PONE-D-19-24366

Interaction between the assembly of the ribosomal subunits: Disruption of 40S ribosomal assembly causes accumulation of extra-ribosomal 60S ribosomal protein uL18/L5

PLOS ONE

Dear Lasse,

Thank you for submitting your manuscript to PLOS ONE. Your manuscript was reviewed by two experts in the ribosome biogenesis field. As you can see from the reviews, their comments were generally very positive, however, they also highlighted a number of inconsistencies that need further clarification. In particular Reviewer #2 raised a number of important points regarding the quantification of the data as well as comparison with previously published work. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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Reviewer #1: Yes

Reviewer #2: Partly

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Reviewer #1: Yes

Reviewer #2: I Don't Know

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: No

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: This manuscript by Rahman, Shamsuzzaman and Lindahl is an interesting follow-up to a previous paper from the Lindahl group showing interdependence of assembly of large and small ribosomal subunits in yeast. Here they demonstrate that ribosomal protein uL18, aka L5, accumulates outside of ribosomes when assembly of either large or small subunits is blocked by depleting a ribosomal protein from one or the other subunit. This was visualized by gradient sedimentation of L5 in extra-ribosomal fractions. Of interest is how varying effects of depletion of different r proteins reflects the order and mechanism of protein and subunit assembly, including effects resulting from post-transcriptional assembly.

The conclusions are sound and the results are interesting. However, the story might be more impactful if the following were added:

(1 Include a figure showing in more detail which SSU and LSU r proteins are assembled early and which much later, and thus why depletion of some might have a greater effect on L5. This is explained in the text but would be clearer to nonaficionados if a figure/cartoon were added.

(2 L5 accumulates in fractions at the top of the gradient. Are 5S rRNA, L11, Rpf2 and Rrs1 also present in the same complex with L5? CO-IP from gradient fractions will demonstrate whether this is the case. This is potentially important since the authors report accumulation of extraribosomal L5 when Rpf2 or Rrs1 are depleted (Fig.4D).

Minor issues

(1 Some figures need to be better labeled and there are numerous places where a word or two need to be added or changed.

Line 56: Aren’t some r proteins not essential, i.e. not completely required for subunit assembly?

Line 119 and elsewhere: a detail perhaps: is the symbol in the figures a star or an asterisk?

Line 151 and elsewhere: the term GAL1/10 promoter should be capitalized and italicized.

Line187: what is meant by Northern blots of fractions were probed? Don’t you mean just “blots”?

Line 367: is ther a word or two missing?

Figures: Does more than one lane of blot data correspond to consecutive gradient fractions, e.g. lanes 3,4 and 5,6 in Fig.1? If so, please explain in the figure legends.

Figure 2B: the text states that L11 was assayed, but it is not shown.

(2 In the Introduction, lines 64-66, the authors need to correct their description of the experiment done by Dehmukh et al. They got trapped by the nomenclature monster! R protein L16 was depleted, which is NOT the same as uL5, as stated. This is important because uL5 aka L11 is in an assembly subcomplex with uL18 aka L5, whereas L16 is not.

Reviewer #2: Rahman et al. report in the manuscript PONE-D-19-24366 on the results of a series of experiments which provide evidence for the extra-ribosomal accumulation of the large ribosomal subunit protein uL18 upon inhibition of small ribosomal subunit assembly in S. cerevisiae. Their conclusions are based on quantitative interpretation of western blotting analysis after sucrose gradient fractionation of cellular extracts prepared from yeast ribosomal protein expression mutants.

In this regard the exact approach chosen might be better described. The authors refer to reference 9, in which an indirect immuno-detection is described using an enzymatic reaction for secondary antibody visualisation. These detection approaches tend to produce saturation artefacts and it might be therefor important to know whether titration of a reference sample was used in the individual western blots to determine the dynamic range of the detected signal. For the sucrose gradient fractionation, in several experiments all fractions were analysed. In these cases the ribosomal proteins could be detected in four to six of the “Top” fractions and in up to eleven of the faster sedimenting “Ribosomes” fractions. In several other analyses only one or two of the “Top” fractions and either two or three of the “Ribosomes” fractions of the sucrose gradients were analysed to deduce the ratio of extra-ribosomal to ribosomal r-proteins as uL18. How was it ensured that these fractions contain all of the extra-ribosomal and ribosomal pools of uL18, or are representative for them?

The western blot results shown in Fig 3A , 3C(iii) and 4D look quite cropped. That complicates interpretation of the data, especially since the uL18/L5 antibody used in this study cross-reacts with a protein running just a bit slower than uL18/L5. The identity and running behavior of the band detected in the “Top” fractions in 3A for the parental strain is, for example, in the actual representation for me ambiguous. Thus, less cropped figures might help the reader in these cases.

