Mammalian GLD-2 homologs are poly(A) polymerases. Kwak JE et al. GLD-2 is a cytoplasmic poly(A) polymerase present in the Caenorhabditis elegans germ line and embryo. It is a divergent member of the DNA polymerase beta nucleotidyl transferase superfamily, which includes CCA-adding enzymes, DNA polymerases and eukaryotic nuclear poly(A) polymerases. The polyadenylation activity of GLD-2 is stimulated by physical interaction with an RNA binding protein, GLD-3. To test whether GLD-3 might stimulate GLD-2 by recruiting it to RNA, we tethered C. elegans GLD-2 to mRNAs in Xenopus oocytes by using MS2 coat protein. Tethered GLD-2 adds poly(A) and stimulates translation of the mRNA, demonstrating that recruitment is sufficient to stimulate polyadenylation activity. We use the same tethered assay to identify human and mouse poly(A) polymerases related to GLD-2. This may provide entrees to previously uncharacterized modes of polyadenylation in mammalian cells.
General function
RNA binding, Translation factor
Comment
Musashi-directed translational activation of target mRNAs is mediated by the polyA] polymerase, Germline Development-2. [Cragle C 2014 et al.
The mRNA binding protein, Musashi, has been shown to regulate translation of select mRNAs and control cellular identity in both stem cells and cancer cells. Within mammalian cells, Musashi has traditionally been characterized as a repressor of translation. However, we have demonstrated that Musashi is an activator of translation in progesterone-stimulated oocytes of the frog Xenopus laevis, and recent evidence has revealed the capability of Musashi to function as an activator of translation in mammalian systems. The molecular mechanism by which Musashi directs activation of target mRNAs has not been elucidated. Here, we report a specific association of Musashi with the noncanonical poly[A] polymerase Germline Development-2 (GLD2) and map the association domain to 30 amino acids within the C-terminal domain of Musashi. We show that loss of GLD2 interaction through deletion of the binding domain, or treatment with antisense oligonucleotides compromises Musashi function. Additionally, we demonstrate that overexpression of both Musashi and GLD2 significantly enhances Musashi function. Finally, we report a similar co-association also occurs between murine Musashi and GLD2 orthologs, suggesting that coupling of Musashi to the polyadenylation apparatus is a conserved mechanism to promote target mRNA translation.
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Cellular localization
Nuclear
Comment
Ovarian function
Oogenesis, Early embryo development
Comment
GLD-2/RNP-8 cytoplasmic poly(A) polymerase is a broad-spectrum regulator of the oogenesis program. Kim KW et al. Regulated polyadenylation is a broadly conserved mechanism that controls key events during oogenesis. Pivotal to that mechanism is GLD-2, a catalytic subunit of cytoplasmic poly(A) polymerase (PAP). Caenorhabditis elegans GLD-2 forms an active PAP with multiple RNA-binding partners to regulate diverse aspects of germline and early embryonic development. One GLD-2 partner, RNP-8, was previously shown to influence oocyte fate specification. Here we use a genomic approach to identify transcripts selectively associated with both GLD-2 and RNP-8. Among the 335 GLD-2/RNP-8 potential targets, most were annotated as germline mRNAs and many as maternal mRNAs. These targets include gld-2 and rnp-8 themselves, suggesting autoregulation. Removal of either GLD-2 or RNP-8 resulted in shortened poly(A) tails and lowered abundance of four target mRNAs (oma-2, egg-1, pup-2, and tra-2); GLD-2 depletion also lowered the abundance of most GLD-2/RNP-8 putative target mRNAs when assayed on microarrays. Therefore, GLD-2/RNP-8 appears to polyadenylate and stabilize its target mRNAs. We also provide evidence that rnp-8 influences oocyte development; rnp-8 null mutants have more germ cell corpses and fewer oocytes than normal. Furthermore, RNP-8 appears to work synergistically with another GLD-2-binding partner, GLD-3, to ensure normal oogenesis. We propose that the GLD-2/RNP-8 enzyme is a broad-spectrum regulator of the oogenesis program that acts within an RNA regulatory network to specify and produce fully functional oocytes.