Maternal factor PABPN1L is essential for maternal mRNA degradation during maternal-to-zygotic transition

AbstractInfertility affects 10% - 15% of families worldwide. However, the pathogenesis of female infertility caused by abnormal early embryonic development is not clear. We constructed a mouse model (Pabpn1l -/-) simulating the splicing abnormality of human PABPN1L and found that the female was sterile and the male was fertile. The Pabpn1l -/- oocytes can be produced, ovulated and fertilized normally, but cannot develop beyond the 2-cell stage. Using RNA-Seq, we found a large-scale upregulation of RNA in Pabpn1l -/- MII oocytes. Of the 2401 transcripts upregulated in Pabpn1l-/- MII oocytes, 1523 transcripts (63.4%) were also upregulated in Btg4 -/- MII oocytes, while only 53 transcripts (2.2%) were upregulated in Ythdf2 -/- MII oocytes. We documented that transcripts in zygotes derived from Pabpn1l -/- oocytes have a longer poly(A) tail than the control group, a phenomenon similar to that in Btg4-/- mice. Surprisingly, the poly(A) tail of these mRNAs was significantly shorter in the Pabpn1l -/- MII oocytes than in the Pabpn1l +/+. These results suggest that PABPN1L is involved in BTG4-mediated maternal mRNA degradation, and may antagonize poly(A) tail shortening in oocytes independently of its involvement in maternal mRNA degradation. Thus, PABPN1L variants could be a genetic marker of female infertility.

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PABPN1L assemble into “ring-like” aggregates in the cytoplasm of MII oocytes and is associated with female infertility†

Biology of Reproduction ◽

10.1093/biolre/ioab203 ◽

2021 ◽

Author(s):

Ying Wang ◽

Tianhao Feng ◽

Mingcong Zhu ◽

Xiaodan Shi ◽

Zerui Wang ◽

...

Keyword(s):

Embryonic Development ◽

Mouse Model ◽

Genetic Marker ◽

Large Scale ◽

Scale Up ◽

Mrna Degradation ◽

Female Infertility ◽

Rna Seq ◽

Maternal Mrna

Abstract Infertility affects 10% - 15% of families worldwide. However, the pathogenesis of female infertility caused by abnormal early embryonic development is not clear. A resent study showed that PABPN1L recruited BTG4 to mRNA 3′-poly(A) tails and was essential for maternal mRNA degradation. Here, we generated an PABPN1L-antibody and found “ring-like” PABPN1L aggregates in the cytoplasm of MII oocytes. PABPN1L-EGFP proteins spontaneously formed“ring-like” aggregates in vitro. This phenomenon is similar with CCR4–NOT catalytic subunit, CNOT7, when it starts deadenylation process in vitro. We constructed two mouse model (Pabpn1l −/− and Pabpn1l tm1a/tm1a) simulating the intron1-exon2 abnormality of human PABPN1L and found that the female was sterile and the male was fertile. Using RNA-Seq, we observed a large-scale up-regulation of RNA in zygotes derived from Pabpn1l−/− MII oocytes. We found that 9222 genes were up-regulated instead of being degraded in the Pabpn1l-♀/+♂zygote. Both the Btg4 and Cnot61 genes are necessary for the deadenylation process and Pabpn1l −/− resembled both the Btg4 and Cnot6l knockouts, where 71.2% genes stabilized in the Btg4-♀/+♂ zygote and 84.2% genes stabilized in the Cnot6l-♀/+♂zygote were also stabilized in Pabpn1l-♀/+♂ zygote. BTG4/CNOT7/CNOT6L was partially co-located with PABPN1L in MII oocytes. The above results suggest that PABPN1L is widely associated with CCR4–NOT-mediated maternal mRNA degradation and PABPN1L variants on intron1-exon2 could be a genetic marker of female infertility. Summary sentence. “Ring-like” PABPN1L aggregates was found in the cytoplasm of MII oocytes and in vitro; intron1-exon2 abnormality of Pabpn1l leads female sterile in mice.

