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

Abstract

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.

Fig 1. LOTR-1 contains both LOTUS and Tudor domains, and colocalizes with Z granules.

A) Top schematic depicts the location of the eLOTUS, mLOTUS and Tudor domain of LOTR-1. Bottom depicts the CRISPR edit used to add an N terminal 3X FLAG tag and GFP. Alleles generated for this study are indicated. B) Predicted 3D model overlap of the eLOTUS and mLOTUS domains of C. elegans LOTR-1 with the Oskar eLOTUS domain from Drosophila melanogaster. The α5 helix (highlighted) is only present in the eLOTUS domain. C) Predicted 3D structure of the Tudor domain (aa 534–670) of LOTR-1 overlapped with TD3 domain of mouse TDRD1. D) GFP::LOTR-1 and PGL-1::RFP in the germline of living worms. E) Super-resolution confocal imaging of GFP::LOTR-1 with PGL-1::RFP (top) and GFP::LOTR-1 with RFP::ZNFX-1 (bottom) in pachytene germ cells. F) Comparison of the center of mass difference from LOTR-1 for PGL-1 or ZNFX-1.

Fig 2. Germ-granule phenotypes in lotr-1 germlines.

A) Live-imaging of young adults shows the distribution of LOTR-1::GFP in the presence and absence of its LOTUS and Tudor domains. B) LOTR-1 distribution during spermatogenesis in the fourth larval stage (L4). Orange boxes indicate region of spermatogenesis. Live imaging of mCherry::PRG-1 and GFP::LOTR-1 in C) L4 stage and D) young adult germlines. E) Live imaging of RFP::ZNFX-1 in the germline of wild-type and lotr-1 mutant adults. F) Comparison of GFP::LOTR-1 and mCherry::PRG-1 expression in the germlines in znfx-1 mutant (top) and in lotr-1; znfx-1 double mutant (bottom) adults. G) RNAi depletion of mip-1 and mip-2 in GFP::LOTR-1 compared to empty vector control RNAi. H) lotr-1 RNAi compared to empty vector control RNAi in adult germlines of GFP-tagged LOTR-1, GLH-1, PGL-3, DEPS-1, MIP-1 and MIP-2 worms. Scale is 20 microns. Quantification provided in S2 Fig.

Fig 3. Quantitative IP-mass spectrometry analysis of LOTR-1 and ZNFX-1 immunoprecipitations.

A) Volcano plots show the significance and enrichment of proteins that immunoprecipitated with 3xFLAG::GFP::LOTR-1 over the lotr-1 deletion expressing GFP::3xFLAG alone, as identified by qMS. B) Venn diagram shows significantly enriched proteins that overlapped between two rounds of LOTR-1 IP-qMS from both embryo and young adult lysates. C) Table showing the change in LOTR-1 association in embryos and young adults when LOTUS or Tudor domains of LOTR-1 were deleted. D) Volcano plots show the significance and enrichment of proteins immunoprecipitated with 3xFLAG::GFP::ZNFX-1 over untagged ZNFX-1, as identified by qMS. E) Venn diagram shows significantly enriched proteins that overlapped between LOTR-1 and ZNFX-1 IP-qMS from both embryo and young adult lysates. F) Table showing the change in ZNFX-1 association in embryos and young adults when lotr-1 was mutated. For C and F, a purple gradient was applied to visualize fold increases (>1, darker) and decreases (<1, lighter) from the C) LOTR-1 and F) ZNFX-1 associations identified in the wild type (WT) replicates.

Fig 4. 26G- and 22G-RNA deregulation in lotr-1 mutants.

A) Relative levels of the indicated small RNA populations normalized to all non-structural reads. B) Differential expression analysis to determine genes and transposons that are significantly depleted or enriched of mapped small RNAs in lotr-1 mutants. The MA plots depict DESeq2 differential results for miRNAs (n = 257), 21U-RNAs over 21ur loci (n = 14328), and 22G-/26G-RNAs over protein coding genes (n = 20222), lincRNAs (n = 172), pseudogenes (n = 1791) and transposons (n = 151); significant changes (>2-fold at 10% FDR) are colored in red with the number of up- and down-regulated hits indicated. C-E) Metagene plots depicting relative (normalized per individual gene as in [71], upper panels) as well as total (lower panels) 22G-RNA coverage over genes whose 22G-RNAs are up-regulated, down-regulated or unaffected in the lotr-1 mutants. Note that the total (cumulative) coverage plots utilize DESeq2-based normalization for library composition and are biased towards genes with strong 22G-RNA levels. TSS/TES, transcription start/end site.

Fig 5. RNAi sensitivity and inheritance in lotr-1 mutants.

A) Sensitivity of two different lotr-1 mutant alleles against pos-1 RNAi, using mut-7(xf125) mutants as a known pos-1 RNAi resistant control. Hatching proportion (y-axis) was scored; horizontal blue lines represent the fitted hatching probabilities. P-values were determined using Likelihood Ratio Tests. Error bars: bootstrap 95% confidence intervals for hatching probabilities. B) Inheritance of RNAi was tested using RNAi against GFP, targeting a transgene expressing a GFP::histone2B fusion (mjIS31) in oocytes [50]. Silencing in the treated P0 generation was fully effective, as was the inheritance of silencing to the F1. C) Transgenerational inheritance assay using RNAi against a GFP::histone2B transgene (ruIs32) in WT and the deletion allele lotr-1(usa1) revealed enhanced silencing up to 10 generations in the absence of LOTR-1. D) A model summarizing how LOTR-1, together with ZNFX-1, could affect 22G RNA production from an RdRP-targeted transcript. We note that all interactions are based on IP-qMS and Y2H experiments. Since these techniques do not report on whether a given interaction reflects a stable complex, or rather a more transient type of interaction, further study will be required to define the precise composition and nature of the associated molecular machinery.