Structural basis for the antagonistic roles of RNP-8 and GLD-3 in GLD-2 poly(A)-polymerase activity

Cytoplasmic polyadenylation drives the translational activation of specific mRNAs in early metazoan development and is performed by distinct complexes that share the same catalytic poly(A)-polymerase subunit, GLD-2. The activity and specificity of GLD-2 depend on its binding partners. In Caenorhabditis elegans, GLD-2 promotes spermatogenesis when bound to GLD-3 and oogenesis when bound to RNP-8. GLD-3 and RNP-8 antagonize each other and compete for GLD-2 binding. Following up on our previous mechanistic studies of GLD-2–GLD-3, we report here the 2.5 Å resolution structure and biochemical characterization of a GLD-2–RNP-8 core complex. In the structure, RNP-8 embraces the poly(A)-polymerase, docking onto several conserved hydrophobic hotspots present on the GLD-2 surface. RNP-8 stabilizes GLD-2 and indirectly stimulates polyadenylation. RNP-8 has a different amino-acid sequence and structure as compared to GLD-3. Yet, it binds the same surfaces of GLD-2 by forming alternative interactions, rationalizing the remarkable versatility of GLD-2 complexes.     

Structural and functional implications of the QUA2 domain on RNA recognition by GLD-1

The STAR family comprises ribonucleic acid (RNA)-binding proteins that play key roles in RNA-regulatory processes. RNA recognition is achieved by a KH domain with an additional α-helix (QUA2) that seems to extend the RNA-binding surface to six nucleotides for SF1 (Homo sapiens) and seven nucleotides for GLD-1 (Caenorhabditis elegans). To understand the structural basis of this probable difference in specificity, we determined the solution structure of GLD-1 KH-QUA2 with the complete consensus sequence identified in the tra-2 gene. Compared to SF1, the GLD-1 KH-QUA2 interface adopts a different conformation resulting indeed in an additional sequence-specific binding pocket for a uracil at the 5′end. The functional relevance of this binding pocket is emphasized by our bioinformatics analysis showing that GLD-1 binding sites with this 5′end uracil are more predictive for the functional response of the messenger RNAs to gld-1 knockout. We further reveal the importance of the KH-QUA2 interface in vitro and that its alteration in vivo affects the level of translational repression dependent on the sequence of the GLD-1 binding motif. In conclusion, we demonstrate that the QUA2 domain distinguishes GLD-1 from other members of the STAR family and contributes more generally to the modulation of RNA-binding affinity and specificity of KH domain containing proteins.     

Self-association of the single-KH-domain family members Sam68, GRP33, GLD-1, and Qk1: role of the KH domain.

Sam68 is a member of a growing family of proteins that contain a single KH domain embedded in a larger conserved domain of approximately 170 amino acids. Loops 1 and 4 of this KH domain family are longer than the corresponding loops in other KH domains and contain conserved residues. KH domains are protein motifs that are involved in RNA binding and are often present in multiple copies. Here we demonstrate by coimmunoprecipitation studies that Sam68 self-associated and that cellular RNA was required for the association. Deletion studies demonstrated that the Sam68 KH domain loops 1 and 4 were required for self-association. The Sam68 interaction was also observed in Saccharomyces cerevisiae by the two-hybrid system. In situ chemical cross-linking studies in mammalian cells demonstrated that Sam68 oligomerized in vivo. These Sam68 complexes bound homopolymeric RNA and the SH3 domains of p59fyn and phospholipase Cgamma1 in vitro, demonstrating that Sam68 associates with RNA and signaling molecules as a multimer. The formation of the Sam68 complex was inhibited by p59fyn, suggesting that tyrosine phosphorylation regulates Sam68 oligomerization. Other Sam68 family members including Artemia salina GRP33, Caenorhabditis elegans GLD-1, and mouse Qk1 also oligomerized. In addition, Sam68, GRP33, GLD-1, and Qk1 associated with other KH domain proteins such as Bicaudal C. These observations indicate that the single KH domain found in the Sam68 family, in addition to mediating protein-RNA interactions, mediates protein-protein interactions.     