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Minor zygotic gene activation is essential for mouse preimplantation development

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1804309115 ◽

2018 ◽

Vol 115 (29) ◽

pp. E6780-E6788 ◽

Cited By ~ 31

Author(s):

Ken-ichiro Abe ◽

Satoshi Funaya ◽

Dai Tsukioka ◽

Machika Kawamura ◽

Yutaka Suzuki ◽

...

Keyword(s):

Dna Replication ◽

Gene Activation ◽

Marked Change ◽

Cell Stage ◽

Maternal Mrna ◽

Maternal To Zygotic Transition ◽

Before And After ◽

H3k4me3 Mark ◽

Zygotic Gene ◽

Zygotic Gene Activation

In mice, transcription initiates at the mid-one-cell stage and transcriptional activity dramatically increases during the two-cell stage, a process called zygotic gene activation (ZGA). Associated with ZGA is a marked change in the pattern of gene expression that occurs after the second round of DNA replication. To distinguish ZGA before and after the second-round DNA replication, the former and latter are called minor and major ZGA, respectively. Although major ZGA are required for development beyond the two-cell stage, the function of minor ZGA is not well understood. Transiently inhibiting minor ZGA with 5, 6-dichloro-1-β-d-ribofuranosyl-benzimidazole (DRB) resulted in the majority of embryos arresting at the two-cell stage and retention of the H3K4me3 mark that normally decreases. After release from DRB, at which time major ZGA normally occurred, transcription initiated with characteristics of minor ZGA but not major ZGA, although degradation of maternal mRNA normally occurred. Thus, ZGA occurs sequentially starting with minor ZGA that is critical for the maternal-to-zygotic transition.

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Comparative RNA-Seq Transcriptome Analysis on Pulmonary Inflammation in a Mouse Model of Asthma–COPD Overlap Syndrome

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.628957 ◽

2021 ◽

Vol 9 ◽

Author(s):

Pei Ma ◽

Shuyi Li ◽

Hui Yang ◽

Jiqiao Yuan ◽

Ziqian Zhang ◽

...

Keyword(s):

Mouse Model ◽

Molecular Mechanisms ◽

Pulmonary Inflammation ◽

Clinical Syndrome ◽

Overlap Syndrome ◽

Mouse Lung ◽

Control Group ◽

Chronic Obstructive ◽

Rna Seq ◽

Immune System Activation

Asthma–chronic obstructive pulmonary disease (COPD) overlap (ACO) is a severe clinical syndrome characterized to describe patients with both asthma and COPD clinical characteristics, which has posed a serious threat to patients’ quality of life and life safety. However, there are many difficulties and uncertainties in its diagnosis and treatment in clinic; especially, its animal model has not been fully and thoroughly established, and the evaluation of therapeutic drugs is still in its infancy. Here, we used ovalbumin (OVA), lipopolysaccharide (LPS), and smoke costimulation to establish an ACO mouse model and then used RNA-seq technology to detect gene expression in mouse lung tissue. The results showed that ACO mice showed an overlap syndrome of asthma and COPD in lung histological changes and the levels of inflammatory cytokines in bronchoalveolar lavage fluid. The RNA-seq analysis results showed that 6,324 differentially expressed genes (DEGs) were screened between the ACO group and the control group, of which 2,717 (42.7%) were downregulated, and 3,607 (57.3%) were upregulated. Metascape analysis results showed that in the ACO model we established, due to the damage of the respiratory system, the accumulated diseased tissue involves lung, spleen, blood, bone marrow, thymus, etc. It has certain characteristics of pneumonia, pulmonary fibrosis, and chronic obstructive airway disease, lung tumors, rheumatoid arthritis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis showed that DEGs were enriched in inflammation, immune system activation and imbalance, cell proliferation, and adhesion migration, and the upstream signaling pathways of inflammation were mainly affected by HLA-DRA, SYK, CTLA4, VAV1, NRAS, and JAK3. In short, our research established a mouse model that can better simulate the clinicopathological characteristics of ACO and suggested the foundations in elucidating the molecular mechanisms for pulmonary inflammation and fibrosis in ACO. This work may help further research and contribute substantially to prevention and clinical treatment of ACO in the future.

