RNA Pol II and Pol III form distinct nuclear foci at the 5S rDNA-SL1 gene cluster.

(A) Co-localization of 5S rDNA and ATTF-6 in embryo nuclei detected by DNA-FISH and immunostaining. DNA-FISH was performed using Cy5-labeled probes targeting the 5S rDNA gene cluster, and immunostaining was conducted with an anti-FLAG antibody to detect ATTF-6::3×FLAG. Nuclei were visualized with DAPI. Dashed lines outline the embryo. Confocal images (60× objective) are shown as maximum-intensity projections spanning the top layer of nuclei in the fixed embryo. Scale bar: 10 μm. (B) Enlarged view of three nuclei within the embryo shown in (A). Scale bar: 2 μm. (C) GFP::Pol II (GFP:: AMA-1) and ATTF-6::RFP foci in a live embryo. Dashed lines outline the embryo. Confocal images (60× objective) are shown as maximum-intensity projections spanning the top layer of nuclei in the embryo. Scale bar: 10 μm. (D) Enlarged view of three nuclei within the embryo shown in (C). Scale bar: 2 μm. (E) Intensity profile of GFP::Pol II (GFP::AMA-1) and ATTF-6::RFP signals along the dotted arrow in nucleus No1. in (D). AU, arbitrary unit. (F) GFP::Pol III (GFP::RPC-1) and ATTF-6::RFP foci in a live embryo. Dashed lines outline the embryo. Confocal images (60× objective) are shown as maximum-intensity projections spanning the top layer of nuclei in the embryo. Scale bar: 10 μm. (G) Enlarged view of three nuclei within the embryo shown in (F). Scale bar: 2 μm. (H) Intensity profile of GFP::Pol III (GFP::RPC-1) and ATTF-6::RFP signals along the dotted arrow in nucleus No. 1 in (G). AU, arbitrary units.

RNA Pol II and Pol III associate with SL1 and 5S rDNA, respectively.

(A) Browser view of ATTF-6, Pol II (AMA-1), and Pol III (RPC-1) ChIP-seq signals on chromosome V. ATTF-6 ChIP-seq signals were normalized to the control IP (Wang et al. 2025)., and AMA-1 and RPC-1 ChIP-signals were normalized to the input (Ikegami and Lieb 2013). The signals represent the average from two biological replicates and are shown in RPKM (Reads Per Kilobase per Million mapped reads). The dashed box highlights the 5S rDNA–SL-1 gene cluster. (B) Browser view zoomed in on the 5S rDNA–SL1 gene cluster. The cluster contains more than 100 tandem repeats of 5S rDNA (yellow arrowhead) and SL1 (green arrowhead) arranged in opposite orientations. (C) An individual example within the 5S rDNA–SL1 gene cluster. The green and yellow arrows indicate the positions and orientations of sls-1.11 and rrn-4.6, respectively. (D) Metagene profiles of ATTF-6, Pol II (AMA-1), and Pol III (RPC-1) ChIP-seq signals around the SL1 snRNA and 5s rRNA genes. The plot is anchored at the 5’ end of the SL1 snRNA genes and spans 500 bp upstream and downstream. Signals (RPKM) were normalized to the control by subtraction and then averaged across two biological replicates. Yellow and green arrows indicate the positions and orientations of the 5S rRNA and SL1 snRNA genes, respectively.

RNA Pol II and Pol III form dynamic foci in vivo.

(A) Table showing foci shape descriptors (top rows), calculated from maximum-intensity projections (60x) for three GFP::ATTF-6, GFP::Pol II, or GFP::Pol III embryos and fluorescence recovery after photobleaching (FRAP) parameters—t1/2 (bottom rows), calculated from models shown in (B) for GFP::ATTF-6, GFP::Pol II, and GFP::Pol III. FRAP parameters for ATTF-6 are labeled as NA (not applicable) because the recovery did not fit the exponential model. (B) Relative intensity of GFP::ATTF-6 (n=4), GFP::Pol II (n=6) and GFP::Pol III (n=4) foci, respectively, after photobleaching in embryos. Intensity was measured from single-plane images every second (sec) before and after photobleaching and normalized to the mean intensity over five timepoints prior to bleaching (time 0). A.U. indicates arbitrary units. Dots represent mean relative intensity, and error bars denote standard deviation. Red and blue curves represent the exponential model of recovery for Pol II or Pol III, respectively. (C) Single-plane time-lapse (100x) images of GFP::ATTF-6 before (pre-bleach) and after photobleaching for the indicated timepoints (seconds). Yellow arrow indicates the photobleached focus; dashed circle indicates the nuclear periphery. Scale bar: 5 µm. (D) Same as in (C) but for GFP::Pol II. Red arrow indicates the photobleached focus. Scale bar: 5 µm. (E) Same as in (C) but for GFP::Pol III. Blue arrow indicates the photobleached focus; dashed circle indicates the nuclear periphery. Scale bar: 5 µm (F-H) Representative maximum intensity projections (60x) of GFP::ATTF-6 (F), GFP::Pol II (G), or GFP::Pol III (H) expressing embryos treated with ptr-2 RNAi and subsequently dissected into either egg buffer (EB) or 5% 1,6-hexanediol. Images depict whole embryos, with each embryo outlined by grey dashed lines (left; scale bar: 10 µm) and the corresponding inset indicated by the white dashed square (right; inset scale bar: 5 µm). Grey dashed circles in each inset outline the nucleus.

