Novel functions of the ubiquitin-independent proteasome system in regulatingXenopusgermline development

Abstract

In most species, early germline development occurs in the absence of transcription with germline determinants subject to complex translational and post-translational regulations. Here, we report for the first time that early germline development is influenced by dynamic regulation of the proteasome system, previously thought to be ubiquitously expressed and to serve 'housekeeping' roles in controlling protein homeostasis. We show that proteasomes are present in a gradient with the highest levels in the animal hemisphere and extending into the vegetal hemisphere of Xenopus oocytes. This distribution changes dramatically during the oocyte-to-embryo transition, with proteasomes becoming enriched in and restricted to the animal hemisphere and therefore separated from vegetally localized germline determinants. We identify Dead-end1 (Dnd1), a master regulator of vertebrate germline development, as a novel substrate of the ubiquitin-independent proteasomes. In the oocyte, ubiquitin-independent proteasomal degradation acts together with translational repression to prevent premature accumulation of Dnd1 protein. In the embryo, artificially increasing ubiquitin-independent proteasomal degradation in the vegetal pole interferes with germline development. Our work thus reveals novel inhibitory functions and spatial regulation of the ubiquitin-independent proteasome during vertebrate germline development.

Single Cell Proteomics by Data-Independent Acquisition To Study Embryonic Asymmetry in Xenopus laevis

“…Immunofluorescence (IF) clearly indicates enrichment of the 20S CP in the animal hemisphere of 8-cell stage embryos, consistent with recent IF findings in fertilized eggs (Hwang et al). 32 Asymmetric distribution of the proteasome during the oocyte to embryo transition in Xenopus may allow stabilization of vegetally localized proteins that are critical for germ cell development (Hwang et al). 32 Furthermore, the protein DDX4/Vasa (also known as Xenopus vasa-like gene 1) was localized to the vegetal hemisphere of the embryo (indicated in yellow in Figure 4A), in line with previous findings by Sindelka et.al.…”

“…32 Asymmetric distribution of the proteasome during the oocyte to embryo transition in Xenopus may allow stabilization of vegetally localized proteins that are critical for germ cell development (Hwang et al). 32 Furthermore, the protein DDX4/Vasa (also known as Xenopus vasa-like gene 1) was localized to the vegetal hemisphere of the embryo (indicated in yellow in Figure 4A), in line with previous findings by Sindelka et.al. 50 DDX4/Vasa belongs to the family of DEAD-box RNA helicases and is a marker for germ cell specification in diverse animal species.…”

“…Some of the interesting hits were validated by immunofluorescence imaging of sectioned embryos to confirm differential expression of 20S CP subunits and DDX4/VASA across animal-vegetal axis. Proteasome subunits were found to be enriched in the animal hemisphere 32 whereas VASA/DDX4 was found to be vegetally localized in Xenopus . This forms the first report showing vegetal enrichment of Vasa enabled by localized sampling and LC-MS/MS.…”

Repurposing Protein Degradation for Optogenetic Modulation of Protein Activities

“…[29][30][31][32][33][34] We have recently discovered a potent 21 amino-acid degradation sequence within Xenopus laevis Dnd protein that promotes rapid ubiquitin-independent proteasomal degradation of Dnd in Xenopus oocytes and mammalian cell lines such as HEK293T and GC-2spd. 35 To determine whether this degradation sequence (here referred to as degron or deg) can be used as a general moiety to mediate degradation of intracellular proteins, we fused tandem arrays of degrons to the C-terminus of firefly luciferase (FLuc) protein (FLuc-tevS-Ndeg, N = 1, 2, and 3). A tobacco etch virus (TEV) protease recognition site (tevS) was sandwiched between FLuc and degron to render TEV-mediated rescue of protein degradation (Figure 1a).…”

“…[42][43][44][45] We previously reported that degradation mediated by the Dnd degron was blocked only when all three types of the ubiquitin-independent proteasome were inhibited simultaneously. 35 Ubiquitin-independent proteasomes are widely present in the majority of cell types. Thus, different from the majority of degrons, which are often recognized by tissue-specific E3 ubiquitin ligases, the Dnd degron can keep the protein of interest at a very low expression level in a wide variety of cells.…”