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


RR2
Reviewer #1 (Remarks to the Author):
 All my concerns have been addressed and the quality of the manuscript has greatly improved. Due to
 the much clearer Figures, the story is much easier to read and follow now. Therefore, the manuscript
 is now ready to be published in Nature Communications. I just noticed one little thing: in
 Supplementary Figure 4d, it is not clear what is shown in the left and what in the right part. I assume
 it is the same arrangement as in Supplementary Figure 4c, but the strains should definitely also be
 indicated in S4d.
Reviewer #2 (Remarks to the Author):

  The authors have made a serious effort to improve the m/s according to the previous points raised by
 all three reviewers. In our case, however, two substantial points remain to be answered.
 In the revised manuscript, the authors still assign the primary role of the Rpf2 CTD to be directly
 involved in the structural H68-70 maturation and “checkpoint” control of the pre-60S biogenesis
 pathway. However, this appears to be overstated, as there is still a lack of convincing support for this
 scenario, as already outlined in our previous review.
 1. The authors should seriously revise their abstract with the focus on the main and clear-cut results
 leaving out the speculative aspects.
 2. The main structural differences of H68-70 are observed just in sub-classes of Nog2-associated
 particles, obtained by using the Rpf2-CTD mutant, Rpf2∆255-344; the yields of pre-ribosomal particles
 purified from the rpf2∆255-344 mutant were significantly lower than from the wild-type cells, as
 authors themselves state. Thus, it is possible that only a small population of pre-ribosomes from these
 mutant cells are in this particular state while another, much larger pool, could be stuck in (an)other
 state(s) and degraded. If true, the Rpf2 C-terminus would have a functional role in other steps of 60S
 assembly as well. It is therefore important to analyze earlier pre-60S particles in this Rpf2∆255-344
 mutant (isolated by appropriate baits), as already suggested during the first round of review.
 3. Along these lines, our previous suggestion to check the stability of the 5S RNP members in their
 Nog2 purifications addressed via Western blot for Myc-Rpf2∆255-344, Rrs1-HA, Rpl5, Rpl11 (shown in
 Supplementary Figures 5b, c), are not convincing, mainly due to the poor quality of these Westerns.
 There are also significant differences between Fig. 4a, Rrs1-HA Western band, and the revised figure
 (now Fig. 5b) with the same Rrs1-HA band. In one case there is a strong signal, in the other case
 there is basically no signal. It further appears that the L5 signal is decreased in the case of the rpf2
 mutant. The authors could perform Northern analysis for the 5S rRNA to address the question of
 whether the 5S RNP is decreased in mutant particles (e.g. for Supplementary figures 7b, f).
 Reviewer #3 (Remarks to the Author):
I would support the publication of this new version of the manuscript as it is without further changes.
 After reviewing this new version of the manuscript and read the letter in response to reviewer
 comments I must say that the authors put a significant amount of thought and effort into addressing
 not only my comments but also those from reviewer 1 and 2.
 I think this new version of the manuscript is significantly improved. The data and structures are much
 more convincing in the new way the figures have been designed.