GLD-3 and control of the mitosis/meiosis decision in the germline of Caenorhabditis elegans

Germ cells can divide mitotically to replenish germline tissue or meiotically to produce gametes. In this article, we report that GLD-3, a Caenorhabditis elegans Bicaudal-C homolog, promotes the transition from mitosis to meiosis together with the GLD-2 poly(A) polymerase. GLD-3 binds GLD-2 via a small N-terminal region present in both GLD-3S and GLD-3L isoforms, and GLD-2 and GLD-3 can be co-immunoprecipitated from worm extracts. The FBF repressor binds specifically to elements in the gld-3S 3′-UTR, and FBF regulates gld-3 expression. Furthermore, FBF acts largely upstream of gld-3 in the mitosis/meiosis decision. By contrast, GLD-3 acts upstream of FBF in the sperm/oocyte decision, and GLD-3 protein can antagonize FBF binding to RNA regulatory elements. To address the relative importance of these two regulatory mechanisms in the mitosis/meiosis and sperm/oocyte decisions, we isolated a deletion mutant, gld-3(q741), that removes the FBF-binding site from GLD-3L, but leaves the GLD-2-binding site intact. Animals homozygous for gld-3(q741) enter meiosis, but are feminized. Therefore, GLD-3 promotes meiosis primarily via its interaction with GLD-2, and it promotes spermatogenesis primarily via its interaction with FBF.     

The DICE-binding activity of KH domain 3 of hnRNP K is affected by c-Src-mediated tyrosine phosphorylation

hnRNP K and hnRNP E1/E2 are RNA-binding proteins comprised of three hnRNP K-homology (KH) domains. These proteins are involved in the translational control and stabilization of mRNAs in erythroid cells. hnRNP E1 and hnRNP K regulate the translation of reticulocyte 15-lipoxygenase (r15-LOX) mRNA. Both proteins bind specifically to the differentiation control element (DICE) in the 3' untranslated region (3'UTR) of the r15-LOX mRNA. It has been shown that hnRNP K is a substrate of the tyrosine kinase c-Src and that tyrosine phosphorylation by c-Src inhibits the binding of hnRNP K to the DICE. Here, we investigate which of the three KH domains of hnRNP E1 and hnRNP K mediate the DICE interaction. Using RNA-binding assays, we demonstrate DICE-binding of the KH domains 1 and 3 of hnRNP E1, and KH domain 3 of hnRNP K. Furthermore, with RNA-binding assays, NMR experiments and in vitro translation studies, we show that tyrosine 458 in KH domain 3 of hnRNP K is important for the DICE interaction and we provide evidence that it is a target of c-Src.     

RNA-binding protein GLD-1/quaking genetically interacts with the mir-35 and the let-7 miRNA pathways in Caenorhabditis elegans

Messenger RNA translation is regulated by RNA-binding proteins and small non-coding RNAs called microRNAs. Even though we know the majority of RNA-binding proteins and microRNAs that regulate messenger RNA expression, evidence of interactions between the two remain elusive. The role of the RNA-binding protein GLD-1 as a translational repressor is well studied during Caenorhabditis elegans germline development and maintenance. Possible functions of GLD-1 during somatic development and the mechanism of how GLD-1 acts as a translational repressor are not known. Its human homologue, quaking (QKI), is essential for embryonic development. Here, we report that the RNA-binding protein GLD-1 in C. elegans affects multiple microRNA pathways and interacts with proteins required for microRNA function. Using genome-wide RNAi screening, we found that nhl-2 and vig-1, two known modulators of miRNA function, genetically interact with GLD-1. gld-1 mutations enhance multiple phenotypes conferred by mir-35 and let-7 family mutants during somatic development. We used stable isotope labelling with amino acids in cell culture to globally analyse the changes in the proteome conferred by let-7 and gld-1 during animal development. We identified the histone mRNA-binding protein CDL-1 to be, in part, responsible for the phenotypes observed in let-7 and gld-1 mutants. The link between GLD-1 and miRNA-mediated gene regulation is further supported by its biochemical interaction with ALG-1, CGH-1 and PAB-1, proteins implicated in miRNA regulation. Overall, we have uncovered genetic and biochemical interactions between GLD-1 and miRNA pathways.     

CeLINC,a fluorescence-based protein–protein interaction assay in Caenorhabditis elegans