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YBX1 Mediates Alternative Splicing and Maternal mRNA Decay During Pre-Implantation Development

10.21203/rs.3.rs-900476/v1 ◽

2021 ◽

Author(s):

Mingtian Deng ◽

Baobao Chen ◽

Zifei Liu ◽

Yongjie Wan ◽

Dongxu Li ◽

...

Keyword(s):

Alternative Splicing ◽

Transcriptional Activity ◽

Mrna Decay ◽

Molecular Mechanisms ◽

Rna Stability ◽

Differentially Expressed ◽

Rna Seq ◽

Cell Stage ◽

Aberrant Expression ◽

Maternal Mrna

Abstract Background: In mammals, maternal gene products decay and zygotic genome activation (ZGA) during maternal to zygotic transition (MZT) is critical for pre-implantation. Y-box binding protein YBX1 plays vital roles in RNA stabilization and transcriptional regulation, but its roles in pre-implantation development remain to be elucidated. The objective of this study is to investigate the role and the molecular mechanisms of YBX1 during MZT.Methods: RNA-seq datasets in mice, human, bovine, and goat embryos were re-analyzed. YBX1 was knocked down by siRNA microinjection. The 8-cell stage embryos were collected for RNA-seq. The differentially expressed genes and alternative splicing (AS) events were identified using DESeq2 and rMATs, respectively. GO/KEGG/GSEA enrichment analysis was performed using clusterProfiler and enrichplot. Furthermore, 5-EU staining was performed to confirm the effect of YBX1 knockdown on transcriptional activity.Results: The expression of YBX1 was increased during MZT in goat, bovine, human, and mice. By microinjection of siRNA against YBX1, we successfully knocked down YBX1, and the embryo development was impaired in YBX1 knockdown embryos. Using RNA-seq, we identified 1623 up-regulated and 3531 down-regulated genes in the 8-cell stage YBX1 knockdown embryos. Of note, the down-regulated genes were enriched in regulation of RNA/mRNA stability and spliceosome, suggesting that YBX1 might medicate RNA stability and AS. To this end, we identified 3284 differential AS events and 1322 differentially expressed maternal mRNAs at the 8-cell stage YBX1 knockdown embryos. Meanwhile, the splicing factors and mRNA decay related showed aberrant expression. Moreover, the transcriptional activity during ZGA in goat and mice was compromised when YBX1 was knocked down.Conclusion: Our results identify that YBX1 serves an important role in maternal mRNA decay, alternative splicing, and the transcriptional activity required for early embryogenesis, which will broaden the current understanding of YBX1 functions during the stochastic reprogramming events.

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Transcriptome Sequencing Reveals Gene Expression Differences in the Response of Malignant Melanomas Cells to Hypoxia

10.21203/rs.3.rs-589732/v1 ◽

2021 ◽

Author(s):

Haiting Xu ◽

Huazhen Liu ◽

Zi Li ◽

Qin Xu ◽

Nan Lin ◽

...

Keyword(s):

Tumor Progression ◽

Luciferase Assay ◽

Control Group ◽

Rna Seq ◽

Positive Effects ◽

Cell Hypoxia ◽

Group A ◽

Hypoxic Group ◽

Lower Expression

Abstract BackgroundMalignant melanoma (MM) is the most deadly type of skin cancer, with 5-year survival rate of less than 16%. HIF-1α overexpression is associated with poor prognosis in many cancers including MM. Hence, we characterized differentially expressed genes (DEGs) in the response of MM cells to normal and hypoxia. MethodsWe first successfully constructed cell hypoxia model and then performed RNA-seq to explore the changes of gene transcription in MM cells during hypoxia. The highest expression of the six genes was detected using qRT-PCR and western blot assays. We explored the binding sites between BIRC7 promoter and HIF-1α by dual-luciferase assay. Cellular function assays were used to observe the role of BIRC7 in the effect of hypoxia on tumor progression. ResultsWe found that compared with the transcriptome data of the control group, a total of 2601 DEGs were identified in the hypoxic group. There were 1517 genes with significantly higher expression and 1084 genes with lower expression in the hypoxic group. Among them, OSCAR, BIRC7, HBA1, SFN, GOLT1A, and BEX2 were significantly up-regulated in the hypoxic group. BIRC7 expression was most significantly up-regulated and a downstream factor of HIF-1α. We highlighted that knockdown of BIRC7 reversed the positive effects of HIF-1α on A875 and M14 cells. ConclusionsOur findings demonstrated that BIRC7 was a downstream factor of HIF-1α and reversed the effect of hypoxia on promoting tumor progression of MM cells.

