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

<div><p>In <i>C</i>. <i>elegans</i>, efficient RNA silencing requires small RNA amplification mediated by RNA-dependent RNA polymerases (RdRPs). RRF-1, an RdRP, and other <i>Mutator</i> complex proteins localize to <i>Mutator</i> foci, which are perinuclear germline foci that associate with nuclear pores and P granules to facilitate small RNA amplification. The <i>Mutator</i> complex protein MUT-16 is critical for <i>Mutator</i> foci assembly. By analyzing small deletions of MUT-16, we identify specific regions of the protein that recruit other <i>Mutator</i> 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 <i>Mutator</i> foci formation. Finally, we establish that MUT-16 foci have many properties consistent with a phase-separated condensate and propose that <i>Mutator</i> 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 <i>C</i>. <i>elegans</i> germ cells may rely on multiple phase-separated compartments through which sorting, processing, and silencing of mRNAs occurs.</p></div

Distinct regions of MUT-16 recruit each of the other <i>Mutator</i> proteins.

<p>(A) Table indicates whether <i>mut-16</i> deletions disrupt MUT-2, RDE-8, NYN-1, MUT-14, RRF-1, and RDE-2 foci. Yes indicates foci present in the majority of animals, No indicates foci absent or severely disrupted, and ND indicates that strain was not constructed or scored. (B-D) MUT-16::mCherry and MUT-2::GFP expression and localization for control strain (B) or when ΔC (C) or ΔK (D) deletions have been introduced into the <i>mut-16</i>::<i>mCherry</i> strain. Scale bars, 5μm. (E) Immunoprecipitation and western blot of MUT-16::mCherry::2xHA (expected sizes between 135–141 kD for MUT-16 deletions and 148 kD for MUT-16 full length) and MUT-2::GFP::3xFLAG (83 kD). Left panels are total lysate from strains indicated above, and right panels are following HA immunoprecipitation. (F) Immunoprecipitation and western blot of MUT-16::mCherry::2xHA (expected sizes between 132–142 kD for MUT-16 deletions and 148 kD for MUT-16 full length) and GFP::3xFLAG::RRF-1 (219 kD). Top two panels are total lysate from strains indicated above, and bottom two panels are following HA immunoprecipitation. The equivalent of ~0.5% of starting material for the input fractions and ~20% of starting material for the IP fractions were loaded onto the gels.</p

Susceptibility of <i>mut-16</i> deletion worms to somatic and germline RNAi.

<p>(A-C) <i>mut-16</i> deletion worms were assayed for their ability to respond to somatic (<i>nhr-23</i> or <i>lin-29</i>) or germline (<i>pos-1</i>) RNAi. For <i>nhr-23</i> RNAi (A) and <i>lin-29</i> RNAi (B), P0 animals were scored as having intermediate RNAi defects (gray bars), or fully penetrant RNAi defects (red bars). For <i>pos-1</i> RNAi (C), the F1 eggs and hatched larvae were counted to calculate % viable progeny from treated P0 animals. Weighted means and standard deviations were calculated from three independent RNAi trials of n = ~20 for <i>nhr-23</i> and <i>lin-29</i>, and n = ~90 F1 eggs from 4 P0 adults for <i>pos-1</i>.</p

MUT-16 and orthologs contain a high degree of predicted disorder.

