Fig 1.
C. elegans gonad and pachytene germ cells.
A. Diagrams representing longitudinal (top) and cross-sectional (bottom left) views of one of the two arms of the adult hermaphrodite gonad. Germ cells are covered by peripheral somatic cells called sheath cells (green). The distal end of the gonad contains proliferating germ cells, and cells exiting the proliferation zone enter meiotic prophase. Hermaphrodite germ cells initially differentiate as sperm which are stored, but all later germ cells differentiate as oocytes. The hermaphrodite sperm are used for self-fertilization until those sperm are depleted, defining the self-fertile period, but hermaphrodites continue to produce oocytes that can be fertilized by male sperm. For most experiments here, worms were grown at 15°C and adults (A1-A5) were synchronized from fourth stage (L4) larvae (arrow in timeline). B. Diagram of pachytene germ cells in cross-sectional (left) and surface (right) views. Each germ cell is connected with the gonad core through an opening called a ring channel. Nuclei have large nucleoli (no) that occupy most of the nuclear volume, and the paired homologous chromosomes (Chr, blue) occupy the space between the nucleolus and the envelope. Perinuclear P granules (Pg, white) are associated with clustered nuclear pores (red) and overlie channels between each set of chromosomes. Cer1 GAG particles concentrate on stable microtubules which surround the germ nuclei and extend for long distances into the gonad core. C. The image shows a single germ nucleus in surface (left) and cross-sectional (right) optical planes; DNA (blue), P granules (PGL-1; green), and nucleoli (FIB-1; red). Note that the perinuclear P granules are aligned above the channels, corresponding to the positions of clustered nuclear pores. In this and selected images below, the DAPI channel is shown in cyan rather than RGB blue for resolution. D.TEM images of three pachytene nuclei; the surrounding cytoplasm is false-colored yellow. Brackets indicate presumptive RNPs in the channels between the compacted chromosomes (Chr). Examples of P granules (Pg) are outlined by dashed lines. Scale bars in microns = (C) 1.0; (D) 0.5.
Fig 2.
A. The protein products of Cer1 are diagrammed at top; the GAG polyprotein contains a nucleocapsid domain (NC) and a capsid-like (CA-like) domain defined in this study. The POL protein contains protease (PR), reverse transcriptase (RT), ribonuclease H (RNH) and integrase (INT) domains; boundaries of each domain are described in S1 Fig, legend. The N-terminal half of CERV shares three peptide sequences with GAG, and one unique peptide; splicing joins the four exons encoding these peptides to a fifth exon encoding the C-terminal half of CERV. The plot represents conservation of aligned Cer1 sequences from C. elegans, C. nigon, C. remanei, C. zanzibari, C. latens, and C. inopinata; the alignment for the GAG and CERV regions is shown in S1, S2, and S3 Figs. The plot was generated using EMBOSS Plotcon [130], which computes local similarity from a sliding window, here 4 amino acids; higher values on the y axis indicate higher conservation. The position of the phosphorylated residue S214 described in the text is indicated (asterisk). The red chevrons at bottom indicate the positions of gRNA-specific oligo probes used for smFISH; oligo sequences are provided in S2 Table. B. The ribbons/slab model at left shows the AlphaFold prediction for a hexamer of the CA-like domain of Cer1 GAG (amino acids 372–541) color coded as per the AlphaFold pLDDT table, a per-residue estimate of confidence on a scale of 0–100 [131]. The middle and right images show the space-filling model of the same hexamer with arbitrary coloring for each subunit. The model has a dome shape; the middle image shows a view inside the dome, and the image at right shows a side view with arrows indicating the relative positions of the N-terminal and NC regions of GAG. C. The left panel shows a near surface plane of a pachytene germ nucleus after smFISH for Cer1 gRNA (red). Four foci of Cer1 gRNA are visible, two on each side of the presumptive LGIII homologs (dotted line). Panel 2 shows a cross-sectional plane through a second germ nucleus; note that the four gRNA foci are coincident with CERV (see below). Arrowheads indicate the relatively low signals from cytoplasmic gRNA. D. Image of a single pachytene germ nucleus in an animal raised at 25°C but shifted to the permissive temperature of 15°C for 45 minutes before fixation; the image shows gRNA, GAG, and CERV as indicated. The gRNA exposure was increased relative to