Peer review of: Coupling of 5S RNP rotation with maturation of functional centers during large ribosomal subunit (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

This is an interesting study employing a combination of modern approaches to study the function of 
the C-terminal extension of ribosome assembly factor Rpf2. The authors provide convincing evidence 
that the Rpf2 CTD is required for correct folding of rRNA helices 68-70, allowing for efficient 
recruitment of Sda1 and consequent 5S RNP rotation. This is a high-quality study deserving 
publication in Nature Communications. There are however some aspects that should be improved, in 
particular: 

- The northern blot analyses in this paper are not very informative. Only two precursors, 27S and 7S 
pre-rRNA are analyzed, and sometimes even the loading control is missing (Supplementary Figure 9). 
Moreover, a direct comparison between depletion of Rpf2 in the rpf2-delta-255-344 strain compared 
to the total depletion of Rpf2 would be interesting. The detection of more precursors would be helpful 
for the interpretation of the actual defect. 

- The authors claim that they tested for stability of the truncated Rpf2 protein indirectly by testing the 
levels of Rrs1, rpL5 and rpL11, which are known to be dependent on stable Rpf2. It is good that they 
can show that these proteins are unaffected, but it would be nice to also have Rpf2 in the analysis. 
The authors argue that there is no antibody available and C-terminal tagging causes growth defects. 
But why don’t they use an N-terminal tag, as they did for the variant under the control of the Gal-
Promoter? This should be easy for the strain in which the truncated variant is provided from a plasmid. 
To be able to distinguish between the protein expressed from the Gal-Promoter and from the pRS315 
plasmid, they could use some epitope tag other than HA, like Flag or Myc. If there is a reason why this 
is not possible, I would suggest to at least re-write the text a bit and start out with saying something 
like “We tested whether the truncated Rpf2 complex affects the stability of Rrs1, Rpl5 or Rpl11.” As a 
final conclusion of the paragraph, they could then argue that considering that these proteins are 
mutually interdependent for their stability, it is likely that also Rpf2 is stable (rather than starting 
already out with Rpf2 stability). 

Minor points 
- The authors refer on multiple occassions to Nog2-TAP particle states 1-3 that they introduced in a 
previous paper but also to pre-60S particle states described by the Beckmann and Klinge groups. The 
many different states discussed in the paper are sometimes confusing. Supplementary Figure 1b is 
helpful for looking up the discussed states, but for example Nog2-TAP particle states 1-3 (which are 
most important for this paper) are not clearly labeled here (there are two Nog2 particles indicated but 
without naming the state). 
To improve the clarity, the authors should include all discussed states in Supplementary Figure 1B 
(and mention all names for particles which have been described by more than one group). For 
example, the text mentions that Arx1/Nog2 State 1/and State F particles are similar – then the Arx1-
particles in the scheme should be labeled Arx1/Nog2 State 1/and State F. 

- The resolution of the images is bad and sometimes the relevant features in the structures are 
difficult to discern and should be better labeled; an example is Figure 4; only the missing densities are 
labeled, and only in State 3. It would be useful to have also labels for the existing densities. Moreover, 
the missing densities of H68-70 should also be labeled in Figure 4b (not only in 4c). Otherwise readers 
might confuse the missing density of H68-70 in 4b (left) with the 5S RNA, which is also in red an 
labeled in Figure 4a. 
I also wonder why the 5S RNP part is not colored in Figure 4b. In Figure 4a (left), representing the 
“perfect fit”, the colors are visible, so shouldn’t they also be visible in the states in 4b where the 5S 
RNP is in the same position? 

- The picture in Figure 8a, indicating conformational changes of H68-71 is in principal nice. However, 
only structures from previous studies are shown. It would be much more interesting to the reader, if 
these were compared with the conformations found in the current study. 

- From the point in the paper where the missing densities of H68-70 are mentioned, the reader 
wonders what is already known about maturation of these helices and if there are known structures in 
which these helices are not yet in their mature conformation. This is nicely summarized in the 
discussion section, but that is too late in my opinion. Also the suggested comparison of the H68-71 
conformations (Figure 8a, see above) should be shown earlier in the manuscript. 

- Figure 3C is a bit difficult to read – it would be helpful to have a color legend directly in the Figure so 
that one does not need to look up which color is what in the Figure legend. 

- Page 12, line 246: Levels of Nop53 were also decreased….indicating that the ITS2…processing may 
be affected. 
The basis for this interpretation should be briefly explained in the text (e.g. reduced exosome 
recruitment…). 

- There are some small spelling mistakes: page 6, line 126: rearagnements; page 23, line 435: 
though (through?); page 26, line 493: Rfp2 instead of Rpf2 

- Methods section: this section is not very detailed. It would be extremely useful to have a list of 
strains and plasmids in the paper, and not just upon request as stated. Also, the methods descriptions 
could be more detailed. Large parts of the methods only refer to previous studies. 


