Proteomic screens of SEL1L-HRD1 ER-associated degradation substrates reveal its role in glycosylphosphatidylinositol-anchored protein biogenesis.

Endoplasmic reticulum-associated degradation (ERAD) plays indispensable roles in many physiological processes; however, the nature of endogenous substrates remains largely elusive. Here we report a proteomics strategy based on the intrinsic property of the SEL1L-HRD1 ERAD complex to identify endogenous ERAD substrates both in vitro and in vivo. Following stringent filtering using a machine learning algorithm, over 100 high-confidence potential substrates are identified in human HEK293T and mouse brown adipose tissue, among which ~88% are cell type-specific. One of the top shared hits is the catalytic subunit of the glycosylphosphatidylinositol (GPI)-transamidase complex, PIGK. Indeed, SEL1L-HRD1 ERAD attenuates the biogenesis of GPI-anchored proteins by specifically targeting PIGK for proteasomal degradation. Lastly, several PIGK disease variants in inherited GPI deficiency disorders are also SEL1L-HRD1 ERAD substrates. This study provides a platform and resources for future effort to identify proteome-wide endogenous substrates in vivo, and implicates SEL1L-HRD1 ERAD in many cellular processes including the biogenesis of GPI-anchored proteins.

The association of cognition with protein energy wasting and synaptic transmission in chronic kidney disease.

INTRODUCTION: In recent years, consciousness impairment in patients with end-stage renal disease (ESRD) has been paid more and more attention, but the cause and mechanism of consciousness state change is not clear. METHODS: As the hippocampus played a crucial role in consciousness, we explored the pathological and electrophysiological changes in chronic kidney disease (CKD) mouse hippocampus. RESULTS: Whole-cell recordings in hippocampal neurons showed that miniature excitatory postsynaptic current (mEPSC) frequency decreased, but the amplitude was unaltered in CKD_8w mice. In addition, α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor-mediated EPSCs (AMPAR-EPSCs) and N-methyl-D-aspartic acid receptor-mediated EPSCs (NMDAR-EPSCs) in hippocampal Schaffer collateral-CA1 synapses displayed a significant decline in CKD_8w mice. Although the ratio of AMPAR-/NMDAR-EPSCs did not change, the paired-pulse ratio (PPR) in CKD_8w mice increased. Intriguingly, the mEPSC frequency and AMPAR-/NMDAR-EPSCs amplitudes were positively associated with body weight, and the mEPSC frequency was negatively correlated with serum creatinine in CKD_8w mice, indicating a potential correlation between cognition and nutritional status in patients with CKD. To confirm the above hypothesis, we collected the clinical data from multiple hemodialysis centers to analyze the correlation between cognition and nutritional status. CONCLUSION: Our analysis indicated that protein energy wasting (PEW) was a possible independent risk factor for consciousness dysfunction in maintenance hemodialysis (MHD) patients. Our results provided a more detailed mechanism underlying the cognitive impairment (CI) in ESRD patients at the synaptic level. Last but not least, our results showed that PEW was a probable new independent risk factor for CI in cases with ESRD.

Ultrasensitive Ribo-seq reveals translational landscapes during mammalian oocyte-to-embryo transition and pre-implantation development.

In mammals, translational control plays critical roles during oocyte-to-embryo transition (OET) when transcription ceases. However, the underlying regulatory mechanisms remain challenging to study. Here, using low-input Ribo-seq (Ribo-lite), we investigated translational landscapes during OET using 30-150 mouse oocytes or embryos per stage. Ribo-lite can also accommodate single oocytes. Combining PAIso-seq to interrogate poly(A) tail lengths, we found a global switch of translatome that closely parallels changes of poly(A) tails upon meiotic resumption. Translation activation correlates with polyadenylation and is supported by polyadenylation signal proximal cytoplasmic polyadenylation elements (papCPEs) in 3' untranslated regions. By contrast, translation repression parallels global de-adenylation. The latter includes transcripts containing no CPEs or non-papCPEs, which encode many transcription regulators that are preferentially re-activated before zygotic genome activation. CCR4-NOT, the major de-adenylation complex, and its key adaptor protein BTG4 regulate translation downregulation often independent of RNA decay. BTG4 is not essential for global de-adenylation but is required for selective gene de-adenylation and production of very short-tailed transcripts. In sum, our data reveal intimate interplays among translation, RNA stability and poly(A) tail length regulation underlying mammalian OET.

Cnot8 eliminates naïve regulation networks and is essential for naïve-to-formative pluripotency transition.

