Two germ granule eIF4E isoforms reside in different mRNPs to hand off C elegans mRNAs from translational repression to activation.

We studied the function of translation factor eIF4E isoforms in regulating mRNAs in germ cell granules/condensates. Translational control of mRNAs plays an essential role in germ cell gene regulation. Messenger ribonucleoprotein (mRNP) complexes assemble on mRNAs as they move from the nucleus into perinuclear germ granules to exert both positive and negative post-transcriptional regulation in the cytoplasm. In C. elegans , germ granules are surprisingly dynamic mRNP condensates that remodel during development. Two eIF4E isoforms (called IFE-1 and IFE-3), eIF4E-Interacting Proteins (4EIPs), RBPs, DEAD-box helicases, polyadenylated mRNAs, Argonautes and miRNAs all occupy positions in germ granules. Affinity purification of IFE-1 and IFE-3 allowed mass spectrometry and mRNA-Seq to identify the proteins and mRNAs that populate stable eIF4E mRNPs. We find translationally controlled mRNAs (e.g. pos-1, mex-3, spn-4, etc.) enriched in IFE-3 mRNPs, but excluded from IFE-1 mRNPs. These mRNAs also require IFE-1 for efficient translation. The findings support a model in which oocytes and embryos utilize the two eIF4Es for opposite purposes on critically regulated germline mRNAs. Careful colocalization of the eIF4Es with other germ granule components suggests an architecture in which GLH-1, PGL-1 and the IFEs are stratified to facilitate sequential interactions for mRNAs. Biochemical characterization demonstrates opposing yet cooperative roles for IFE-3 and IFE-1 to hand-off of translationally controlled mRNAs from the repressed to the activated state, respectively. The model involves eIF4E mRNPs shuttling mRNAs through nuclear pore-associated granules/condensates to cytoplasmic ribosomes.

The Caenorhabditis elegans TDRD5/7-like protein, LOTR-1, interacts with the helicase ZNFX-1 to balance epigenetic signals in the germline.

LOTUS and Tudor domain containing proteins have critical roles in the germline. Proteins that contain these domains, such as Tejas/Tapas in Drosophila, help localize the Vasa helicase to the germ granules and facilitate piRNA-mediated transposon silencing. The homologous proteins in mammals, TDRD5 and TDRD7, are required during spermiogenesis. Until now, proteins containing both LOTUS and Tudor domains in Caenorhabditis elegans have remained elusive. Here we describe LOTR-1 (D1081.7), which derives its name from its LOTUS and Tudor domains. Interestingly, LOTR-1 docks next to P granules to colocalize with the broadly conserved Z-granule helicase, ZNFX-1. The Tudor domain of LOTR-1 is required for its Z-granule retention. Like znfx-1 mutants, lotr-1 mutants lose small RNAs from the 3' ends of WAGO and mutator targets, reminiscent of the loss of piRNAs from the 3' ends of piRNA precursor transcripts in mouse Tdrd5 mutants. Our work shows that LOTR-1 acts with ZNFX-1 to bring small RNA amplifying mechanisms towards the 3' ends of its RNA templates.

Quantification of tissue-specific protein translation in whole C. elegans using O-propargyl-puromycin labeling and fluorescence microscopy.

The regulation of gene expression via protein translation is critical for growth, development, and stress response. While puromycin-based techniques have been used to quantify protein translation in C. elegans, they have been limited to using lysate from whole worms. To achieve tissue-specific quantification of ribosome activity in intact C. elegans, we report the application of O-propargyl-puromycin in a cuticle defective mutant followed by conjugation of an azide fluorophore for detection using fluorescent confocal microscopy. We apply this technique to quantify translation in response to heat shock, cycloheximide, or knockdown of translation factors. Furthermore, we demonstrate that O-propargyl-puromycin can be used to quantify translation between tissues or within a tissue like the germline. This technique is expected to have a broad range of applications in determining how protein translation is altered in different tissues in response to stress or gene knockdowns or with age.