Caenorhabditis elegans germ granules accumulate hundreds of low translation mRNAs with no systematic preference for germ cell fate regulators

In animals with germ plasm, embryonic germline precursors inherit germ granules, condensates proposed to regulate mRNAs coding for germ cell fate determinants. In C. elegans, mRNAs are recruited to germ granules by MEG-3, a sequence non-specific RNA-binding protein that forms stabilizing interfacial clusters on germ granules. Using fluorescent in situ hybridization, we confirmed that 441 MEG-3-bound transcripts distribute in a pattern consistent with enrichment in germ granules. 13 are related to transcripts reported in germ granules in Drosophila or Nasonia. The majority, however, are low-translation maternal transcripts required for embryogenesis that are not maintained preferentially in the nascent germline. Granule enrichment raises the concentration of certain transcripts in germ plasm but is not essential to regulate mRNA translation or stability. Our findings suggest that only a minority of germ granule-associated transcripts contribute to germ cell fate in C. elegans and that th..., For single molecule fluorescence in situ hybridization (smFISH), embryos were extruded from adult animals and subjected to freeze-crack on 0.01% poly-lysine coated slides, followed by fixation in -20°C methanol overnight. Slides were washed once in 1:1 methanol:PBSTw, five times in PBSTw (1X PBS + 0.1% Tween-20), and fixed in 4% paraformaldehyde (PFA) in PBS for 1 hour at room temperature. Fixed slides were washed four times in PBSTw, twice in 2x SSC, once in wash buffer (10% formamide, 2x SSC), and blocked in hybridization buffer (10% formamide, 2x SSC, 200ug/ml BSA, 2mM Ribonucleoside Vanadyl Complex, 0.2mg/ml yeast total RNA, 10% dextran sulfate) for 30 minutes at 37°C in a humid chamber. Slides were incubated with probes in hybridization buffer at 37°C overnight, washed twice in wash buffer at 37°C for 30 min, twice in 2x SSC, once in PBSTw, and twice in PBS before mounting in ProLong Glass Antifade Mountant with NucBlue Stain and cured overnight.
For the initial in situ screen, ima..., , # *Caenorhabditis elegans* germ granules accumulate hundreds of low translation mRNAs with no systematic preference for germ cell fate regulators

[https://doi.org/10.5061/dryad.02v6wwq98](https://doi.org/10.5061/dryad.02v6wwq98)

*in situ* screen of MEG-3-bound RNAs identified in Lee et al., 2020. Images were collected across 7 stages (2-cell, 4-cell, 8-cell, 28-cell, ~40-cell, ~60-cell, ~100-cell). 2-cell, 4-cell, 8-cell and 28-cell embryos were scored manually for granules visible in P1, P2, P3, and P4 blastomeres (P blastomeres are readily identified at these stages based on their position in the embryo). Transcripts positive for granules were classified as Group I or II, and transcripts negative for granules were classified as Group III. 

The distinction between Group I and Group II transcripts was made by examining ~40-cell, ~60-cell, ~100-cell stage embryos. Transcripts that showed consistent enrichment in one cell (40-cell and 60-cell stages) and two cells (100-cell stage) acro...