Kim, Seongseop et al. (2013) International Worm Meeting "The SACY-1 DEAD-box RNA helicase genetically interacts with components of the spliceosome."

Tsukamoto, Tatsuya, Greenstein, David, Kim, Seongseop

[International Worm Meeting, 2013]

In C. elegans, major sperm protein triggers oocyte meiotic maturation through a mechanism involving somatic Gas-adenylate cyclase-protein kinase A (PKA) signaling, gap-junctional communication, and translational regulation by OMA-1/2. Previously we identified sacy-1 as a negative regulator of meiotic maturation, functioning in the germ line downstream of PKA signaling. Genetic analysis established that sacy-1 also functions in the hermaphrodite sperm-to-oocyte switch and is required for gamete maintenance. To isolate additional alleles of sacy-1, we screened for mutations that fail to complement sacy-1(tn1385rf) for the suppression of fog-2(lf) self-sterility. From 15,577 mutagenized haploid genomes, we isolated five new sacy-1 missense alleles affecting conserved residues in the DEAD-box helicase domain. sacy-1(tn1481), which affects the ATP-binding Q-motif, exhibits a masculinization of the germ line phenotype. This result is consistent with the conclusion that sacy-1 promotes the oocyte fate. sacy-1(tn1479) exhibits gamete degeneration and sterility similar to the sacy-1(tm5503) likely null allele; however, sacy-1(tn1479) differs from sacy-1(tm5503) in that the majority of the former, but not the latter, bursts as adults. This result suggests that sacy-1(tn1479) may possess a poisoning activity. Finally, sacy-1(tn1480) exhibits temperature-sensitive sterility. We conducted a genome-wide RNAi screen for sacy-1 enhancers to address its molecular function. This screen identified three enhancer loci, mog-2, emb-4, and cacn-1. RNAi of each of these three loci caused sterility (emb-4 and cacn-1) or embryonic lethality (mog-2) in sacy-1(tn1385rf) but not in the wild type under the RNAi conditions utilized. Orthologs of sacy-1 and these enhancer loci were identified biochemically as components of spliceosomal complexes. Whether the multiple sacy-1 germline phenotypes result from defects in pre-mRNA splicing or translational regulation remains to be determined.

Spike, Caroline et al. (2015) International Worm Meeting "The NHL-TRIM Protein LIN-41 is a Major Determinant of the Extended Meiotic Prophase of C. elegans Oocytes."

Greenstein, David, Tsukamoto, Tatsuya, Spike, Caroline

[International Worm Meeting, 2015]

An extended meiotic prophase is a hallmark of oogenesis in sexually reproducing animals. Arrest in meiotic prophase I coincides with the period of growth that presumably enables oocytes to acquire the cytoplasmic components needed to produce healthy progeny and to gain competence to complete meiosis. Meiotic resumption (also called meiotic maturation) involves the transition to metaphase I, and is triggered by maturation-promoting factor, which consists of the Cdk1 catalytic subunit and the cyclin B regulatory subunit. Because full-grown oocytes of most animals are transcriptionally quiescent, translational regulation is a major control point. Biochemical and genetic results suggest that LIN-41 controls mRNA translation to coordinate and control oocyte growth and meiotic maturation downstream of intercellular signaling. In the absence of LIN-41, pachytene-stage oocytes cellularize prematurely and fail to progress to diplotene. Instead, these cells activate CDK-1, enter M phase, and attempt to segregate chromosomes. Translational derepression of the CDK-1 activator CDC-25.3 contributes to premature M-phase entry in lin-41 mutant oocytes. LIN-41 copurifies with regulators of cytoplasmic polyadenylation, including the GLD-2 poly(A) polymerase and its cofactor GLD-3, which are thought to function as translational activators, suggesting that LIN-41 might regulate translational activation and repression. Consistent with this idea, the premature M-phase entry defect in lin-41 mutants requires gld-2 function-lin-41 gld-2 double mutant oocytes arrest in pachytene. Genetic analysis using classical and CRISPR-Cas9-induced alleles suggests the NHL domain, which constitutes a novel RNA-binding domain, is a chief determinant of LIN-41 function. Our results suggest that LIN-41 functions as a molecular "stop sign" that imposes prophase arrest. Further, genetic analysis also reveals that LIN-41 is not simply an arbiter of the meiotic maturation decision; its proper function is also required to form high-quality oocytes and to prevent aneuploidy. Upon the onset of meiotic maturation, LIN-41 is degraded in a CDK-1-dependent manner. Thus LIN-41 controls and coordinates oocyte growth and meiotic maturation to enable successful fertilization.

