Dynamic RNA Polymerase compartments organize the transcription of gene clusters
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eLife Assessment
This manuscript explores the dynamic behaviors of Pol II and Pol III puncta that encompass the SL1 and 5S genes, following up on the authors' prior studies on ATTF-6. The authors show that ATTF-6 is required for RNA Pol II but not RNA Pol III foci, demonstrating that within the gene cluster, the regulation of RNA Pol II and RNA Pol III remain distinct from each other. The study is useful for analyzing understudied gene families, but it is incomplete and needs additional edits and experiments.
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Abstract
Spatial organization of transcription machinery is emerging as a key regulator of gene expression, yet how RNA polymerases are organized at gene clusters remains unclear. Here, we show that RNA polymerases II and polymerase III form distinct nuclear foci at the 5S ribosomal DNA (rDNA)–Spliced leader 1 (SL1) cluster in C. elegans. Within this cluster, polymerase II binds to the SL1 gene, while polymerase III associates with 5S rDNA. Both polymerase foci display dynamic but distinct behaviors within the nucleus. The assembly of these polymerase foci is regulated across the cell cycle. ATTF-6, an AT-hook transcription factor, is essential for polymerases II foci formation but dispensable for polymerases III foci. While Pol III foci are largely resistant to temperature changes, Pol II foci are temperature-sensitive, and their dissolution correlates with reduced SL1 expression. Together, these results reveal a spatial and temporal regulation of two RNA polymerases that organize gene cluster transcription.
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eLife Assessment
This manuscript explores the dynamic behaviors of Pol II and Pol III puncta that encompass the SL1 and 5S genes, following up on the authors' prior studies on ATTF-6. The authors show that ATTF-6 is required for RNA Pol II but not RNA Pol III foci, demonstrating that within the gene cluster, the regulation of RNA Pol II and RNA Pol III remain distinct from each other. The study is useful for analyzing understudied gene families, but it is incomplete and needs additional edits and experiments.
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Reviewer #1 (Public review):
This study examines how two types of RNA polymerases organize themselves within the nucleus of C. elegans cells, building directly on the same group's prior publication and largely functioning as a companion to that earlier work. While the observation that the two polymerases occupy distinct but neighboring locations at the same genomic region adds nuance to our understanding of gene cluster regulation, the manuscript would benefit from more clearly delineating which findings are new versus continuations of previously published work. Protein localization, gene expression effects, and genomic mapping data appear to overlap substantially with the earlier paper.
The condensate claims would also benefit from additional experimental support. Demonstrating fusion events and concentration-dependent assembly are now …
Reviewer #1 (Public review):
This study examines how two types of RNA polymerases organize themselves within the nucleus of C. elegans cells, building directly on the same group's prior publication and largely functioning as a companion to that earlier work. While the observation that the two polymerases occupy distinct but neighboring locations at the same genomic region adds nuance to our understanding of gene cluster regulation, the manuscript would benefit from more clearly delineating which findings are new versus continuations of previously published work. Protein localization, gene expression effects, and genomic mapping data appear to overlap substantially with the earlier paper.
The condensate claims would also benefit from additional experimental support. Demonstrating fusion events and concentration-dependent assembly are now standard expectations in the field. Additionally, one measurement reported appears inconsistent with a condensate model, warranting further discussion.
Some findings would benefit from more interpretive context. Why does polymerase clustering fluctuate with the cell cycle? What are the functional implications of ATTF-6 being required for one polymerase's foci but not the others?
The elevated-temperature experiments are intriguing but difficult to interpret, as the temperature used is well-established as a broad stress trigger in this organism. Acknowledging this and considering additional controls would help clarify whether the observed effects are specific to foci behavior.
Finally, the manuscript would be strengthened by adding quantification to some figures and revising the model diagram to better reflect what the current data support.
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Reviewer #2 (Public review):
Summary:
The researchers analyzed GFP-tagged RNA Pol II and RNA Pol III catalytic subunits RPB-1 and RPC-1, and showed that they form foci in early embryo nuclei that overlap with the 5S rDNA loci and foci by ATTF-6-RFP. They showed foci are round, dissolve upon hexanediol incubation, and are detected during S phase, removed during, and re-established after mitosis. The researchers performed FRAP and showed fast exchange of polymerases, unlike ATTF-6. They show that, unlike RNA Pol III, RNA Pol II foci are dependent on ATTF-6 and temperature sensitive. The researchers propose that the two polymerases form distinct foci with different biochemical dependencies. This study shows that, although closely located within a gene cluster, the regulation of RNA Pol II and RNA Pol III is independent.
Strengths:
The …
Reviewer #2 (Public review):
Summary:
The researchers analyzed GFP-tagged RNA Pol II and RNA Pol III catalytic subunits RPB-1 and RPC-1, and showed that they form foci in early embryo nuclei that overlap with the 5S rDNA loci and foci by ATTF-6-RFP. They showed foci are round, dissolve upon hexanediol incubation, and are detected during S phase, removed during, and re-established after mitosis. The researchers performed FRAP and showed fast exchange of polymerases, unlike ATTF-6. They show that, unlike RNA Pol III, RNA Pol II foci are dependent on ATTF-6 and temperature sensitive. The researchers propose that the two polymerases form distinct foci with different biochemical dependencies. This study shows that, although closely located within a gene cluster, the regulation of RNA Pol II and RNA Pol III is independent.