Fig 2C to visualize the cytoplasmic foci of gRNA (arrowheads). The increased exposure typically obscures the four foci of nuclear gRNA, such that there appears to be only two, larger foci (asterisks). Most of the perinuclear gRNA and GAG foci overlie nuclear channels, where perinuclear P granules are localized. Note that CERV is localized to the nuclear foci of gRNA, but not the perinuclear, cytoplasmic foci of gRNA. E. Low magnification of the pachytene region of an A1 gonad, showing GAG and gRNA dispersed throughout the gonad core; the image is a 3-micron Z-projection. The insets at bottom show that many of the brighter foci of gRNA (arrowheads) colocalize with GAG particles while the dimmer foci (arrows) do not. F. Panels 1 and 2 shows linear arrays of cytoplasmic gRNA foci (arrowheads) near pachytene germ nuclei; most of the cytoplasmic but not nuclear (asterisks) foci of gRNA colocalize with GAG particles, which have been shown to form linear arrays through association with microtubules (Fig 1B and [24]). Small arrays are evident beginning at the A2 stage (panels 1 and 2), but additional large, linear aggregates occur in A3 and older animals (panel 3). Panel 4 shows gRNA in the post-pachytene region of the gonad where oogonia increase in size and cellularize as oocytes. gRNA and coincident GAG particles in this region become heavily concentrated around nuclei (see also S1 Video). The oogonia and oocytes also contain dispersed, cytoplasmic gRNA without coincident GAG particles; cytoplasm signals in the boxed regions are shown with increased exposures in the insets. G. Arrested oocytes in the proximal arm of an A6 fog-2(q72) gonad, shown as a 4-micron Z-projection; the inset shows a higher magnification of the boxed region in three oocytes. Cytoplasmic gRNA and coincident GAG particles are concentrated at the cortical region of each oocyte; note that there is relatively little cytoplasmic gRNA that is not associated with GAG. H. Wild-type gonad exposed to gRNA(RNAi) for 48 hrs beginning on A1. Panel 1 shows a surface view of pachytene germ nuclei, and panel 2 shows a longitudinal view of the gonad at lower magnification. The cytoplasmic foci of gRNA (arrowheads) are generally coincident with GAG particles (see also S5 Fig). Scale bars in microns = (C, D) 1.0; (E-H) 5.0.
Fig 3.
CERV structure and function in gRNA export.
A. The left image is the AlphaFold-predicted structural model for the complete CERV protein (aa1-517), with the pLDDT color-coded confidence scores as in Fig 2A. The H, M, and G domains and the cysteine-rich loop described in the present study are indicated. Both images at right show different views of a space-filling model of the CERV hexamer; only the indicated amino acids are depicted in the model, and the subunit coloring is arbitrary. The central ring (top right, base view) is built from multimers of the M domain with the conserved Cys loops near the central axis (see also S6 Fig). The G domains extend radially from the M domains by flexible spokes (see also S4 Fig). H domains from adjacent subunits are predicted to bind together in coiled coils that project at variable angles from the ring (bottom right). B. Western blot of protein extracts from the indicated strains: cerv(stop) = WM746, gfp:cerv/gag = WM638, and gag:gfp = WM743 (see S1 Table for details). The blot was probed sequentially with α-GAG, mAbP3C6, and α-ACTIN. Note that the single band recognized by mAbP3C6 in the gfp:cerv/gag extract is GFP:CERV; this strain does not express GAG:GFP (see analysis in S7 Fig). C. Panel 1 (top row) shows A2 pachytene germ nuclei stained for CERV (mAbP3C6); the inset at right shows CERV, gRNA, and DNA (blue and cyan) at higher magnification. Panel 2 shows A4 pachytene germ nuclei stained for CERV. The intense, nuclear foci of CERV have disappeared; CERV is dispersed in the nucleoplasm and present in irregular aggregates at the center of nucleoli (arrows). Note that the level of gRNA in the nuclear foci (double arrows) has decreased and is comparable to the signal from cytoplasmic foci of gRNA (see also S8 Fig). D. Germ nuclei from A2 adults with a CERV-specific stop mutation. Panel 1 is an 8 micron Z-projection, showing there is no gRNA detectable in the cytoplasm (compare with the cytoplasmic gRNA visible in Fig 3G, which is a 3 micron Z-projection of similar germ nuclei). The lack of CERV expression is shown in the right half of panel 1. Panel 2 is a 3 micron projection showing the absence of GAG particles in the gonad core. E. Pachytene germ nuclei in a wild-type hermaphrodite cultured at the non-permissive