Novel LOTUS-domain proteins are organizational hubs that recruit C. elegans Vasa to germ granules

Abstract

We describe MIP-1 and MIP-2, novel paralogous C. elegans germ granule components that interact with the intrinsically disordered MEG-3 protein. These proteins promote P granule condensation, form granules independently of MEG-3 in the postembryonic germ line and balance each other in regulating P granule growth and localization. MIP-1 and MIP-2 each contain two LOTUS domains and intrinsically disordered regions and form homo- and heterodimers. They bind and anchor the Vasa homolog GLH-1 within P granules and are jointly required for coalescence of MEG-3, GLH-1, and PGL proteins. Animals lacking MIP-1 and MIP-2 show temperature-sensitive embryonic lethality, sterility, and mortal germ lines. Germline phenotypes include defects in stem cell self-renewal, meiotic progression, and gamete differentiation. We propose that these proteins serve as scaffolds and organizing centers for ribonucleoprotein networks within P granules that help recruit and balance essential RNA processing machinery to regulate key developmental transitions in the germ line.

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

“…However, three LOTUS containing proteins have recently been identified: MIP-1, MIP-2, and D1081.7. [71] We find that D1081.7 is in germ granules and interacts with both MIP-1 and MIP-2. D1081.7 is the only known C. elegans protein to pair LOTUS and Tudor domains, similar to both TDRD5 and TDRD7, so we have named it LOTUS and TudoR containing protein 1 (LOTR-1).…”

“…RNAi depletion of mip-1 and mip-2 together causes the dispersal of PGL-1, PGL-3, and GLH-1. [71] Similarly, GFP-tagged LOTR-1 granules are reduced in size and less prominent around the nuclear periphery following mip-1; mip-2 RNAi (Fig 2F). These results suggest that the LOTUS-domain proteins MIP-1, and MIP-2 affect the localization of LOTR-1 at germ granules.…”

“…Bait-prey interactions were done on SC-Leu-Trp-Ade plates or SC-Leu-Trp-His plates supplemented with varying concentrations of 3-amino-1,2,4-triazole, as previously described. [71] Reporters for small RNA activity Experiments were performed as previously described. [82,84,86,115]…”

“…Paired LOTUS domains reflect the arrangement of TDRD5 and TDRD7 in mammals and two proteins recently described in C. elegans called MIP-1 and MIP-2 (S1 Fig). [71] Default BLAST parameters fail to uncover the Tudor domain within D1081.7, but it readily appears in domain-enhanced searches and was previously identified using multiple sequence alignment. [12] The Tudor domain (aa 534-670) is most similar to Tudor domain 3 (TD3) of mouse TDRD1, which is known to bind symmetrically dimethylated arginine (sDMA) marks through a hydrophobic pocket that is created by the arrangement of four aromatic residues (Fig 1C, F767, Y774, Y791, and Y794).…”

“…Next, LOTR-1 localization was examined in the absence of two other recently characterized LOTUScontaining proteins, MIP-1 and MIP-2. [71] MIP-1 and MIP-2 are MEG-3 interacting proteins that localize to P granules throughout development and promote germ granule condensation. Although MIP-1 and MIP-2 lack Tudor domains, they each contain two eLOTUS domains that could indicate some functional redundancy with LOTR-1.…”