Project description:An extended meiotic prophase is a hallmark of oogenesis. Hormonal signaling activates the CDK1/cyclin B kinase to promote oocyte meiotic maturation, which involves nuclear and cytoplasmic events. Nuclear maturation encompasses nuclear envelope breakdown, meiotic spindle assembly, and chromosome segregation. Cytoplasmic maturation involves major changes in oocyte protein translation and cytoplasmic organelles and is poorly understood. In the nematode Caenorhabditis elegans, sperm release the major sperm protein (MSP) hormone to promote oocyte growth and meiotic maturation. Large translational regulatory ribonucleoprotein (RNP) complexes containing the RNA-binding proteins OMA-1, OMA-2, and LIN-41 regulate meiotic maturation downstream of MSP signaling. To understand the control of translation during meiotic maturation, we purified LIN-41-containing RNPs and characterized their protein and RNA components. Protein constituents of LIN-41 RNPs include essential RNA-binding proteins, the GLD-2 cytoplasmic poly(A) polymerase, the CCR4-NOT deadenylase complex, and translation initiation factors. RNA sequencing defined mRNAs associated with both LIN-41 and OMA-1, as well as sets of mRNAs associated with either LIN-41 or OMA-1. Genetic and genomic evidence suggests that GLD-2, which is a component of LIN-41 RNPs, stimulates the efficient translation of many LIN-41-associated transcripts. We analyzed the translational regulation of two transcripts specifically associated with LIN-41 that encode the RNA regulators SPN-4 and MEG-1. We found that LIN-41 represses translation of spn-4 and meg-1, whereas OMA-1 and OMA-2 promote their expression. Upon their synthesis, SPN-4 and MEG-1 assemble into LIN-41 RNPs prior to their functions in the embryo. This study defines a translational repression-to-activation switch as a key element of cytoplasmic maturation.
Project description:An extended meiotic prophase is a hallmark of oogenesis. Hormonal signaling activates the CDK1/cyclin B kinase to promote oocyte meiotic maturation, which involves nuclear and cytoplasmic events. Nuclear maturation encompasses nuclear envelope breakdown, meiotic spindle assembly, and chromosome segregation. Cytoplasmic maturation involves major changes in oocyte protein translation and cytoplasmic organelles and is poorly understood. In the nematode Caenorhabditis elegans, sperm release the major sperm protein (MSP) hormone to promote oocyte growth and meiotic maturation. Large translational regulatory ribonucleoprotein (RNP) complexes containing the RNA-binding proteins OMA-1, OMA-2, and LIN-41 regulate meiotic maturation downstream of MSP signaling. To understand the control of translation during meiotic maturation, we purified LIN-41-containing RNPs and characterized their protein and RNA components. Protein constituents of LIN-41 RNPs include essential RNA-binding proteins, the GLD-2 cytoplasmic poly(A) polymerase, the CCR4-NOT deadenylase complex, and translation initiation factors. RNA sequencing defined mRNAs associated with both LIN-41 and OMA-1, as well as sets of mRNAs associated with either LIN-41 or OMA-1. Genetic and genomic evidence suggests that GLD-2, which is a component of LIN-41 RNPs, stimulates the efficient translation of many LIN-41-associated transcripts. We analyzed the translational regulation of two transcripts specifically associated with LIN-41 that encode the RNA regulators SPN-4 and MEG-1. We found that LIN-41 represses translation of spn-4 and meg-1, whereas OMA-1 and OMA-2 promote their expression. Upon their synthesis, SPN-4 and MEG-1 assemble into LIN-41 RNPs prior to their functions in the embryo. This study defines a translational repression-to-activation switch as a key element of cytoplasmic maturation.
Project description:The GLD-2 class of poly(A) polymerases regulate the timing of translation of stored transcripts by elongating the poly(A) tails of target mRNAs in the cytoplasm. WISPY is a GLD-2 enzyme that acts in the Drosophila female germline and is required for the completion of the egg-to-embryo transition. Though a handful of WISPY target mRNAs have been identified during both oogenesis and early embryogenesis, we aimed to discover the full range of WISPY targets at each stage. To globally identify these targets, we carried out microarray analysis to look for maternal mRNAs whose poly(A) tails fail to elongate in the absence of WISP function. We examined the polyadenylated portion of the maternal transcriptome in both stage 14 (mature) oocytes and in early embryos that had completed egg activation. Our analysis shows that the poly(A) tails of thousands of maternal mRNAs fail to elongate in wisp-deficient oocytes and embryos. Furthermore, we have identified specific classes of genes that are highly regulated in this manner at each stage. Our study shows that cytoplasmic polyadenylation is a major regulatory mechanism during oocyte maturation and egg activation.
Project description:The GLD-2 class of poly(A) polymerases regulate the timing of translation of stored transcripts by elongating the poly(A) tails of target mRNAs in the cytoplasm. WISPY is a GLD-2 enzyme that acts in the Drosophila female germline and is required for the completion of the egg-to-embryo transition. Though a handful of WISPY target mRNAs have been identified during both oogenesis and early embryogenesis, we aimed to discover the full range of WISPY targets at each stage. To globally identify these targets, we carried out microarray analysis to look for maternal mRNAs whose poly(A) tails fail to elongate in the absence of WISP function. We examined the polyadenylated portion of the maternal transcriptome in both stage 14 (mature) oocytes and in early embryos that had completed egg activation. Our analysis shows that the poly(A) tails of thousands of maternal mRNAs fail to elongate in wisp-deficient oocytes and embryos. Furthermore, we have identified specific classes of genes that are highly regulated in this manner at each stage. Our study shows that cytoplasmic polyadenylation is a major regulatory mechanism during oocyte maturation and egg activation. Four groups of comparisons: WT vs. wisp total RNA from stage 14 oocytes, WT vs. wisp total RNA from fertilized eggs, WT vs. wisp poly(A)+ RNA from stage 14 oocytes, WT vs. wisp poly(A)+ RNA from fertilized eggs. Each comparison consisted of three independent RNA extractions and each experiment was done with dye-swap pairs as two technical replicates.
Project description:Comparision of mRNA abundance and translation efficiency mediated by cytoplasmic poly(A) polymerases in C. elegans comparision of different adults treated with RNAi