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Transcriptome Analysis of Rat Lungs Exposed to Moxa Smoke after Acute Toxicity Testing

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2021/5107441 ◽

2021 ◽

Vol 2021 ◽

pp. 1-15

Author(s):

Xiaoyu Xu ◽

Wen Deng ◽

Wanqing Zhang ◽

Junhua Zhang ◽

Muchen Wang ◽

...

Keyword(s):

Acute Toxicity ◽

Toxicity Testing ◽

Alveolar Epithelial Cells ◽

Control Group ◽

Rna Seq ◽

Alveolar Epithelial ◽

Cell Components ◽

Group A ◽

Tolerable Dose ◽

Live Group

The increasing use of moxibustion has led to a debate concerning the safety of this treatment in human patients. Inhalation of cigarette smoke induces lung inflammation and granulomas, the proliferation of alveolar epithelial cells, and other toxic effects; therefore, it is important to assess the influence of inhaled moxa smoke on the lungs. In the present study, a novel poisoning cabinet was designed and used to assess the acute toxicity of moxa smoke in rats. We evaluated pathological changes in rat lung tissue and analyzed differentially expressed genes (DEGs) using RNA-seq and transcriptomic analyses. Our results show that the maximum tolerable dose of moxa smoke was 290.036 g/m³ and LC50 was 537.65 g/m³. Compared with that of the control group, the degree of inflammatory cell infiltration in the lung tissues of group A rats (all dead group) was increased, while that in group E rats (all live group) remained unchanged. GO and KEGG enrichment analyses showed that the DEGs implicated in cell components, binding, and cancer were significantly enriched in the experimental groups compared with the profile of the control group. The expressions of MAFF, HSPA1B, HSPA1A, AOC1, and MX2 determined using quantitative real-time PCR were similar to those determined using RNA-seq, confirming the reliability of RNA-seq data. Overall, our results provide a basis for future evaluations of moxibustion safety and the development of moxibustion-based technology.

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The ATTEND trial: An alternative explanation with implications for future recovery and rehabilitation clinical trials

International Journal of Stroke ◽

10.1177/1747493017743061 ◽

2017 ◽

Vol 13 (2) ◽

pp. 112-116 ◽

Cited By ~ 6

Author(s):

Carolee Winstein

Keyword(s):

Clinical Trials ◽

Large Scale ◽

Optimal Recovery ◽

Stroke Recovery ◽

Control Group ◽

Experimental Intervention ◽

The Past ◽

Group A ◽

Intervention Trials ◽

The Individual

Over the past decade, ATTEND is one of only a handful of moderate to large-scale nonpharmacologic stroke recovery trials with a focus on rehabilitation. While unique in some respects, its test of superiority for the experimental intervention returned negative/neutral results, with no differences in outcome between the experimental intervention and an appropriate control group – a result not uncommon to the majority of moderate to large stroke rehabilitation intervention trials (i.e. six out of eight conducted in the past decade). The authors offer a number of potential explanations for the negative outcome, all of which have merit. We choose not to dwell on these possibilities, but rather offer a radically different explanation, one which has implications for future rehabilitation clinical trials. Our premise is that the process of neurorehabilitation is complex and multifaceted, but most importantly, for success, it requires a genuine collaboration between the patient and the clinician or caregiver to effect optimal recovery. This collaborative relationship must be defined by the unique perspective of each patient. By doing so, we acknowledge the importance of the individual patient’s values, goals, perspectives, and capacity. Rehabilitation scientists can design what arguably is a scientifically sound intervention that is evidence-based and even with preliminary data supporting its efficacy, but if the patient does not value the target outcome, does not fully engage in the therapy, or does not expect the intervention to succeed, the likelihood of success is poor. We offer this opinion, not to be critical, but to suggest a paradigm shift in the way in which we conduct stroke recovery and rehabilitation trials.

