer complex in mammalian cells and expand the knowledge of mRNA acetylation and ac4C site preferences.

Polyene macrolactams are a special group of natural products with great diversity, unique structural features, and a wide range of biological activities. Herein, a cryptic gene cluster for the biosynthesis of putative macrolactams was disclosed from a sponge-associated bacterium, Streptomyces sp. DSS69, by genome mining. Cloning and heterologous expression of the whole biosynthetic gene cluster led to the discovery of weddellamycin, a polyene macrolactam bearing a 23/5/6 ring skeleton. A negative regulator, WdlO, and two positive regulators, WdlA and WdlB, involved in the regulation of weddellamycin production were unraveled. The fermentation titer of weddellamycin was significantly improved by overexpression of wdlA and wdlB and deletion of wdlO. Notably, weddellamycin showed remarkable antibacterial activity against various Gram-positive bacteria including MRSA, with MIC values of 0.10–0.83 μg/mL, and antifungal activity against Candida albicans, with an MIC value of 3.33 μg/mL. Weddellamycin also displayed cytotoxicity against several cancer cell lines, with IC50 values ranging from 2.07 to 11.50 µM.

Revascularization treatment is a critical measure for tissue engineering therapies like spinal cord repair. As multipotent stem cells, mesenchymal stem cells (MSCs) have proven to regulate the lesion microenvironment through feedback to the microenvironment signals. The angiogenic capacities of MSCs have been reported to be facilitated by vein endothelial cells in the niche. As emerging evidence demonstrated the roles of exosomes in cell-cell and cell-microenvironment communications, to cope with the ischemia complication for treatment of traumatic spinal cord injury, the study extracts the microenvironment factors to stimulate angiogenic MSCs through using exosomes (EX) derived from hypoxic preconditioned (HPC) human umbilical vein endothelial cells (HUVEC). The HPC treatment with a hypoxia time segment of only 15 min efficiently enhanced the function of EX in facilitating MSCs angiogenesis activity. MSCs stimulated by HPC-EX showed significant tube formation within 2 h, and the in vivo transplantation of the stimulated MSCs elicited effective nerve tissue repair after rat spinal cord transection, which could be attributed to the pro-angiogenic and anti-inflammatory impacts of the MSCs. Through the simulation of MSCs using HPC-tailored HUVEC exosomes, the results proposed an efficient angiogenic nerve tissue repair strategy for spinal cord injury treatment and could provide inspiration for therapies based on stem cells and exosomes.

INTRODUCTION:The R-loop is a naturally formed three-strand nucleic acid structure that recently has been reported to participate in multiple biological processes and helped answer some previously unexplained scientific questions. Meiosis process involves multiple chromatin-related events such as DNA double-stranded breaks (DSB) formation, repairing and transcriptional dynamics. OBJECTIVES:Explore the regulatory roles and physiological functions of R-loops in the mammalian meiosis process. METHODS:In our study, using genome-wide S9.6 CUT & Tag seq, we first mapped the genomic distribution and dynamic changes of R-loop during the meiotic process in mice, from spermatogonia to secondary spermatocytes. And we further explore the role of R-loop in physiological conditions by constructing conditional knockout mice of Rnaseh1, which deleted the R-loop endonuclease before meiosis entry. RESULTS:R-loop predominantly distributes at promoter-related regions and varies across different meiotic stages. By joint analysis with the corresponding transcriptome, we found that the R-loop was closely related to transcription during the meiotic process. The high frequency of promoter-related R-loop in meiotic cells is usually accompanied by high transcription activity, and we further verified this in the leptotene/zygotene to the pachytene transition process. Moreover, the lack of RNase H1 caused sterility in male mice with R-loop accumulation and abnormal DSB repair in spermatocytes. Further analysis showed that abnormal R-loop accumulation in the leptotene/zygotene stages influenced transcriptional regulation in the pachytene stage. CONCLUSION:The mutual regulation of the R-loop and transcription plays an essential role in spermatogenesis. And R-loop is also important for the normal repair process of DSB during meiosis.