An RNA-Binding Multimer Specifies Nematode Sperm Fate

Abstract

FOG-3 is a master regulator of sperm fate in Caenorhabditis elegans and homologous to Tob/BTG proteins, which in mammals are monomeric adaptors that recruit enzymes to RNA binding proteins. Here, we determine the FOG-3 crystal structure and in vitro demonstrate that FOG-3 forms dimers that can multimerize. The FOG-3 multimeric structure has a basic surface potential, suggestive of binding nucleic acid. Consistent with that prediction, FOG-3 binds directly to nearly 1,000 RNAs in nematode spermatogenic germ cells. Most binding is to the 3' UTR, and most targets (94%) are oogenic mRNAs, even though assayed in spermatogenic cells. When tethered to a reporter mRNA, FOG-3 represses its expression. Together these findings elucidate the molecular mechanism of sperm fate specification and reveal the evolution of a protein from monomeric to multimeric form with acquisition of a distinct mode of mRNA repression.

Keywords: FOG-3; RNA binding protein; Tob/BTG; multimerization; protein evolution; sperm fate.

Figure 1.. FOG-3 Is a Divergent Tob/BTG Protein

(A) FOG-3 is a terminal regulator of the sperm fate, and hence essential for sperm fate specification in both hermaphrodite larvae and males. (B) Linear diagram of FOG-3 protein. Predicted Tob/BTG fold is N-terminal (gray). C terminus is abbreviated (dashed lines). Vertical lines mark missense mutation sites (also see Figure S1): green, conserved in all orthologs; cyan, conserved in nematode orthologs; magenta, missense mutation generated in this study. Horizontal bars below show extents of subunits in crystal dimer, termed FOG-3A (light blue) and FOG-3B (dark blue). (C) Asymmetric unit of the FOG-3 crystal structure (PDB: 5TD6). FOG-3A (chain A, light blue) and FOG-3B (chain B, dark blue). Arrows highlight the nematode-specific linker-helix extension. (D) FOG-3 and human Tob/BTG structures. RMSD of human Tob1 (orange, PDB: 2Z15) compared with FOG-3A and FOG-3B was 1.062Å and 1.086Å_, respectively. Arrows highlight the linker-helix extension. (E) Location of FOG-3 missense mutants in the crystal structure. Color scheme matches that in (B).

Figure 2.. FOG-3 Forms Higher-Order, Multi-dimer Assemblies

(A) Crystal packing of the FOG-3 dimer. FOG-3A and FOG-3B are represented in light and dark blue, respectively. (B) Model for multimerization of FOG-3 dimers, as observed in the crystal. Image was generated by extending the crystal symmetry. Subunits are colored as in (A). (C) Negative-stain electron microscopy of recombinant FOG-3 1–137. Note the presence of long rods (arrow). Scale bar, 50 nm. (D) Coomassie-stained gel of modified FOG-3 recombinant protein incubated with increasing amounts of BS3 crosslinker. ‘‘–’’ represents no BS3 included. Different exposure shown in Figure S3I. (E–G) Mutation of a key residue, E126K, transforms the germline from spermatogenic to oogenic. The E126K allele, fog-3(q847), was maintained over a balancer (nT1). (E) Heterozygous or homozygous adult males were analyzed for sperm or oocytes. (F and G) Representative DIC images of adult males, heterozygous (F) or homozygous (G) for the E126K mutation. See Figures S3L–S3N for similar results in hermaphrodites.

Figure 3.. FOG-3 in Spermatogenic Germ Cells Binds Directly to 3′ UTRs of Oogenic mRNAs

(A) FOG-3 iCLIP enriches for mRNAs. (B) Most FOG-3-bound mRNAs belong to the oogenesis program, which includes RNA expressed only in oogenic germlines (light pink) and in both oogenic and spermatogenic germlines (dark pink), as categorized previously (Noble et al., 2016). (C) Distribution of FOG-3 iCLIP sequence reads within mRNAs. Transcript lengths are normalized so that 5′ UTRs, coding sequences (CDS), and 3′ UTRs (50, 1,000, and 200 nt, respectively) are reported as arbitrary units (AU). (D) Many FOG-3 targets possess multiple binding peaks in their 3′ UTRs, as shown in (E). (E) Examples of FOG-3 binding peaks across 3′ UTRs. x axis, 3′ UTR with coding region in pink and 3′ end marked by red line; y axis, number of mapped reads. Peaks are marked by black dots; their heights correspond to number of mapped reads.

Figure 4.. FOG-3 Represses mRNA Reporter Expression When Tethered in Nematodes

(A) Summary of the protein-mRNA tethering assay. With lN22, FOG-3 can bind and regulate the protein expression of a GFP::histone reporter transcript containing 3xboxB hairpins in its 3′ UTR. (B–I) Maximum intensity projections from confocal images of representative male adult germlines expressing both modified FOG-3 and the GFP reporter. FOG-3::3xFLAG (B–E) or FOG-3::lN22::3xFLAG (F–I) with the GFP::histone reporter were imaged by fluorescent confocal microscopy for (B and F) FLAG, (C and G) GFP, (D and H) FLAG and GFP overlay, and (E and I) DNA (DAPI). DNA images reduced in size 2.5-fold. Dashed white line outlines male germlines. Scale bar, 20 μm. (J) Model of the FOG-3 molecular mechanism. Multimers of FOG-3 dimers bind 3′ UTRs and promote the sperm fate by repressing mRNAs in the oogenic program. FOG-3 may find its targets by interacting with a distinct sequence-specific seed protein, one candidate being FOG-1 (see Discussion)..