Mammalian early epiblasts at different phases are characterized by naïve, formative, and primed pluripotency states, involving extensive transcriptome changes. Here, we report that deadenylase Cnot8 of Ccr4-Not complex plays essential roles during the transition from naïve to formative state. Knock out (KO) Cnot8 resulted in early embryonic lethality in mice, but Cnot8 KO embryonic stem cells (ESCs) could be established. Compared with the cells differentiated from normal ESCs, Cnot8 KO cells highly expressed a great many genes during their differentiation into the formative state, including several hundred naïve-like genes enriched in lipid metabolic process and gene expression regulation that may form the naïve regulation networks. Knockdown expression of the selected genes of naïve regulation networks partially rescued the differentiation defects of Cnot8 KO ESCs. Cnot8 depletion led to the deadenylation defects of its targets, increasing their poly(A) tail lengths and half-life, eventually elevating their expression levels. We further found that Cnot8 was involved in the clearance of targets through its deadenylase activity and the binding of Ccr4-Not complex, as well as the interacting with Tob1 and Pabpc1. Our results suggest that Cnot8 eliminates naïve regulation networks through mRNA clearance, and is essential for naïve-to-formative pluripotency transition.

In vitro investigation of mammalian peri-implantation embryogenesis†.

The embryos attach and invade the uterus, establishing the connection with their mother in peri-implantation development. During this period, the pluripotent epiblast cells of the embryo undergo symmetry breaking, cell lineage allocation, and morphogenetic remodeling, accompanied by the dramatic changes of transcriptomic, epigenomic, and signaling pathways, and preparing the stage for their differentiation and gastrulation. The progress in mouse genetics and stem cell biology has advanced the knowledge of these transformations, which are still largely hindered by the hard accessibility of natural embryos. To gain insight into mammalian peri-implantation development, much effort has been made in the field. Recently, advances in the prolonged in vitro culture of blastocysts, the derivation of multiple pluripotent stem cells, and the construction of stem cell-based embryo-like models have opened novel avenues to investigate peri-implantation development in mammals, especially humans. Combining with other emerging new technologies, these new models will substantially promote the comprehension of mammalian peri-implantation development, thus accelerating the progress of reproductive and regenerative medicine.

Formative pluripotent stem cells show features of epiblast cells poised for gastrulation.

The pluripotency of mammalian early and late epiblast could be recapitulated by naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), respectively. However, these two states of pluripotency may not be sufficient to reflect the full complexity and developmental potency of the epiblast during mammalian early development. Here we report the establishment of self-renewing formative pluripotent stem cells (fPSCs) which manifest features of epiblast cells poised for gastrulation. fPSCs can be established from different mouse ESCs, pre-/early-gastrula epiblasts and induced PSCs. Similar to pre-/early-gastrula epiblasts, fPSCs show the transcriptomic features of formative pluripotency, which are distinct from naïve ESCs and primed EpiSCs. fPSCs show the unique epigenetic states of E6.5 epiblast, including the super-bivalency of a large set of developmental genes. Just like epiblast cells immediately before gastrulation, fPSCs can efficiently differentiate into three germ layers and primordial germ cells (PGCs) in vitro. Thus, fPSCs highlight the feasibility of using PSCs to explore the development of mammalian epiblast.

Effects of pituitary-specific overexpression of FSHα/ß on reproductive traits in transgenic boars.

BACKGROUND: Follicle-stimulating hormone (FSH) is a gonadotropin synthesized and secreted by the pituitary gland. FSH stimulates follicle development and maturation in females. It also plays an important role in spermatogenesis in males, including humans and mice. However, the effects of FSH on male pigs are largely unknown. In this study, we generated transgenic pigs to investigate the effects of FSHα/ß overexpression on reproductive traits in boars. RESULTS: After five transgenic F0 founders were crossed with wide-type pigs, 193 F1 animals were obtained. Of these, 96 were confirmed as transgenic. FSHα and FSHß mRNAs were detected only in pituitary tissue. Transgenic boars exhibited significantly higher levels of FSHα and FSHß mRNA, serum FSH, and serum testosterone, compared to full-sib non-transgenic boars. Significant increases in testis weight, vas deferens diameter, seminiferous tubule diameter, and the number of Leydig cells were observed, suggesting that the exogenous FSHα/ß affects reproductive traits. Finally, transgenic and non-transgenic boars had similar growth performance and biochemical profiles. CONCLUSIONS: Pituitary-specific overexpression of FSHα/ß genes is likely to impact reproductive traits positively, as indicated by enhancements in serum testosterone level, testis weight, the development of vas deferens, seminiferous tubules, and Leydig cells in transgenic boars. A high level of serum FSH induces secretion of serum testosterone, possibly by boosting the number of Leydig cells, which presumably increases the libido and the frequency of sexual activity in transgenic boars. Our study provides a preliminary foundation for the genetic improvement of reproductive traits in male pigs.

Self-assembling of submicrometer three-dimensional photonic crystals in concave microzones etched on silicon substrates.

By vertical sedimentation and oblique titration, silica microspheres were grown in different shapes of concave microzones that were etched on a (100) p-silicon substrate. Through scanning electron microscope observation and optical reflective spectra measurement, sedimentation of microspheres in those microzones was compared. An index was introduced to judge the efficiency of sedimentation. The comparison demonstrates that regular hexagons and triangles facilitate the growth of photonic crystals the most.