Differential Nanos 2 protein stability results in selective germ cell accumulation in the sea urchin

Abstract

Nanos is a translational regulator required for the survival and maintenance of primordial germ cells. In the sea urchin, Strongylocentrotus purpuratus (Sp), Nanos 2 mRNA is broadly transcribed but accumulates specifically in the small micromere (sMic) lineage, in part because of the 3′UTR element GNARLE leads to turnover in somatic cells but retention in the sMics. Here we found that the Nanos 2 protein is also selectively stabilized; it is initially translated throughout the embryo but turned over in the future somatic cells and retained only in the sMics, the future germ line in this animal. This differential stability of Nanos protein is dependent on the open reading frame (ORF), and is independent of the sumoylation and ubiquitylation pathways. Manipulation of the ORF indicates that 68 amino acids in the N terminus of the Nanos protein are essential for its stability in the sMics whereas a 45 amino acid element adjacent to the zinc fingers targets its degradation. Further, this regulation of Nanos protein is cell autonomous, following formation of the germ line. These results are paradigmatic for the unique presence of Nanos in the germ line by a combination of selective RNA retention, distinctive translational control mechanisms (Oulhen et al., 2013), and now also by defined Nanos protein stability.

Methods to label, isolate, and image sea urchin small micromeres, the primordial germ cells (PGCs)

“…Calcein labeling can be combined with other live labeling techniques, including the expression of exogenous mRNAs (Campanale & Hamdoun, 2012; Oulhen & Wessel, 2016) described below, the isolation of PGCs by fluorescence-activated cell sorting (FACS, Swartz et al, 2014), and in combination with additional dyes that label cellular membranes and organelles in live cells. These include red shifted dyes such as the plasma membrane marker FM4–64, Lysotraker, Mitotraker, or with red shifted versions of fluorescent dextran-labeled endosomes.…”

“…That is, Nanos 2 is transcribed broadly in the embryo, but through post-transcriptional and post-translational activities, Nanos 2 mRNA and protein only accumulate in the small micromeres. The basic understanding of how these mechanisms work enabled engineering of molecular tools for efficient and selective expression only in the small micromeres in vivo (Campanale et al, 2014; Oulhen & Wessel, 2013, 2016). For instance, simply injecting RNA encoding S. purpuratus Vasa linked to GFP results in selective fluorescence of the small micromeres (Figs.…”

“…The Sp nanos2 ORF itself is sufficient to restrict the expression of nanos2 protein in the small micromeres (Oulhen & Wessel, 2016). A GFP reporter will accumulate selectively in the PGCs when designed as follows: Sp nanos2 5′UTR fused to the GFP ORF followed by Sp nanos2 ORF and Sp nanos2 3′UTR ΔGNARLE.…”

A quiet space during rush hour: Quiescence in primordial germ cells

“…Both Nanos mRNA and protein appear to accumulate selectively in the PGCs, yet this is accomplished largely post-transcriptionally and even post-translationally, respectively (e.g. (Oulhen and Wessel, 2016a; Oulhen et al, 2013; Swartz et al, 2014). The gene encoding Nanos 2 appears to be broadly transcribed throughout the embryo in most, if not all, cell types.…”

“…So too, the Nanos 2 protein accumulates largely post-translationally (Oulhen and Wessel, 2016b). When an mRNA encoding Nanos2 is forced to broadly translate throughout the embryo (lacking GNARLE), the Nanos 2 protein is detected only within the germ line.…”