Reviewer #2 (Remarks to the Author): 

In the manuscript “Coupling of 5S RNP rotation with maturation of functional centers during middle 
stages of large ribosomal subunit assembly”, Woolford and coworkers performed a combined genetic 
and structural analysis in the yeast Saccharomyces cerevisiae to investigate how the 5S RNP and 
nearby regions on the pre-60S particle mature depending on the C-terminal tail of the Rpf2 assembly 
factor. 

While this study contributes to a better understanding of ribosome biogenesis by revealing some 
specific aspects regarding the role of the C-terminal extension of Rfp2 for 5S RNP rotation within the 
pre-60S subunit, it remains a bit short on what this really means in terms of the exact role of the C-
terminus of Rpf2 for these middle stage 60S assembly steps. In particular, ‘the checkpoint theory in 
subunit maturation’ as written in the abstract appears to be very overstated. 

The main focus was a structural comparison between wild-type Nog2 particles and particles isolated 
from the rpf2 mutant lacking the C-extension under growth-retarded conditions. It was observed that 
some distinct 25S rRNA helices (H68-70) and a few assembly factors were affected in this mutant, 
which prompted the authors to speculate on the mechanistic implications. 
It was surprising that the deletion of the Rpf2 C-terminus did not affect the overall association of Rpf2 
and the 5S RNP with the pre-60S particle. However, the way how this was analysed cannot fully 
distinguish between different scenarios. Therefore, it is important to rigorously show that the deletion 
of the Rpf2 C-terminus only affects the middle stage biogenesis steps (which is a major conclusion of 
this study), but not the first steps of 5S RNP assembly into the early pre-60S particles. 

Specific points 
It is crucial to analyze the middle stage pre-60S particle, e.g. purified by Nop7, suggested to contain 
Rpf2C, Rrs1, Rpl5, Rpl11 and the other biogenesis factors in stoichiometric amounts, by SDS-PAGE 
and Coomassie staining. Rpf2C could be easily HA-tagged like it was done for wild-type Rpf2 and 
hence should be clearly visible later on gels. 

Supplementary Figure 4a and 4c: These experiments lack both an important control to demonstrate 
that depletion of Rpf2-HA per se has no effect on the composition of Nop7-TAP and Nog2-TAP 
particles. 

Supplementary Figure 4a: the authors refer to a control already published in 2007 (“Assembly factors 
Rpf2 and Rrs1 recruit 5S rRNA and ribosomal proteins rpL5 and rpL11 into nascent ribosomes”), but 
re-depicted in Figure 3. Comparing this new Nop7-TAP purification under GAL condition with the old 
but same purification, there is a significant difference in the pattern of co-enriched bands. It would be 
important to perform side-by-side a new control under the same conditions, which only would allow to 
directly compare these results. 
Supplementary Figure 4c: please include a SDS-PAGE reference for the western from the Nog2-TAP 
purification, similar to Figure 4a. 

Page 6, lanes 135-137: Include references 

Page 7, lane 156: Properly “folded” seems not the best term for an unstructured, flexible protein 
extension. 

Page 9, lane 189-196: Despite it was previously shown that protein levels of the analysed proteins are 
interdependent, it would be necessary to show the stability of the Rpf2 truncated protein by a western 
blot analysis of cell lysates (apparently Supplementary Fig. 4a does not include whole cell lysates but 
only eluates). Moreover, L5 and L11 westerns (Supplementary Fig. 4a) should be improved. 

Page 12, lanes 253-255: It is mentioned that classes C2 and C3 were combined since the difference 
was “only” in presence/absence of a few assembly factors. Still, it would be important to mention, 
which assembly factors in fact were present/absent, compared to wild-type particles. 

Page 16, lanes 321-322: It is not evident that the Rpf2 CTD is directly involved in H68-70 maturation 
as suggested in the text. It could be also indirect via affecting the positioning of other factors 
necessary for this maturation (e.g. the first two alpha-helices of Nog2 that interact with this rRNA 
region). 

Supplementary Fig. 2a: It is rather surprising that Rpf2 co-purifies the 90S factor Mpp10. This remains 
to be further addressed in this study. Therefore, the authors should not only analyse Tsr1-TAP and 
late cytoplasmic Lsg1-TAP, but also perform affinity-purification of “middle stage” (e.g. Nog2, Nsa2) 
and nucleolar factors (e.g. Nsa1) that are not present on 90S particles. Also loading controls (bait 
protein and r-protein westerns) and/or Coomassie staining would be important to validate the results. 

The title of Supplementary Fig. 2 should be revised, as this figure does not give information at which 
stage pre-60S maturation is affected (“yet truncation of its CTD affects subunit assembly at later 
steps”). Since there is also a nucleolar L25-GFP signal (which normally is not the case in mutants with 
a later nucleoplasmic defect), it is not obvious (at least from this figure) whether there is also an early 
pre-60S biogenesis defect. 