<p>(A) Graph comparing disorder tendency of <i>C</i>. <i>elegans</i> MUT-16 (isoform b, WP:CE40347) in red to orthologs in <i>C</i>. <i>remanei</i> (RP:RP48608) in light gray, <i>C</i>. <i>briggsae</i> (BP:CBP44329) in medium gray, and <i>C</i>. <i>japonica</i> (JA:JA63728) in dark gray using IUPRED and long disorder parameters (<a href="http://iupred.enzim.hu/" target="_blank">http://iupred.enzim.hu/</a>). Scores above 0.5 indicate disorder. Residue positions correspond to the <i>C</i>. <i>elegans</i> MUT-16 sequence, but all proteins are similar in length. (B) Q, N, and P residues in <i>C</i>. <i>elegans</i> MUT-16 were counted in amino acid 100-mers, starting at position one, shifting 10 residues at a time, and displayed as stacked columns. Indicated residue positions are the mid-point of the 100-mer. (C) Schematic of known dependencies of <i>Mutator</i> foci formation. The requirements for localization of RRF-1, RDE-8, NYN-1, NYN-2, and SMUT-1 were not known prior to this work. (D, E) Diagram (D) and table (E) of MUT-16 deletions generated by CRISPR. Bars in (D) are drawn to scale relative to residue positions in (A,B).</p

Concentration dependence of <i>Mutator</i> foci.

<p>(A) GFP expression in negative control (wild-type) and <i>mut-16p</i>::<i>gfp</i> L4 stage animals. The same exposure time was used for both images. Scale bar, 50μm. (B) Quantification of GFP fluorescence in the soma and the germline of <i>mut-16p</i>::<i>gfp</i> animals. Fluorescence intensity was calculated using ImageJ by tracing the germline and soma of 31 L4 stage animals and measuring the mean gray value. P values were calculated using a paired t-test (*** indicates p<0.001). (C) Top row: MUT-16::GFP overexpressed from the <i>myo-3</i> promoter forms foci in muscle cells marked with muscle-specific mCherry. Yellow arrow indicates the location of the nucleus. Middle row: Endogenously expressed MUT-16::GFP in muscle cells marked with muscle-specific mCherry does not form foci. White arrows point to MUT-16 foci in the neighboring germ cells. Bottom row: Overexpression of the GFP protein alone in muscle cells does not form foci. Scale bars, 10μm. (D) MUT-16::GFP in muscle cells labeled with <i>myo-3</i>::<i>mCherry</i>, where <i>myo-3p</i>::<i>mut-16</i>::<i>gfp</i> was injected at the indicated concentrations. Microscope exposure settings were changed as follows to account for variation in brightness between lines—top left: 0.02 sec exposure with neutral density filter at 10% transmittance; top right: 0.2 sec exposure; bottom left: 0.2 sec exposure; bottom right: 0.4 sec exposure. Scale bars, 10μm. (E) Top row: MUT-16::GFP overexpressed from the <i>myo-3</i> promoter forms foci. No mCherry proteins are expressed in this strain. Middle and bottom rows: Overexpression of MUT-16::GFP in muscle cells recruits MUT-15::mCherry (middle row) and RDE-2::mCherry (bottom row). Scale bars, 5μm.</p

The C-terminal region of MUT-16 is necessary for <i>Mutator</i> foci formation.

<p>(A) Table indicates whether MUT-16 foci are present or absent in each <i>mut-16</i> deletion strain. Yes indicates foci present in the majority of animals, No indicates foci absent or severely disrupted, and Weak (for ΔC) indicates an intermediate phenotype where the fluorescence intensity of cytoplasmic MUT-16 appeared reduced relative to the other deletion lines. (B-E) Live imaging of MUT-16::mCherry expression and localization for control strain (B) or when ΔC (C), ΔL (D), or ΔE-I (E) deletions have been introduced into the <i>mut-16</i>::<i>mCherry</i> strain. Scale bars, 5μm. (F) MUT-16 western blot to assess protein levels in full-length and <i>mut-16</i> deletion strains. Expected sizes for MUT-16::mCherry::2xHA are 148 kD (full-length), 139 kD (ΔA), 137 kD (ΔB), 134 kD (ΔC), 138 kD (ΔD), 137 kD (ΔE), 138 kD (ΔF), 141 kD (ΔG), 132 kD (ΔH-I), 135 kD (ΔJ), 141 kD (ΔK), 138 kD (ΔL), 105 kD (ΔE-I), and 85 kD (ΔE-K). Approximately 200 synchronous adult animals were loaded per lane and actin was used as a loading control.</p