Abstract
PUMILIO/FBF (PUF) proteins have a conserved function in stem cell regulation. Caenorhabditis elegans PUF-8 protein inhibits the translation of target mRNAs by interacting with PUF binding element (PBE) in the 3′ untranslated region (3′ UTR). In this work, an in silico analysis has identified gld-2 [a poly(A) polymerase] as a putative PUF-8 target. Biochemical and reporter analyses showed that PUF-8 specifically binds to a PBE in gld-2 3′ UTR and represses a GFP reporter gene carrying gld-2 3′ UTR in the C. elegans mitotic germ cells. GLD-2 enhances meiotic entry at least in part by activating GLD-1 (a KH motif-containing RNA-binding protein). Our genetic analyses also demonstrated that heterozygous gld-2(+/−) gld-1(+/−) genes in the absence of PUF-8 are competent for meiotic entry (early differentiation), but haplo-insufficient for the meiotic division (terminal differentiation) of spermatocytes. Indeed, the arrested spermatocytes return to mitotic cells via dedifferentiation, which results in germline tumors. Since these regulators are broadly conserved, we thus suggest that similar molecular mechanisms may control differentiation, dedifferentiation, and tumorigenesis in other organisms, including humans.
Highlights
During development, stem cells must make a number of major fate decisions – the initial decision to either proliferate or differentiate, followed by whether to remain in a differentiating state or revert to being undifferentiated as occurs in regeneration or tumorigenesis
C. elegans 3 untranslated region (3 UTR) sequences were obtained from BioMart, and we identified 800 genes (3.6%) harboring at least one PUF binding element (PBE) in their 3 UTRs (Figure 1D and Supplementary Table S1)
We have focused on P granule-associated proteins (GO ID: 0043186) that function in reproduction (GO ID: 0000003) and meiosis (GO ID: 0051321) (Figure 1E and Supplementary Table S3)
Summary
Stem cells must make a number of major fate decisions – the initial decision to either proliferate or differentiate, followed by whether to remain in a differentiating state or revert to being undifferentiated as occurs in regeneration or tumorigenesis. Germ cells progress from GSCs at the distal end, through meiotic prophase as they move proximally to become differentiated gametes (sperm and oocytes) at the proximal end (Figure 1A). This developmental process requires a battery of RNA regulators (Kimble and Crittenden, 2002; Figure 1B). The PUF protein binds a specific regulatory element in its target mRNA 3 untranslated regions (3 UTRs) and inhibits the expression of its target mRNAs by recruiting translational repressor complexes (Wickens et al, 2002) These include cytoplasmic Ccr4p-Pop2p-Not deadenylase complex (Goldstrohm et al, 2007) and Ago-eEF1A translational complex (Friend et al, 2012)
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