temperature of 25°C. The gonad has prominent nuclear, but not cytoplasmic, foci of gRNA, and GAG is not expressed. Note that CERV is present in the nucleoplasm but not concentrated at the nuclear gRNA foci (arrows). F. A2 adult male gonad at 15°C. The gonad has nuclear, but not cytoplasmic, foci of gRNA, and GAG is not expressed. Note that CERV is present in the nucleoplasm but not concentrated on the nuclear gRNA (arrows). G. A2 adult hermaphrodite gonad at 15°C, showing the boundary (arrowhead) between the proliferation zone (inset 1) where gRNA is not exported, and the pachytene region (inset 2) where gRNA is exported and GAG is expressed. Note that the appearance of gRNA and GAG in the cytoplasm corresponds to where CERV first concentrates on the nuclear foci of gRNA. These images are 3-micron Z-projections, so signals from a few cytoplasmic foci of gRNA are artificially superimposed on the nuclei. Scale bars in microns = (A-G) 1.0.
Fig 4.
CERV phosphorylation and the cysteine-rich loop are required for gRNA export.
A.The peptide sequence at top left shows the beginning of the CERV M; residues in bold are invariant in diverse species of Caenorhabditis (see also S3 Fig). Most of this region is contained in a flexible cysteine-rich loop (AlphaFold model at right) which faces the central axis of the predicted ring multimers (Fig 3A; see S6 Fig for additional analysis of the M domain). The Cys loop brings multiple Cys residues into close proximity: predicted distances between the cysteine sulfur atoms are C195-C193 (3.3 Å), C193-C200 (3.8 Å), C200-C269 (3.9 Å), and C269-C195 (3.3 Å) (ChimeraX [132]). The blot (inset) shows protein extracts from the following strains: WT, WM746 [cerv(stop)], and JJ2706 [(cerv(S214A)]; see S1 Table for strain details. The blot was probed with mAbP3C6, which stains a prominent CERV band and a weaker GAG band in the wild-type extract. The cerv-specific STOP mutation eliminates both the CERV and GAG band, while the S214A substitution eliminates only the GAG band. B. Immunostained gonads from a homozygous mutant with a CERV S214A substitution (panel 1), and from a heterozygous strain with the same mutation plus a wild-type copy (panel 2). C. Immunoprecipitation assays of FLAG-tagged CERV and FLAG-tagged GAG followed by Western Blot analysis. Extracts are from WT worms, a strain with a FLAG tag on the N-terminus of CERV (WM894), and a strain with a FLAG tag on the C-terminus of GAG (WM895); see S1 Table for strain details. The extracts were immunoprecipitated with an anti-FLAG antibody and blotted; a duplicate blot is shown at right after treating the same extracts with phosphatase. Both blots were stained with the α-pS/T-P antiserum; the arrowhead points to a band at the predicted size of CERV that is absent after phosphatase treatment. D. Germ nuclei in an A2 wild-type gonad (panel 1) and a CERV S214A mutant (panel 2) stained for CERV and phospho-S/T-P. Note that the S214A mutant nuclei fail to stain with α-pS/T-P and CERV is present in the nucleoplasm but not concentrated into foci. E. Panel 1 shows different types of germ nuclei as listed after immunostaining for CERV and α-pS/T-P. Note that CERV only concentrates into foci after hermaphrodites are shifted to the export-permissive temperature of 15°C, where CERV becomes phosphorylated. Panel 2 shows the gonad boundary (arrowhead) where CERV first concentrates at the nuclear foci of gRNA, and panel 3 shows the subsequent, post-pachytene boundary where the CERV foci disappear. Some CERV in the post-pachytene region localizes to nucleolar inclusions (arrows) that stain positively for ubiquitin (panel 4, red). F. Mutant gonad with serine substitutions at each of the cysteines C193, C195, and C200 in the M domain of CERV (cer1(zu527) and strain JJ2700, Fig 4A). Panel 1 shows that nuclear, but not cytoplasmic foci of gRNA are present, and that CERV does not concentrate on the nuclear foci. Panel 2 shows that CERV appears to be phosphorylated at S214. Arrows indicate perinuclear foci of CERV that occur frequently in this strain. G. Mutant gonad with an R194A substitution in the M domain of CERV (cer1(zu531) and strain JJ2704). Panel 1 shows that gRNA is present in nuclear but not cytoplasmic foci, and that GAG is not expressed. Panel 2 shows that the nuclei contain bright foci of phosphorylated CERV. Panel 3 is a 4-micron Z-projection to visualize entire nuclear volumes, and shows that nuclei have more than the two expected CERV foci and that none of the CERV foci are coincident with the nuclear gRNA foci. Scale bars in microns (B,D-G) 1.0 micron.
