Ubiquitin A-52 residue ribosomal protein fusion product 1 (Uba52) is essential for preimplantation embryo development

Abstract

Ubiquitin A-52 residue ribosomal protein fusion product 1 (Uba52), a ubiquitin-ribosomal fusion gene, is a major source of ubiquitin protein for covalent modification of proteinaceous substrates recycled by ubiquitin-proteasome system (UPS). Its role in early embryo development has not been studied. Using the CRISPR/Cas9 gene editing tool, the objective of this study was to determine if UBA52 protein is required for mammalian embryogenesis. Matured metaphase II porcine oocytes were injected with CRISPR Cas9+guide RNAs (Uba52 gRNA) or Cas9 without gRNAs as control, followed by in vitro fertilization (IVF) and embryo culture to day 7. Injection of Cas9+gRNAs affected embryo development. On day 4 of embryo culture, the proportion of 2-, 4- and 8-cell stage embryos was significantly different between the Uba52 gRNA and control group (P<0.05), with more 8-cell stage embryos in the control and more 4- and 2-cell stage embryos in the Uba52g RNA group. This delay in the development of Uba52 gRNA embryos occurred at the transition from the 4- to 8-cell stages, around the time of major zygotic genomic activation. The percentage of blastocyst formation on day 7 and the cell number per blastocyst were significantly lower in the Uba52 gRNA group than in the control (P<0.05). Genotyping by PCR and DNA gel electrophoresis analysis showed that 91.8% of embryos that failed to develop to blastocyst had either a monoallelic or a biallelic modification of the Uba52 gene. In comparison, only 24.4% of embryos that reached blastocyst had a monoallelic modification and biallelic editing was not found in any of the blastocysts. Based on immuno-labeling intensity, both UBA52 and proteasome protein levels on days 4 and 7 of culture were significantly lower in the Uba52 gRNA group than in the control (P<0.05), in agreement with UBA52 western blotting-densitometry of day 4 embryos. Morphological examination of blastomere nuclei revealed abnormal nuclear structure in the Uba52 gRNA group, such as reduced size, irregular shapes, nucleus fragmentation and uneven DNA distribution at all stages of embryo development. Nuclear morphology studies of embryos injected with Cas9+gRNAs and co-injected with plasmid DNA encoding nuclear localized GFP further supported these observations. In conclusion, our data indicate that the Uba52 gene is essential in early embryogenesis.

Proteomic profile of pre‐implantational ovine embryos produced in vivo

“…Ubiquitin plays roles in covalent modification of proteinaceous substrates further degraded by ubiquitin‐proteasome system (UPS). Uba52 gene modification/knockout causes development arrest prior to embryo implantation in porcine (Mao et al., 2018) and mutation of Uba52 gene is lethal to the mouse embryo, affecting embryo ubiquitin levels, ribosome assembly, cell cycle progression and overall protein synthesis (Kobayashi et al., 2016).…”

Single‐cell transcriptome analysis of human oocyte ageing

“…However, whether abnormal expression of SKP1 will affect chromosome separation in human oocytes remains to be further verified. In addition, other ubiquitination‐related genes such as UBA52 is instrumental to the regulation of physiological ubiquitination and embryonic development, and modification/knockout of the UBA52 gene causes embryonic developmental arrest before implantation 34,35 . Thus, downregulated expression of UBA52 (1.4‐|log2FC|) may influence the formation of ubiquitin and oocyte development.…”

Clinical Relevance of Secreted Small Noncoding RNAs in an Embryo Implantation Potential Prediction at Morula and Blastocyst Development Stages

“…BTRC (b-transducing repeat containing) protein, as a part of the E3 ligase complex, promotes ubiquitination of phosphorylated b-catenin, which participates in the adherens junctions, providing an anchor between the cytoskeleton and cadherins, and plays a role as a transcriptional coactivator of the expression of a growing number of target genes [59]. Ubiquitin A-52 residue ribosomal protein fusion product 1 (Uba52), being the major source of ubiquitin protein for covalent modification of proteins in the proteasome system, is essential in early embryogenesis, enabling cell cycle progressing, mitosis, RNA translation and processing, protein catabolism, and chromatin remodeling [60]. In the case of the Uba52 gene modified embryos using the CRISPR/Cas9 gene editing tool, delay in embryo development, abnormal blastomere nuclear structure, and decreased percentage of blastocyst formation were revealed.…”