Interactions among proteins are fundamental for life and determining whether two particular proteins physically interact can be essential for fully understanding a protein’s function. We present Caenorhabditis elegans light-induced coclustering (CeLINC), an optical binary protein–protein interaction assay to determine whether two proteins interact in vivo. Based on CRY2/CIB1 light-dependent oligomerization, CeLINC can rapidly and unambiguously identify protein–protein interactions between pairs of fluorescently tagged proteins. A fluorescently tagged bait protein is captured using a nanobody directed against the fluorescent protein (GFP or mCherry) and brought into artificial clusters within the cell. Colocalization of a fluorescently tagged prey protein in the cluster indicates a protein interaction. We tested the system with an array of positive and negative reference protein pairs. Assay performance was extremely robust with no false positives detected in the negative reference pairs. We then used the system to test for interactions among apical and basolateral polarity regulators. We confirmed interactions seen between PAR-6, PKC-3, and PAR-3, but observed no physical interactions among the basolateral Scribble module proteins LET-413, DLG-1, and LGL-1. We have generated a plasmid toolkit that allows use of custom promoters or CRY2 variants to promote flexibility of the system. The CeLINC assay is a powerful and rapid technique that can be widely applied in C. elegans due to the universal plasmids that can be used with existing fluorescently tagged strains without need for additional cloning or genetic modification of the genome.     

Solution Structure of the QUA1 Dimerization Domain of pXqua,the Xenopus Ortholog of Quaking

The STAR protein family member Quaking is essential for early development in vertebrates. For example, in oligodendrocyte cells it regulates the splicing, localization, translation and lifetime of a set of mRNAs that code for crucial components of myelin. The Quaking protein contains three contiguous conserved regions: a QUA1 oligomerization element, followed by a single-stranded RNA binding motif comprising the KH and QUA2 domains. An embryonic lethal point mutation in the QUA1 domain, E48G, is known to affect both the aggregation state and RNA-binding properties of the murine Quaking ortholog (QKI). Here we report the NMR solution structure of the QUA1 domain from the Xenopus laevis Quaking ortholog (pXqua), which forms a dimer composed of two perpendicularly docked α-helical hairpin motifs. Size exclusion chromatography studies of a range of mutants demonstrate that the dimeric state of the pXqua QUA1 domain is stabilized by a network of interactions between side-chains, with significant roles played by an intra-molecular hydrogen bond between Y41 and E72 (the counterpart to QKI E48) and an inter-protomer salt bridge between E72 and R67. These results are compared with recent structural and mutagenesis studies of QUA1 domains from the STAR family members QKI, GLD-1 and Sam68.     

Discovery of two distinct aminoacyl-tRNA synthetase complexes anchored to the Plasmodium surface tRNA import protein

Aminoacyl-tRNA synthetases (aaRSs) attach amino acids to their cognate transfer RNAs. In eukaryotes, a subset of cytosolic aaRSs is organized into a multisynthetase complex (MSC), along with specialized scaffolding proteins referred to as aaRS-interacting multifunctional proteins (AIMPs). In Plasmodium, the causative agent of malaria, the tRNA import protein (tRip), is a membrane protein that participates in tRNA trafficking; we show that tRip also functions as an AIMP. We identified three aaRSs, the glutamyl-tRNA synthetase (ERS), glutaminyl-tRNA synthetase (QRS), and methionyl-tRNA synthetase (MRS), which were specifically coimmunoprecipitated with tRip in Plasmodium berghei blood stage parasites. All four proteins contain an N-terminal glutathione-S-transferase (GST)–like domain that was demonstrated to be involved in MSC assembly. In contrast to previous studies, further dissection of GST-like interactions identified two exclusive heterotrimeric complexes: the Q-complex (tRip–ERS–QRS) and the M-complex (tRip–ERS–MRS). Gel filtration and light scattering suggest a 2:2:2 stoichiometry for both complexes but with distinct biophysical properties and mutational analysis further revealed that the GST-like domains of QRS and MRS use different strategies to bind ERS. Taken together, our results demonstrate that neither the singular homodimerization of tRip nor its localization in the parasite plasma membrane prevents the formation of MSCs in Plasmodium. Besides, the extracellular localization of the tRNA-binding module of tRip is compensated by the presence of additional tRNA-binding modules fused to MRS and QRS, providing each MSC with two spatially distinct functions: aminoacylation of intraparasitic tRNAs and binding of extracellular tRNAs. This unique host–pathogen interaction is discussed.     