Formation of Pol II and Pol III foci is regulated across the cell cycle.

(A) Dynamics of GFP::Pol II foci during cell division in an early embryo. A time-lapse imaging series of an embryo expressing GFP::Pol II and the chromatin marker H2B::mCherry. The circular dashed line outlines the embryo, and the dashed square highlights the dividing cell. The bottom panel shows zoomed-in views of the dividing cell, grouped into S phase and M phase. Arrowheads indicate GFP::Pol II foci in the nucleus of the dividing cell. Confocal images (60× objective) are shown as maximum-intensity projections spanning the top layer of nuclei in the embryo. Scale bars: 10 μm (top panel) and 2 μm (bottom panel). (B) Same analysis as in (A), but with GFP::Pol III.

Pol II nor Pol III are dispensable for assembly of ATTF-6 foci.

(A) Fluorescence micrographs of nuclei in live embryos expressing GFP::ATTF-6 and RFP::Pol II. Embryos were dissected from worms treated with control (L4440) or Pol II (ama-1) RNAi. The circular dashed line outlines the embryo. Images are shown as maximum-intensity projections spanning the top nuclear layer. The right panel presents individual enlarged views of three nuclei selected from the embryo. Confocal images were acquired with a 60× objective. Scale bars: 10 μm (embryo) and 2 μm (nuclei). (B) Quantification of GFP::ATTF-6 foci numbers in embryo nuclei. Embryos were dissected from worms treated with control (L4440) or Pol II (ama-1) RNAi. Nuclei from three independent embryos were counted. Error bars represent the standard deviation (SD). Statistical significance was determined using a two-tailed Student’s t-test (ns: p > 0.05). (C-D) Analyses as in (A-B), but using embryos dissected from worms treated with Pol III (rpc-1) RNAi.

SNPC-4 and ATTF-6 are required for Pol II foci, but not Pol III foci formation.

(A) Live embryos expressing GFP::Pol II and ATTF-6::AID::RFP, dissected from worms treated with or without 1 mM auxin. The right panel presents individual enlarged views of three nuclei selected from each embryo. Confocal images (60× objective) are shown as maximum-intensity projections spanning the top nuclear layer. Scale bars: 10 μm (embryo) and 2 μm (nuclei). (B) Same auxin treatment conditions as in (A), but using embryos expressing GFP::Pol III and ATTF-6::AID::RFP. (C) Live embryos expressing GFP::Pol II and ATTF-6::AID::RFP, dissected from worms treated with control (L4440), snpc-4, or attf-6 RNAi. Embryos show the merged GFP and RFP signals. The right panels present individual enlarged views of three nuclei selected from each embryo, shown as separate GFP, RFP, and merged channels. Confocal images (60× objective) are shown as maximum-intensity projections spanning the top nuclear layer. Scale bars: 10 μm (embryo) and 2 μm (nuclei). (D) Same RNAi conditions as in (C), but showing embryos expressing GFP::Pol III together with ATTF-6::AID::RFP.

Pol II foci are temperature-sensitive and regulate SL1 expression.

(A) Representative maximum intensity projections (60x) of GFP::ATTF-6 expressing embryos cultured at 20°C (top) or shifted to 32°C for either 2 hours (middle) or 3 hours (bottom). Whole embryos are shown on the left (scale bar: 10 µm), outlined by grey dashed ovals. The corresponding insets to the right are indicated by the white dashed squares. Insets to the right show single nuclei outlined by grey dashed circles (Inset scale bar: 3 µm). Heating conditions are further described in the Materials and Methods. (B) Same conditions as in (A), but for GFP::Pol II expressing embryos. (C) Same conditions as in (A), but for GFP::Pol III expressing embryos (D) Ethidium bromide staining of total RNAs from wild-type embryos cultured at 20°C, or shifted to 32°C for either 2 hours or 3 hours in M9 buffer, respectively. rRNAs and tRNAs are indicated. (E) Northern blotting of SL1 RNA (top) or 5S rRNA (bottom) from wild-type embryos cultured at 20°C, or shifted to 32°C for either 2 hours or 3 hours, respectively. Relative SL1 abundance upon heating was normalized to 5S rRNA abundance. (F) RT-qPCR analysis of Pol II transcripts (SL1, act-3, and gpd-2) and the Pol III transcript 5S rRNA in embryos. Transcript fold change upon heat stress was calculated relative to 20°C and normalized to 18S rRNA as the internal control. Bar plots show mean +/− SD; dots represent technical replicates. Statistical significance was assessed using Student’s two-sample t-test. * indicates p ≤0.05, ** p ≤0.01, and ns indicates not significant. (G) Working model showing Pol II and Pol III occupy the 5S rDNA–SL1 locus but assemble into distinct nuclear compartments. ATTF-6 promotes Pol II foci formation to drive SL1 transcription while Pol III independently engages nearby 5S rRNA genes.