Strengths:
The researchers provide high-quality images that support the main results. The researchers' use of auxin-inducible and RNAi depletion work is validated in the same embryos by fluorescent analysis of the target protein.
Weaknesses:
Although the researchers propose the hypothesis that the RNA Pol II and RNA Pol III form distinct condensates, alternative hypotheses are not presented, and the criteria by which the other possibilities are ruled out are not discussed.
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Reviewer #3 (Public review):
Wang et al demonstrate that RNA polymerase II and RNA polymerase III form distinct nuclear foci at the 5S rDNA-SL1 gene cluster in C. elegans. By ChIP, Pol II is highly enriched at the SL1 gene, whereas Pol III is enriched at the 5S rRNA gene. Both polymerase foci are spherical, show rapid exchange in FRAP experiments, and assemble in a cell-cycle-dependent manner, predominantly during S phase. The transcription factors ATTF-6 and SNPC-4 are required for the formation of Pol II foci but are dispensable for Pol III foci. Pol II foci, but not Pol III foci, are temperature-sensitive and dissolve upon heat stress; dissolution correlates with a strong reduction of SL1 transcription, whereas 5S rRNA levels remain largely unaffected.
Overall, this is a clean, well-organized, and well-controlled study, and I only …
Reviewer #3 (Public review):
Wang et al demonstrate that RNA polymerase II and RNA polymerase III form distinct nuclear foci at the 5S rDNA-SL1 gene cluster in C. elegans. By ChIP, Pol II is highly enriched at the SL1 gene, whereas Pol III is enriched at the 5S rRNA gene. Both polymerase foci are spherical, show rapid exchange in FRAP experiments, and assemble in a cell-cycle-dependent manner, predominantly during S phase. The transcription factors ATTF-6 and SNPC-4 are required for the formation of Pol II foci but are dispensable for Pol III foci. Pol II foci, but not Pol III foci, are temperature-sensitive and dissolve upon heat stress; dissolution correlates with a strong reduction of SL1 transcription, whereas 5S rRNA levels remain largely unaffected.
Overall, this is a clean, well-organized, and well-controlled study, and I only have two comments.
(1) Roundness measurements, FRAP, and sensitivity to 1,6-hexanediol are indicative but not sufficient to show that these foci are condensates. They could, for example, also be scaffolded /chromatin-anchored assemblies (see https://pubmed.ncbi.nlm.nih.gov/36526633/). Please either provide better evidence or rephrase/tone down the condensate statements.
(2) Image quantification is only provided for Figure 5, but should also be reported for Figures 6 and 7. In addition to the foci number, also, e.g., intensity over background (similar to partition coefficient) should be quantified.
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Author response:
Reviewer #1:
We appreciate the reviewer’s suggestions. In the revision, we will clarify which results are new and better position this work relative to our earlier publication. We will also expand the discussion of the functional implications of polymerase clustering and its cell-cycle dynamics.
Regarding the condensate interpretation, we agree that the current evidence is suggestive but not definitive. In the revised manuscript, we will clarify how our measurements relate to commonly used criteria for condensate assemblies and revise the text to avoid overstating this interpretation. We will also add quantification to additional figures and revise the model diagram to more accurately reflect the conclusions supported by the data.
Reviewer #2:
We thank the reviewer for the positive assessment of the imaging quality. …
Author response:
Reviewer #1:
We appreciate the reviewer’s suggestions. In the revision, we will clarify which results are new and better position this work relative to our earlier publication. We will also expand the discussion of the functional implications of polymerase clustering and its cell-cycle dynamics.
Regarding the condensate interpretation, we agree that the current evidence is suggestive but not definitive. In the revised manuscript, we will clarify how our measurements relate to commonly used criteria for condensate assemblies and revise the text to avoid overstating this interpretation. We will also add quantification to additional figures and revise the model diagram to more accurately reflect the conclusions supported by the data.
Reviewer #2:
We thank the reviewer for the positive assessment of the imaging quality. We agree that the manuscript would benefit from a broader discussion of possible models for the observed polymerase foci. In the revision, we will expand the discussion to include alternative interpretations, such scaffolded assemblies as suggested by the reviewer 3, and further clarify the properties of the RNA Pol II and RNA Pol III foci.
Reviewer #3:
We thank the reviewer for the positive evaluation of the study and the helpful suggestions. We agree that the current evidence is indicative but not sufficient to definitively demonstrate condensate formation. In the revision, we will revise the language and discuss alternative interpretations, including scaffolded assemblies. We will also provide additional quantifications for the relevant figures.
Overall, we appreciate the reviewers’ suggestions and believe that the planned revisions will improve the clarity and impact of the manuscript.
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