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Suppression of the transcription factor MSX1 gene delays bovine preimplantation embryo development in vitro

Reproduction ◽

10.1530/rep-09-0312 ◽

2010 ◽

Vol 139 (5) ◽

pp. 857-870 ◽

Cited By ~ 14

Author(s):

D Tesfaye ◽

A Regassa ◽

F Rings ◽

N Ghanem ◽

C Phatsara ◽

...

Keyword(s):

Transcription Factor ◽

Mrna Expression ◽

Large Scale ◽

Target Genes ◽

Preimplantation Embryos ◽

Control Group ◽

Cleavage Rate ◽

Cell Stage ◽

Preimplantation Embryo Development

This study was conducted to investigate the effect of suppressing transcription factor geneMSX1on the development ofin vitroproduced bovine oocytes and embryos, and identify its potential target genes regulated by this gene. Injection of long double-stranded RNA (LdsRNA) and small interfering RNA (siRNA) at germinal vesicle stage oocyte reducedMSX1mRNA expression by 73 and 37% respectively at metaphase II stage compared with non-injected controls. Similarly, injection of the same anti-sense oligomers at zygote stage reducedMSX1mRNA expression by 52 and 33% at 8-cell stage compared with non-injected controls. Protein expression was also reduced in LdsRNA- and siRNA-injected groups compared with non-injected controls at both stages. Blastocysts rates were 33, 28, 20 and 18% in non-injected control, scrambled RNA (scRNA), LdsRNA- and siRNA-injected groups respectively. Cleavage rates were also significantly reduced in Smartpool siRNA (SpsiRNA)-injected group (53.76%) compared with scRNA-injected group (57.76%) and non-injected control group (61%). Large-scale gene expression analysis showed that 135 genes were differentially regulated in SpsiRNA-injected group compared with non-injected controls, of which 54 and 81 were down- and up-regulated respectively due to suppression ofMSX1. Additionally, sequence homology mapping and gene enrichment analysis with known human pathway information identified several functional modules that were affected due to suppression ofMSX1. In conclusion, suppression ofMSX1affects oocyte maturation, embryo cleavage rate and the expression of several genes, suggesting its potential role in the development of bovine preimplantation embryos.

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Establishment of a Real-Time, Quantitative, and Reproducible Mouse Model of Staphylococcus Osteomyelitis Using Bioluminescence Imaging

Infection and Immunity ◽

10.1128/iai.06166-11 ◽

2011 ◽

Vol 80 (2) ◽

pp. 733-741 ◽

Cited By ~ 35

Author(s):

Haruki Funao ◽

Ken Ishii ◽

Shigenori Nagai ◽

Aya Sasaki ◽

Tomoyuki Hoshikawa ◽

...

Keyword(s):

Mouse Model ◽

Real Time ◽

Bioluminescence Imaging ◽

Serum Levels ◽

Infected Group ◽

Control Group ◽

Content Type ◽

Reactive Protein ◽

Chemotactic Protein ◽

Group A

ABSTRACTOsteomyelitis remains a serious problem in the orthopedic field. There are only a few animal models in which the quantity and distribution of bacteria can be reproducibly traced. Here, we established a real-time quantitative mouse model of osteomyelitis using bioluminescence imaging (BLI) without sacrificing the animals. A bioluminescent strain ofStaphylococcus aureuswas inoculated into the femurs of mice. The bacterial photon intensity (PI) was then sequentially measured by BLI. Serological and histological analyses of the mice were performed. The mean PI peaked at 3 days, and stable signals were maintained for over 3 months after inoculation. The serum levels of interleukin-6, interleukin-1β, and C-reactive protein were significantly higher in the infected mice than in the control mice on day 7. The serum monocyte chemotactic protein 1 level was also significantly higher in the infected group at 12 h than in the control group. A significantly higher proportion of granulocytes was detected in the peripheral blood of the infected group after day 7. Additionally, both acute and chronic histological manifestations were observed in the infected group. This model is useful for elucidating the pathophysiology of both acute and chronic osteomyelitis and to assess the effects of novel antibiotics or antibacterial implants.

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