Fig 5.
A. Panel 1 is a 3-micron Z-projection of an A4 gonad, showing nuclei with CERV rods in addition to nuclei with CERV foci. Note that the apoptotic cell (x) does not have a CERV rod. Panel 2 compares the sizes of nuclear rods, seen in both longitudinal (left) and cross-sectional (right) profiles, with the sizes of cytoplasmic GAG particles (red). The arrowhead indicates one end of a rod that appears to protrude slightly from the nucleus; additional examples of protruding rods are shown in panel 4. Panel 3 illustrates that rods are associated with the periphery of the nucleolus (FIB-1, red). Panel 5 shows a rod-containing nucleus with perinuclear P granules, which are lost in early apoptosis. Panel 6 shows that the rods are highly phosphorylated, similar to CERV foci. Panel 7 shows CERV rods in an apoptosis-defective ced-3(n717) mutant, and panel 8 shows CERV rods in the wild strain MY16 with a Cer1 insertion on LGX [20]. B. Panel 1 shows serial optical sections of a single nucleus, demonstrating that the rings of CERV represent cross sections of CERV rods (see also S2 Video). Note that both ends of the CERV rod appear to fill the nuclear channel, but the middle of the rod (at z = -0.6 microns) shifts asymmetrically toward the nucleolus. Panels 2 and 3 indicate the paired LGIII homologs (dotted line) between the two nuclear foci of gRNA (asterisks), and show that the CERV rod localizes to only one of the two flanking nuclear channels (arrows). Increased exposures (+ exp) show gRNA in some rods (panels 3 and 4, arrowheads). C. The plot shows the total number of rod-containing germ nuclei per gonad (left vertical axis) and the percentage of gonads with at least one rod-containing nucleus (right vertical axis, grey bars). Mated animals in the wild-type and fog-2 mutant series were marked and mixed with wild-type males 24 hours before processing; mating was confirmed for each gonad by sperm in the spermatheca, but this experiment did not determine when mating occurred. P-values were calculated using nonparametric Mann-Whitney U test and graphing was performed using GraphPad Prism software version 9.5. **** P< 0.0001, ***P ≤ 0.01, ns = not significant. D. Gonad of a mated wild-type animal at A5 showing large numbers of CERV rods, all of which stained positively with α-pS/T-P. Scale bars in microns = (A,B) 1.0; (D) 5.0.
Fig 6.
Candidate intermediate stages in the formation of CERV rods.