Supplementary Fig. 2c: it would be necessary to show the polysome profiles with (N-terminally) 
epitope tagged Rpf2 and Rpf2∆255-344 and assess them by western blotting, in order to reveal how 
well the mutant is binding to the particles and to compare the free/pre-60S-bound levels of wild-type 
vs mutant protein. In addition, a western blot against Nog2 could clarify whether Nog2 pre-60S 
association in the rpf2 mutant is indeed completely unaffected as mentioned in the manuscript (e.g. 
on page 12, lanes 238-240). 

Figure 3: On the silver-stained gel only the bands corresponding to Nog2-TAP, Arx1, Sda1, Rea1 are 
indicated. Can also other major bands be indicated for a better understanding on the protein pattern? 
On page 12, lanes 246-247, it is mentioned that Nop53 is decreased and ITS2 processing is therefore 
affected. This is not apparent from the stained gel, where Nop53 does not significantly change, in 
contrast to the iTRAQ data suggested. What is the explanation for this discrepancy? 



Reviewer #3 (Remarks to the Author): 

This manuscript explores the effect of removing the C-terminal end of assembly factor Rpf2 in the 
maturation of the functional centers of assembling 60S subunits in yeast. To this end, the authors 
follow mainly a cryo-EM structural approach that leads to the obtention of a structure of an immature 
60S subunit assembled in a strain expressing a variant of the assembly factor Rpf2. A comparison of 
the obtained structure with previously reported structures of other assembly intermediates allows the 
authors to infer on the roles of the C-terminal end of Rpf2 in ribosomal assembly. 

The quality of the structural work is excellent and certainly at the level expected from readers in 
Nature Communications. The text is well written, and descriptions of the structures are clear. 
However, the main issue I see in the present form of the manuscript is that the design of the figures 
makes very difficult to visualize the highlighted structural differences and convey a clear message. It 
will be important that the design of the structural figures is improved to make a convincing case and 
solidify the conclusions derived from the structures. Without access to the cryo-EM maps and 
molecular models derived from these maps is not possible to validate some of the conclusions and 
statements made by the authors only based on the provided figures. 

I will provide now some specific feedback in the figures to illustrate this point and hopefully help the 
authors in creating a more efficient set of figures: 

1. I think it is a great idea to add a supplementary figure to assist the comprehension of the process 
in the introduction describing the assembly of the 5S RNP and stable association with the ribosomal 
particle. However, I found very difficult to correlate the text on page 4, which refers to Suppl Fig. 1b 
to what is shown in this panel. Unless the reader is familiar with the maturation process of the 60S 
subunit, this figure is not very helpful. 

2. Figure 4 illustrates the docking of the molecular model of State 1 Nog2 particle into the cryo-EM 
map obtained for the pre-ribosomal 60S particle from the C-terminal deletion mutant of rpf2 and on 
maps for State 1 and 3 of Nog2 particles. The difference between the 5S RNP not rotated in Stage 1 
and rotated in stage 3 is not visible in the provided views in panel A. Panel C, left is also confusing. I 
can see there is no density representing the C-terminal end of Rpf2, but the image is very crowded. 
Overall this figure would benefit and become clearer if the authors were to make use of a higher level 
of transparencies for the cryo-EM map and also an efficient use of segmentation tools for example in 
the Chimera program. This would contribute to declutter the images and allow to easily see what the 
text is describing. 

3. Similarly, I don’t find Fig 5A particularly effective in showing the altered configuration of H75 in the 
three different structures. Panel C could also be improved by zooming into the relevant regions 
showing differences (GTPase domain and the C-terminal domain). 

4. Fig 7 is quite clear. However, I think using a light grey colour for the non-coloured regions of the 
maps will make a clearer figure. A good way to also show the differences between Class c2 and C3 
would have been difference map analysis. The authors should consider this approach to highlight the 
differences between both maps. 
5. Suppl. Fig 8 would also benefit from using a light grey colour for the map and density segmentation 
tools could be used to easily visualize the different positions adopted by L1. 

A few additional comments non-related to the figure design follows: 

1. Supplementary figures should include the FSC plots and local resolution analysis of the presented 
structures. 

2. It is not mentioned anywhere in the manuscript that the obtained cryo-EM maps and derived 
molecular models have been properly deposited in the EMDB and PDB. EMDB and PDB IDs of these 
depositions are not provided. This is a requirement for publication in this and any other reputable 
journal. 

3. In the 3D classification of the cryo-EM maps shown in Supple Fig 5b, the authors do not comment 
in what could be the origin of the ‘bad particles’. What makes them bad and what do they represent? 
They represent about 31-32% of the data. The data set classified in Suppl. Fig 7b only shows a ~2% 
of bad particles. Could this difference be explained somehow? If these are particles damaged by the 
water-air interface, why the percentage of damaged particles is much smaller in the dataset in Suppl. 
Fig. 7.