Kwah, Ji Kent et al. (2021) International Worm Meeting "Role of spe-11 and oops-1 in early embryogenesis and eggshell formation"

Jaramillio-Lambert, Aimee, Tsukamoto, Tatsuya, Kwah, Ji Kent, Greenstein, David

[International Worm Meeting, 2021]

Gametes provide a variety of cellular and genetic materials during their fusion to form a zygote. Although the majority of proteins required for embryogenesis are maternally provided and present in the oocyte, sperm contributions are also essential to this process. In the model organism C. elegans, SPE-11 is a sperm protein identified to be paternal-effect embryonic lethal. It was experimentally shown that homozygous mutant sperm (from hermaphrodite or male) will always lead to non-viable embryos regardless of the source of oocyte. However, oocytes from a spe-11 mutant can be successfully fertilized when sperm from a wild type male is provided therefore confirming SPE-11 function can only be provided by the paternal gamete. spe-11 mutant single cell embryos are defective in a myriad of ways such as inability to complete the meiotic divisions and cytokinesis, improper spindle formation, and weak eggshell formation. Currently, little is known about the role and function of the SPE-11 protein as it has no known homology to any known domains. Recently, an interacting partner of SPE-11 was identified and given the name oocyte partner of SPE-11 (OOPS-1). As these two proteins were found to physically interact, we asked if mutant versions had similar phenotypes. Similar to spe-11 mutants, we found that deletion mutants of oops-1 are embryonic lethal indicating that the OOPS-1 protein is required for the embryogenesis process. We are using fluorescently labeled histones and meiotic spindles to image oocyte meiosis in vivo in wild-type, spe-11(hc90), and oops-1(tn1898) strains. Interestingly, while both spe-11 and oops-1 mutants have defects in oocyte meiosis, the two mutants have slightly different phenotypes. As previously published, in spe-11(hc90) mutant embryos, the chromosomes segregate at anaphase I and II but fail to form polar bodies. In contrast, it appears that oops-1(tn1898) can complete meiosis I but cannot enter anaphase II. The exact timing and the analysis of other meiotic division phenotypes is still ongoing. Alongside this, we are also investigating the integrity of the embryo eggshell in these mutants.

Jha, Sweta et al. (2021) International Worm Meeting "UPS modulation and autophagy."

Jha, Sweta, Holmberg, Carina

[International Worm Meeting, 2021]

The ubiquitin-proteasome system (UPS) and the autophagy-lysosomal pathway (ALP) are the two main eukaryotic intracellular proteolytic systems involved in maintaining proteostasis. Autophagy is responsible for degrading defective cellular organelles and long-lived proteins in the cytosol and is involved in cell growth, survival, development and death. UPS mostly degrades soluble and short-lived proteins in the nucleus and cytosol and affects various cellular processes. These two proteolytic systems are essential components of the cellular protein quality control system. Several studies have reported an interplay between the UPS and ALP, however it still remains largely unknown how these systems communicate in a multicellular organism. We have recently shown that downregulation of autophagy genes elicits tissue-specific effects on UPS function in C. elegans1. Here, we address how genetic or pharmacological modulation of the proteasome and the proteasome-associated DUBs affects autophagy. We use transgenic C. elegans expressing the autophagy reporters GFP::LGG-12 or mCherry::GFP::LGG-13 and analyze the accumulation of LGG-1 puncta and autophagic flux. Our preliminary data show that downregulation of the proteasome-associated DUBs ubh-4, usp-14 and rpn-11 as well as some proteasomal subunits affect the number of puncta positive for autophagosomes in hypodermal seam cells, intestinal cells as well as in pharynx. To nvestigate a conserved function of the UPS on autophagy in human cells, we are utilizing a HeLa cell line expressing the fluorescence probe GFP::LC3::RFP::LC3G4. A better understanding of the multilayered crosstalk between UPS and ALP in vivo may facilitate development of therapeutic options for various disorders linked to dysfunction in proteostasis. References Jha, S., Holmberg, C.I. Tissue-Specific Impact of Autophagy Genes on the Ubiquitin-Proteasome System in C. elegans. Cells 2020, 9, 1858. Melendez A, Talloczy Z, Seaman M, Eskelinen E-L, Hall DH, Levine B. Autophagy genes are essential for dauer develop-ment and life-span extension in C. elegans. Science 2003. 301:1387-91 Chang J.T., Kumsta C., Hellman A.B., Adams L.M., Hansen M. (2017). Spatiotemporal regulation of autophagy during caenorhabditis elegans aging. Elife. pii: e18459 Kaizuka T., Morishita H., Hama Y., Tsukamoto S., Matsui T., Toyota Y., Kodama A., Ishihara T., Mizushima T., Mizushima N. (2016). An autophagic flux probe that releases an internal control. Mol Cell. 64(4):835-849.