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. 2021 Sep 10;12(1):5364.
doi: 10.1038/s41467-021-25413-w.

In situ cryo-electron tomography reveals gradient organization of ribosome biogenesis in intact nucleoli

Philipp S Erdmann  1   2 , Zhen Hou #  3 , Sven Klumpe #  3 , Sagar Khavnekar #  3 , Florian Beck  3 , Florian Wilfling  3   4 , Jürgen M Plitzko  3 , Wolfgang Baumeister  5
Affiliations

In situ cryo-electron tomography reveals gradient organization of ribosome biogenesis in intact nucleoli

Philipp S Erdmann et al. Nat Commun. .

Abstract

Ribosomes comprise a large (LSU) and a small subunit (SSU) which are synthesized independently in the nucleolus before being exported into the cytoplasm, where they assemble into functional ribosomes. Individual maturation steps have been analyzed in detail using biochemical methods, light microscopy and conventional electron microscopy (EM). In recent years, single particle analysis (SPA) has yielded molecular resolution structures of several pre-ribosomal intermediates. It falls short, however, of revealing the spatiotemporal sequence of ribosome biogenesis in the cellular context. Here, we present our study on native nucleoli in Chlamydomonas reinhardtii, in which we follow the formation of LSU and SSU precursors by in situ cryo-electron tomography (cryo-ET) and subtomogram averaging (STA). By combining both positional and molecular data, we reveal gradients of ribosome maturation within the granular component (GC), offering a new perspective on how the liquid-liquid-phase separation of the nucleolus supports ribosome biogenesis.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Structural and molecular organization of the C. reinhardtii nucleolus.
A The C. reinhardtii nucleolus is clearly visible as a dense structure decorated with large particles (white arrow & zoom in). Some of these particles (black arrows) are also found floating in the nucleoplasm. B, C Using reference-free template matching, pre-60S particles (blue) and SSU processomes (red) are detected (n = 85 tomograms). D Their 3D organization is consistent with a sphere decorated by pre-ribosomes (n = 26 tomograms for which spheres could be fit). E The small subunit precursors show a characteristically different probability density function (PDF) than the pre-60S, with a sharper peak around the nucleolar surface (n = 26 tomograms; Fisher-Pitman permutation test with significantly smaller variance for the SSU: p = 1.0E–06). LSU Large Subunit, SSU Small Subunit, NE Nuclear envelope, NPC Nuclear Pore Complex. Source data are provided as a Source Data file.
Fig. 2
Fig. 2. Image-based analysis of the nucleolar organization.
A Individual tomograms can be denoised and filtered to allow segmentation of nuclear particles (bottom). Analysis of their apparent mass (top) shows an increase in apparent mass per particle toward the surface of the nucleolus starting at ~0.75 of the normalized distance. Before and after that, the average mass remains relatively constant until the nuclear envelope is reached. B Spectral analysis of particle mass vs. normalized distance over all tomograms with sphere fits shows two main features: 1. There are constantly high levels of particles up to a size of ~2.5 MDa, except around the surface of the nucleoli (dashed line). 2. At this distance, a lack of smaller masses is compensated by a burst of large complexes, consistent with the detection of pre-ribosomal particles such as the SSU processome and the pre-60S. NE Nuclear envelope. Source data are provided as a Source Data file.
Fig. 3
Fig. 3. Structural heterogeneity of in situ SSU processome particles.
A–C Three distinct SSU processome class averages (Class 1–3) with different similarity to published structures are detected. D There is a clear trend from Class 1 to Class 3 toward a more compact arrangement of the 5′ and central domains. This is particularly evident when all three STA structures are aligned and superimposed, and indicates a sequence of maturation (Supplementary Movie 2). E The relative abundance of each class is subject to external stimuli. While Class 2 dominates during logarithmic (log) and stationary growth (stat), Class 1 becomes the predominant form when treated with diazaborine (DAZ), a drg1 AAA-ATPase inhibitor, and when ribogenesis is stimulated (dark). PDB model for A–C: 5WYJ. See Supplementary Fig. 6 for a structural comparison with other maps. Source data are provided as a Source Data file.
Fig. 4
Fig. 4. Sub-Nucleolar Partitioning of Pre-Ribosomes.
A The SSU processome classes show significant differences in their spatial distribution depending on their level of maturation (n = 26 tomograms; two-sided Fisher-Pitman permutation test of mean: p1,2 = 1 E−6, p1,3 = 1.07 E−04, p2,3 = 4.1 E−02; n = 1809 particles; data are represented as boxplots where the red cross indicates the mean, the middle line is the median, the lower and upper hinges correspond to the first and third quartiles, top and bottom whiskers indicate maximum and minimum, respectively. Circles show outlier points. See Supplementary Fig. 7d for pre-60S results. B, C Plotting the probability density function (PDF) for the large and small subunit precursors reveals discrete behaviors. There is a sharp peak for SSU Class 2 & 3 at the nucleolar surface and Class 1 displays higher probabilities at smaller radii. Likewise, pre-60S Class 1 shows a peak at even lower distances, implying that particles can still be detected further inside the nucleolus and that template matching is not restricted to its periphery (n = 26 tomograms). D The data suggests a model, in which pre-ribosomal particles are separated in maturation gradients and thus provide molecular order to the nucleolus and ribosome biogenesis. SSU Small subunit, LSU large subunit, GC Granular Component, NPC Nuclear Pore Complex. Source data are provided as a Source Data file.
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