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. 2023 Aug 28;19(8):e1010854.
doi: 10.1371/journal.pgen.1010854. eCollection 2023 Aug.

rRNA transcription is integral to phase separation and maintenance of nucleolar structure

Soma Dash  1 , Maureen C Lamb  1 , Jeffrey J Lange  1 , Mary C McKinney  1 , Dai Tsuchiya  1 , Fengli Guo  1 , Xia Zhao  1 , Timothy J Corbin  1 , MaryEllen Kirkman  1 , Kym Delventhal  1 , Emma L Moore  1 , Sean McKinney  1 , Rita Shiang  2 , Paul A Trainor  1   3
Affiliations

rRNA transcription is integral to phase separation and maintenance of nucleolar structure

Soma Dash et al. PLoS Genet. .

Abstract

Transcription of ribosomal RNA (rRNA) by RNA Polymerase (Pol) I in the nucleolus is necessary for ribosome biogenesis, which is intimately tied to cell growth and proliferation. Perturbation of ribosome biogenesis results in tissue specific disorders termed ribosomopathies in association with alterations in nucleolar structure. However, how rRNA transcription and ribosome biogenesis regulate nucleolar structure during normal development and in the pathogenesis of disease remains poorly understood. Here we show that homozygous null mutations in Pol I subunits required for rRNA transcription and ribosome biogenesis lead to preimplantation lethality. Moreover, we discovered that Polr1a-/-, Polr1b-/-, Polr1c-/- and Polr1d-/- mutants exhibit defects in the structure of their nucleoli, as evidenced by a decrease in number of nucleolar precursor bodies and a concomitant increase in nucleolar volume, which results in a single condensed nucleolus. Pharmacological inhibition of Pol I in preimplantation and midgestation embryos, as well as in hiPSCs, similarly results in a single condensed nucleolus or fragmented nucleoli. We find that when Pol I function and rRNA transcription is inhibited, the viscosity of the granular compartment of the nucleolus increases, which disrupts its phase separation properties, leading to a single condensed nucleolus. However, if a cell progresses through mitosis, the absence of rRNA transcription prevents reassembly of the nucleolus and manifests as fragmented nucleoli. Taken together, our data suggests that Pol I function and rRNA transcription are required for maintaining nucleolar structure and integrity during development and in the pathogenesis of disease.

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Conflict of interest statement

The authors have declared that no competing interests exist

Figures

Fig 1
Fig 1. Loss of function of Pol I subunits results in nucleolar defects.
Bright field images of WT, Polr1a-/-, Polr1b-/-, Polr1c-/- and Polr1d-/- embryos at E2.5 (A) indicate that the mutant embryos are indistinguishable from WT embryos at 8–16 cell stage. However, by E3.5 (B), Pol I mutant embryos are arrested in association with blastomere fragmentation while the WT embryos progress to the blastocyst stage. Immunostaining with Y10b to visualize rRNA (C) indicates that rRNA expression is reduced in all the mutant embryos. While Fbl (D) expression is variable, it is expressed in the NPBs of all the mutants. Ncl (E) and Treacle (F, I) expression are increased in the NPBs of all the mutants. In addition, the number of NPBs per blastomere is reduced in all the mutants (G), while the volume per NPB increases (H). The data is represented as mean+/-SEM. Scale bar for A-F = 12.5 μm. * indicates p<0.05, ns indicates p>0.05.
Fig 2
Fig 2. Nucleolar organization is altered in the Polr1c-/- mutants.
(A) Immunoelectron microscopy with antibodies against Y10b (large particles) and Ncl (small particles) on Polr1c-/- mutant embryos indicate that the number of FC and DFC are significantly reduced in Polr1c-/- mutants compared to control embryos. Scale bar = 200 nm. (A’) is higher magnification image of the FC/DFC shows lower levels of both Y10b (magenta) and Ncl (black) in the Polr1c-/- mutants. Scale bar = 75 nm. Quantification (Mean+/-SEM) indicates that while Y10b is significantly reduced in Polr1c-/- mutants (B), Ncl is not (C). 3D-immuno FISH with Ncl antibody (D’), and 5’ETS region of the 47S rDNA (D) indicates rDNA localization to the condensed nucleolus. Scale bar = 15 μm. (E) ChIP with Treacle antibody pulls down a significantly higher amount of rDNA promoter in tamoxifen treated Polr1cfx/fx;Cre-ERT2 MEFs compared to DMSO treated MEFs. B2M promoter was used as an internal control (E’). Data is represented as mean +/- upper and lower limits. * indicates p<0.05, ** indicates p<0.01, ns indicates p>0.05.
Fig 3
Fig 3. Pol I inhibition alters nucleolar structure and phase separation properties.
(A, B) 16-cell stage DMSO and BMH-21 treated mouse embryos immunostained with Fbl, Y10b, Treacle and Ncl, and counterstained with DAPI and Phalloidin. The yellow arrow indicates a condensed nucleolus phenotype, while the red arrow points to a fragmented nucleolus phenotype. Scale bar = 20μm. (C, D) E8.0 embryos treated with DMSO and BMH-21 immunostained with Fbl and Treacle, and counterstained with DAPI. Scale bar = 50μm. (C’-D”) High magnification images of cells from insets in C and D to show nucleolar disruption in BMH-21 treated embryos. Scale bar = 2.5μm (E-G) Stills from live imaging video of fluorescently tagged hiPSCs treated with BMH-21 showing the nucleolus condensing. Scale bar = 10μm. (H-K) Analysis of live imaging videos showing changes in the number of UBF (H) and FBL (I) puncta, NPM1 area (J), and NPM1 sphericity (K). n = 15 cells. Data is represented as mean (line) +/- 95% CI (shading). (L-M) Stills from half FRAP time-lapse video of pre-bleach, bleach, and recovery of NPM1 in control (L) and BMH-21 treated (M) cells. (N-O) Graphs of Tau values for half FRAP (N) and whole FRAP (O) showing both Tau1 and Tau2 significantly increase upon BMH-21 treatment (Student’s t-test). The data is represented as mean+/-SD. ** indicates p<0.01, **** indicates p<0.0001.
Fig 4
Fig 4. BMH-21 inhibition of Pol I function prevents nucleolar reassembly following cell division.
(A-L) Stills from time-lapse imaging of control (A-F) and BMH-21 treated (G-L) hiPSCs progressing through the cell cycle. UBF = grey. FBL = green. NPM1 = blue. Yellow lines denote dividing cells. Scale bar = 10μm. (M-P) Confocal images of hiPSCs treated with BMH-21 (M), synchronized in G2/M with VM-26 (N), synchronized with VM-26 and released for 3 hours in regular media (O), and synchronized with VM-26 and released for 3 hours with BMH-21 treated media (P). Red lines denote cells with fragmented nucleoli. (Q) Graph of the percentage of cells with fragmented nucleoli indicating that BMH-21 treatment just prior to cell division increases percentage of cell with fragmented nucleoli. Data is represented as mean+/-SD. One-way ANOVA. **** indicates p<0.0001.
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