Distinct regions of the intrinsically disordered protein MUT-16 mediate assembly of a small RNA amplification complex and promote phase separation of Mutator foci

Abstract

In C. elegans, efficient RNA silencing requires small RNA amplification mediated by RNA-dependent RNA polymerases (RdRPs). RRF-1, an RdRP, and other Mutator complex proteins localize to Mutator foci, which are perinuclear germline foci that associate with nuclear pores and P granules to facilitate small RNA amplification. The Mutator complex protein MUT-16 is critical for Mutator foci assembly. By analyzing small deletions of MUT-16, we identify specific regions of the protein that recruit other Mutator complex components and demonstrate that it acts as a scaffolding protein. We further determine that the C-terminal region of MUT-16, a portion of which contains predicted intrinsic disorder, is necessary and sufficient to promote Mutator foci formation. Finally, we establish that MUT-16 foci have many properties consistent with a phase-separated condensate and propose that Mutator foci form through liquid-liquid phase separation of MUT-16. P granules, which contain additional RNA silencing proteins, have previously been shown to have liquid-like properties. Thus, RNA silencing in C. elegans germ cells may rely on multiple phase-separated compartments through which sorting, processing, and silencing of mRNAs occurs.

Author summary

Small RNAs are a driving force behind the regulation of both essential genes and deleterious transcripts. The Mutator complex is critical to the amplification of high levels of small RNAs and it requires the protein MUT-16 for its assembly. Here we investigate the function of MUT-16 by generating small deletions in the mut-16 gene. Through analysis of the subsequently altered protein, we demonstrate that MUT-16 functions as a scaffold, bringing together many other proteins required for small RNA biogenesis and amplification. Furthermore, we identified a fragment of MUT-16 that is sufficient to promote assembly of MUT-16 into foci that are dynamic and responsive to environmental conditions. We propose that these Mutator foci behave like liquid droplets within the cell, similar to the immiscibility of oil droplets in water. Mutator foci localize to the periphery of germ cell nuclei near P granules, which also have liquid-like properties and contain many factors involved in RNA silencing. Thus, our data suggest that RNA silencing is mediated by compartments of RNAs and proteins in liquid-like assemblies at the periphery of germ cell nuclei.