A. A4 germ nucleus showing the major nuclear foci of gRNA and CERV (asterisks) with additional signals (arrowheads) in a nuclear channel flanking LGIII (dotted line). B. Panels 1 and 2 show near-surface views of germ nuclei with extended, flattened streaks (arrowheads) of gRNA and CERV extending unidirectionally (panel 1) and bidirectionally (panel 2) from the main foci (arrowheads). Panel 3 shows an example of a CERV streak that extends in a channel which is nearly orthogonal to LGIII; a summary reconstruction of the streak and chromosomes is shown at right. Panels 4–7 show multiple examples of flattened streaks of CERV and gRNA at the periphery of the nucleolus. C. Panel 1 shows a single focal plane (left) and a 3-micron Z-projection of a group of germ nuclei. The CERV streak at top left remains rectangular in the projected view, but the bottom two streaks (arrowheads) are seen to be much larger oval-shaped "caps" of CERV. Note the main nuclear foci of gRNA and CERV (asterisks) at the perimeters of the caps. Panel 2 shows a germ nucleus with a CERV cap (indicated by the 3-micron Z-projection at right) where the nucleolus is stained for FIB-1/fibrillarin (red). Note that the CERV cap extends below and between multiple nuclear channels (arrowheads), suggesting that the cap is associated primarily with the surface of the nucleolus. D. Panel 1 shows dispersed nucleolar components in A1 nuclei, and panel 2 shows segregation of the same components in A5 nucleoli. E. TEM images of nucleoli in A1 or A5 wild-type germ nuclei, as listed. Note that the nucleoli in the A5 nuclei have variable patterns of segregation. F. Images comparing nucleolar components in A2 wild-type nuclei with the same components in A2 fog-2 "female" nuclei. Note that the A2 fog-2 nucleoli resemble older, A5 wild-type nucleoli (see Fig 6D). Scale bars in microns = A-C (1.0), D, F(2.0), E(1.0).
Fig 7.
TEM of CERV-containing nuclear structures.
A. A2 germ nuclei expressing APEX2-CERV. The low magnification images at top show prominent electron-dense regions that appear to be in nuclear channels next to P granules (dotted outlines). Panel 2 shows one such region at high magnification; note the electron-lucent fibrils (black arrows) within the electron-dense region. Panel 3 shows additional examples of the electron-lucent fibrils within APEX2-CERV foci. B. A2 control nuclei. Panel 1 shows a nucleus with two clusters of electron-dense fibrils (outlined in red), and panel 2 shows similar regions in other nuclei. White arrowheads indicate electron-lucent regions around the fibrils. C. Examples of aligned fibrils in A2 germ nuclei expressing APEX2-CERV; arrows indicate individual, electron-lucent fibrils. D. Panel 1 shows three examples of individual fibrils oriented perpendicular to the nuclear envelope; nuclear pores are indicated by small white arrowheads. Panel 2 shows groups of aligned fibrils near the envelope. Panel 3 is a high magnification of a single fibril; note zig-zag appearance (black arrowheads), with striations or flanges that are perpendicular to the long axis of the fibril (see also S9 Fig). E. Examples of flattened streaks or caps of APEX2-CERV at the perimeter of nucleoli (no) in A5 germ cells. Note that the streaks contain numerous electron-lucent fibrils (black arrows) which are aligned in a plane parallel to the nucleolar surface. F. Control A5 gonads showing electron-dense fibrils (black arrows) surrounded by an electron-lucent matrix (white arrowheads) at the surfaces of nucleoli.
Fig 8.
A. Panels 1–3 show longitudinal or cross-sections of APEX2-CERV rods in A5 germ nuclei. Note the electron-lucent fibrils in the rods (black arrows). The top row in panel 2 shows two examples of rods associated with small protrusions (arrowheads) of the nuclear envelope (compare with arrowheads in Fig 5A, panels 2 and 4). In the cross-sectional images (panel 3), note the curvature of the fibrils (black arrows) within the electron-dense matrix (white arrowhead). B. Longitudinal or cross-sections of rods in A5 control nuclei; an example of serial sections through a rod is provided in S9 Fig. The rods consist of electron-dense fibrils (black arrows) surrounded by an electron-lucent matrix (white arrowheads). The cross-sectional images of rods in panel 2 are shown as insets at higher magnification; note the curvature of fibrils at the perimeter of the rods. One of the nuclei in panel 2 has a rare double rod which is also seen in immunostained preparations (S8 Fig). Panel 3 shows additional examples of cross-sections through rods. C. Speculative model for CERV rod formation, shown in cross-sectional view. CERV and gRNA are proposed to form flattened streaks or caps on the surfaces of nucleoli. Unknown events cause the streaks to roll lengthwise into cylinders, such that gRNA molecules which were previously parallel to the nucleolar surface now become curved. Scale bars in microns as listed.