Structure of a construct of a human poly(C)-binding protein containing the first and second KH domains reveals insights into its regulatory mechanisms

Poly(C)-binding proteins (PCBPs) are important regulatory proteins that contain three KH (hnRNP K homology) domains. Binding poly(C) D/RNA sequences via KH domains is essential for multiple PCBP functions. To reveal the basis for PCBP-D/RNA interactions and function, we determined the structure of a construct containing the first two domains (KH1-KH2) of human PCBP2 by NMR. KH1 and KH2 form an intramolecular pseudodimer. The large hydrophobic dimerization surface of each KH domain is on the side opposite the D/RNA binding interface. Chemical shift mapping indicates both domains bind poly(C) DNA motifs without disrupting the KH1-KH2 interaction. Spectral comparison of KH1-KH2, KH3, and full-length PCBP2 constructs suggests that the KH1-KH2 pseudodimer forms, but KH3 does not interact with other parts of the protein. From NMR studies and modeling, we propose possible modes of cooperative binding tandem poly(C) motifs by the KH domains. D/RNA binding may induce pseudodimer dissociation or stabilize dissociated KH1 and KH2, making protein interaction surfaces available to PCBP-binding partners. This conformational change may represent a regulatory mechanism linking D/RNA binding to PCBP functions.     

Paip2A inhibits translation by competitively binding to the RNA recognition motifs of PABPC1 and promoting its dissociation from the poly(A) tail

Eukaryotic mRNAs possess a poly(A) tail at their 3′-end, to which poly(A)-binding protein C1 (PABPC1) binds and recruits other proteins that regulate translation. Enhanced poly(A)-dependent translation, which is also PABPC1 dependent, promotes cellular and viral proliferation. PABP-interacting protein 2A (Paip2A) effectively represses poly(A)-dependent translation by causing the dissociation of PABPC1 from the poly(A) tail; however, the underlying mechanism remains unknown. This study was conducted to investigate the functional mechanisms of Paip2A action by characterizing the PABPC1–poly(A) and PABPC1–Paip2A interactions. Isothermal titration calorimetry and NMR analyses indicated that both interactions predominantly occurred at the RNA recognition motif (RRM)2–RRM3 regions of PABPC1, which have comparable affinities for poly(A) and Paip2A (dissociation constant, K_d = 1 nM). However, the K_d values of isolated RRM2 were 200 and 4 μM in their interactions with poly(A) and Paip2A, respectively; K_d values of 5 and 1 μM were observed for the interactions of isolated RRM3 with poly(A) and Paip2A, respectively. NMR analyses also revealed that Paip2A can bind to the poly(A)-binding interfaces of the RRM2 and RRM3 regions of PABPC1. Based on these results, we propose the following functional mechanism for Paip2A: Paip2A initially binds to the RRM2 region of poly(A)-bound PABPC1, and RRM2-anchored Paip2A effectively displaces the RRM3 region from poly(A), resulting in dissociation of the whole PABPC1 molecule. Together, our findings provide insight into the translation repression effect of Paip2A and may aid in the development of novel anticancer and/or antiviral drugs.     

Highly conserved amino acids in Pax and Ets proteins are required for DNA binding and ternary complex assembly

Combinatorial association of DNA-binding proteins on composite binding sites enhances their nucleotide sequence specificity and functional synergy. As a paradigm for these interactions, Pax-5 (BSAP) assembles ternary complexes with Ets proteins on the B cell-specific mb-1 promoter through interactions between their respective DNA-binding domains. Pax-5 recruits Ets-1 to bind the promoter, but not the closely related Ets protein SAP1a. Here we show that, while several different mutations increase binding of SAP1a to an optimized Ets binding site, only conversion of Val68 to an acidic amino acid facilitates ternary complex assembly with Pax-5 on the mb-1 promoter. This suggests that enhanced DNA binding by SAP1a is not sufficient for recruitment by Pax-5, but instead involves protein–protein interactions mediated by the acidic side chain. Recruitment of Ets proteins by Pax-5 requires Gln22 within the N-terminal β-hairpin motif of its paired domain. The β-hairpin also participates in recognition of a subset of Pax-5-binding sites. Thus, Pax-5 incorporates protein–protein interaction and DNA recognition functions in a single motif. The Caenorhabditis elegans Pax protein EGL-38 also binds specifically to the mb-1 promoter and recruits murine Ets-1 or the C.elegans Ets protein T08H4.3, but not the related LIN-1 protein. Together, our results define specific amino acid requirements for Pax–Ets ternary complex assembly and show that the mechanism is conserved between evolutionarily related proteins of diverse animal species. Moreover, the data suggest that interactions between Pax and Ets proteins are an important mechanism that regulates fundamental biological processes in worms and humans.     

Interaction of poly(rC)-binding protein 2 domains KH1 and KH3 with coxsackievirus RNA

Recombinant hnRNP K-homology (KH) domains 1 and 3 of the poly(rC)-binding protein (PCBP) 2 were purified and assayed for interaction with coxsackievirus B3 RNA in electrophoretic mobility shift assays using in vitro transcribed RNAs which represent signal structures of the 5′-nontranslated region. KH domains 1 and 3 interact with the extended cloverleaf RNA and domain IV RNA of the internal ribosome entry site (IRES). KH1 but not KH3 interacts with subdomain IV/C RNA, whereas KH3 interacts with subdomain IV/B. All in vitro results are consistent with yeast three-hybrid experiments performed in parallel. The data demonstrate interaction of isolated PCBP2 KH1 and KH3 domains to four distinct target sites within the 5′-nontranslated region of the CVB3 genomic RNA.     

Molecular Insights into the Coding Region Determinant-binding Protein-RNA Interaction through Site-directed Mutagenesis in the Heterogeneous Nuclear Ribonucleoprotein-K-homology Domains

The ability of its four heterogeneous nuclear RNP-K-homology (KH) domains to physically associate with oncogenic mRNAs is a major criterion for the function of the coding region determinant-binding protein (CRD-BP). However, the particular RNA-binding role of each of the KH domains remains largely unresolved. Here, we mutated the first glycine to an aspartate in the universally conserved GXXG motif of the KH domain as an approach to investigate their role. Our results show that mutation of a single GXXG motif generally had no effect on binding, but the mutation in any two KH domains, with the exception of the combination of KH3 and KH4 domains, completely abrogated RNA binding in vitro and significantly retarded granule formation in zebrafish embryos, suggesting that any combination of at least two KH domains cooperate in tandem to bind RNA efficiently. Interestingly, we found that any single point mutation in one of the four KH domains significantly impacted CRD-BP binding to mRNAs in HeLa cells, suggesting that the dynamics of the CRD-BP-mRNA interaction vary over time in vivo. Furthermore, our results suggest that different mRNAs bind preferentially to distinct CRD-BP KH domains. The novel insights revealed in this study have important implications on the understanding of the oncogenic mechanism of CRD-BP as well as in the future design of inhibitors against CRD-BP function.     

GLD-4-Mediated Translational Activation Regulates the Size of the Proliferative Germ Cell Pool in the Adult C. elegans Germ Line

To avoid organ dysfunction as a consequence of tissue diminution or tumorous growth, a tight balance between cell proliferation and differentiation is maintained in metazoans. However, cell-intrinsic gene expression mechanisms controlling adult tissue homeostasis remain poorly understood. By focusing on the adult Caenorhabditis elegans reproductive tissue, we show that translational activation of mRNAs is a fundamental mechanism to maintain tissue homeostasis. Our genetic experiments identified the Trf4/5-type cytoplasmic poly(A) polymerase (cytoPAP) GLD-4 and its enzymatic activator GLS-1 to perform a dual role in regulating the size of the proliferative zone. Consistent with a ubiquitous expression of GLD-4 cytoPAP in proliferative germ cells, its genetic activity is required to maintain a robust proliferative adult germ cell pool, presumably by regulating many mRNA targets encoding proliferation-promoting factors. Based on translational reporters and endogenous protein expression analyses, we found that gld-4 activity promotes GLP-1/Notch receptor expression, an essential factor of continued germ cell proliferation. RNA-protein interaction assays documented also a physical association of the GLD-4/GLS-1 cytoPAP complex with glp-1 mRNA, and ribosomal fractionation studies established that GLD-4 cytoPAP activity facilitates translational efficiency of glp-1 mRNA. Moreover, we found that in proliferative cells the differentiation-promoting factor, GLD-2 cytoPAP, is translationally repressed by the stem cell factor and PUF-type RNA-binding protein, FBF. This suggests that cytoPAP-mediated translational activation of proliferation-promoting factors, paired with PUF-mediated translational repression of differentiation factors, forms a translational control circuit that expands the proliferative germ cell pool. Our additional genetic experiments uncovered that the GLD-4/GLS-1 cytoPAP complex promotes also differentiation, forming a redundant translational circuit with GLD-2 cytoPAP and the translational repressor GLD-1 to restrict proliferation. Together with previous findings, our combined data reveals two interconnected translational activation/repression circuitries of broadly conserved RNA regulators that maintain the balance between adult germ cell proliferation and differentiation.     

Histone H2A and H4 N-terminal Tails Are Positioned by the MEP50 WD Repeat Protein for Efficient Methylation by the PRMT5 Arginine Methyltransferase

The protein arginine methyltransferase PRMT5 is complexed with the WD repeat protein MEP50 (also known as Wdr77 or androgen coactivator p44) in vertebrates in a tetramer of heterodimers. MEP50 is hypothesized to be required for protein substrate recruitment to the catalytic domain of PRMT5. Here we demonstrate that the cross-dimer MEP50 is paired with its cognate PRMT5 molecule to promote histone methylation. We employed qualitative methylation assays and a novel ultrasensitive continuous assay to measure enzyme kinetics. We demonstrate that neither full-length human PRMT5 nor the Xenopus laevis PRMT5 catalytic domain has appreciable protein methyltransferase activity. We show that histones H4 and H3 bind PRMT5-MEP50 more efficiently compared with histone H2A(1–20) and H4(1–20) peptides. Histone binding is mediated through histone fold interactions as determined by competition experiments and by high density histone peptide array interaction studies. Nucleosomes are not a substrate for PRMT5-MEP50, consistent with the primary mode of interaction via the histone fold of H3-H4, obscured by DNA in the nucleosome. Mutation of a conserved arginine (Arg-42) on the MEP50 insertion loop impaired the PRMT5-MEP50 enzymatic efficiency by increasing its histone substrate K_m, comparable with that of Caenorhabditis elegans PRMT5. We show that PRMT5-MEP50 prefers unmethylated substrates, consistent with a distributive model for dimethylation and suggesting discrete biological roles for mono- and dimethylarginine-modified proteins. We propose a model in which MEP50 and PRMT5 simultaneously engage the protein substrate, orienting its targeted arginine to the catalytic site.     

A specific role for the C-terminal region of the Poly(A)-binding protein in mRNA decay

mRNA poly(A) tails affect translation, mRNA export and mRNA stability, with translation initiation involving a direct interaction between eIF4G and the poly(A)-binding protein Pab1. The latter factor contains four RNA recognition motifs followed by a C-terminal region composed of a linker and a PABC domain. We show here that yeast mutants lacking the C-terminal domains of Pab1 display specific synthetic interactions with mutants in the 5′-3′ mRNA decay pathway. Moreover, these mutations impair mRNA decay in vivo without significantly affecting mRNA export or translation. Inhibition of mRNA decay occurs through slowed deadenylation. In vitro analyses demonstrate that removal of the Pab1 linker domain directly interferes with the ability of the Pop2–Ccr4 complex to deadenylate the Pab1-bound poly(A). Binding assays demonstrate that this results from a modulation of poly(A) packaging by the Pab1 linker region. Overall, our results demonstrate a direct involvement of Pab1 in mRNA decay and reveal the modular nature of this factor, with different domains affecting various cellular processes. These data suggest new models involving the modulation of poly(A) packaging by Pab1 to control mRNA decay.     

The RNA-binding proteins PUF-5, PUF-6, and PUF-7 reveal multiple systems for maternal mRNA regulation during C. elegans oogenesis

In metazoans, many mRNAs needed for embryogenesis are produced during oogenesis and must be tightly regulated during the complex events of oocyte development. In C. elegans, translation of the Notch receptor GLP-1 is repressed during oogenesis and is then activated specifically in anterior cells of the early embryo. The KH domain protein GLD-1 represses glp-1 translation during early stages of meiosis, but the factors that repress glp-1 during late oogenesis are not known. Here, we provide evidence that the PUF domain protein PUF-5 and two nearly identical PUF proteins PUF-6 and PUF-7 function during a specific period of oocyte differentiation to repress glp-1 and other maternal mRNAs. Depletion of PUF-5 and PUF-6/7 together caused defects in oocyte formation and early embryonic cell divisions. Loss of PUF-5 and PUF-6/7 also caused inappropriate expression of GLP-1 protein in oocytes, but GLP-1 remained repressed in meiotic germ cells. PUF-5 and PUF-6/7 function was required directly or indirectly for translational repression through elements of the glp-1 3' untranslated region. Oogenesis and embryonic defects could not be rescued by loss of GLP-1 activity, suggesting that PUF-5 and PUF-6/7 regulate other mRNAs in addition to glp-1. PUF-5 and PUF-6/7 depletion, however, did not perturb repression of the maternal factors GLD-1 and POS-1, suggesting that subsets of maternal gene products may be regulated by distinct pathways. Interestingly, PUF-5 protein was detected exclusively during mid to late oogenesis but became undetectable prior to completion of oocyte differentiation. These results reveal a previously unknown maternal mRNA control system that is specific to late stages of oogenesis and suggest new functions for PUF family proteins in post-mitotic differentiation. Multiple sets of RNA-binding complexes function in different domains of the C. elegans germ line to maintain silencing of Notch/glp-1 and other mRNAs.