For several of the experiments (Fig 3A +B) I could not find the exact incubation times in glucose containing medium which were applied to repress the expression of individual r-proteins. In the materials and methods section it is stated that cultures were shifted for 6 to 21 hours to glucose containing medium. In one experiment (Fig. 2) the control strain is shifted to glucose containing medium for a different time period (16 hours) than the two r-protein expression mutants analysed (6 hours and 8 hours). Why was that done, and can we be sure that this might not affect the outcome of the experiments? At least from the data shown in Fig 3C for Pgal-eS31 I got the impression that the level of free uL18/L5 decreases with prolonged incubation times in glucose containing medium.

Recently, changes in levels of proteins in several large and small subunit r-protein expression mutants were measured by mass spectrometry proteome wide (https://doi.org/10.1016/j.molcel.2018.10.032). Did the authors analyse these published datasets for a possible excess of uL18/L5 over other large subunits proteins as ul4/L4 or uL5/L11? That might give further support to the authors conclusions by an analysis performed with an independent experimental approach.

A few other comments and remarks:

1) Line 52: “..also requires in excess of 250 ribosomal assembly factors”

Does that refer to the stochiometry of components?

2) Line 54: “..while others are specific to the formation of one the ribosomal subunits”

of one of ?

3) Line 110: “..western blots probed anti-uL18 revealed..”

probed with?

4) Line 124: “…but no band corresponding asterisked band in…”

??

5) Fig 1, labelling: Ribs or Rbs ?

6) Line 163: Why was the strain Pgal-e43 chosen for these experiments?

7) Line 164: “A band co-migrating with the ribosomal uL18 band ..”

was that inferred by some other detection method, as for example by mass spectrometry, or by the expected running behavior of uL18? Does the running behaviour of the detected band fit with the predicted size of uL18?

8) Line 166: “.. we confirmed that band that..”

that the band?

9) Line 174: “This was anticipated, since repression of the 60S r-protein uL5 is known to provoke a buildup of extra-ribosomal uL18..”

uL5 is together with uL18 part of the 5S RNP, in this regard I am not sure whether that specific example allows a straight forward prediction for what is to be expected upon expression shut down of an unrelated large subunit r-protein as eL43.

10) Line 177, Fig 2.: “Repression of several 40S r-protein genes causes … “

Several 40S r-protein genes? In Fig 2 is just an analysis shown for eS4.

11) Line 177, Fig 2.: “..causes accumulation of extra-ribosomal uL18/L5, but not extra-ribosomal uL5/L11 …”

How was the specificity analysed of the antibody used for detection of uL5?

12) Line 187: “(C) Northern blots of fractions from ..”

Western blots of ..?

13) Line 195: “.. was found outside in the ribosome peaks..”

outside of ?

14) Line 198: “.. parent whether it grows glucose and galactose medium..”

parent strain? grows in ?

15) Line 199: “..the results in Fig 2 shows that..”

show?

16) Lines 202 - 205: Why were these large and small subunit protein expression mutants chosen?

17) Line 260: “No uL18 band was seen at the top of the gradient 260 after cycloheximide inhibition of protein synthesis (Fig 4A(ii))..”

The western signals shown in Fig 4A (ii) look a bit blurry. Was a technical replicate of this experiment performed?

18) Line 296: “To investigate if extra-ribosomal uL18 also accumulates when rRNA synthesis th is inhibited by the TOR-targeting drug rapamycin”

th ? Rapamycine has also quite a drastic effect on the r-protein mRNA levels and thus on r-protein production. A conditional mutant of a gene coding for an RNA polymerase I subunit might have addressed the question asked more clearly.

19) Lines 310 and following: I was wondering if the authors also analysed the possible accumulation of uL18 bound 5S rRNA?

20) Line 332: “In the extremes, abolishment of eS4synthesis..”

eS4 synthesis?

21) Line 332 and following: “In the extremes, abolishment of eS4synthesis generates a response similar to that seen during repression of two 60S r-protein genes, while extra-ribosomal uL18 is borderline detectable during abrogation of eS31 synthesis (Fig 3). This gradient correlates with the abundance of 40S r-proteins in the 90S ribosomal particle, an early 40S assembly intermediate [19], suggesting that preventing early steps of pre-40S precursor assembly have the strongest effect on accumulation of extra-ribosomal uL18.”

Was the subunit imbalance phenotype compared between expression mutants of eS4 and eS31? The respective penetrance of the small subunit synthesis phenotype might well correlate with the observed effects on uL18. The authors suggest in the abstract and here in the discussion that specifically inhibition of early steps in small subunit assembly leads to the observed effects on uL18. That assumption would have been much strengthened if other 40S r-protein expression mutants with late 40S maturation phenotypes which were not detected in early 40S precursors would have been analysed (e.g. expression mutants of uS2, uS3, uS5 or others). I would also see the authors attempt to interprete the data from reference 19, Fig 7, in regard to the “abundance” of r-proteins in 90S pre-ribosomes as inappropriate, since the authors of reference 19 for probably good reasons use a cut-off to just deduce the presence of the respective proteins.

22) Line 348: “Since two 60S r-proteins (uL4 and uL24) bind to Domain 1 ..”

It is unclear to me why to list these two r-proteins here, but not other r-proteins which clearly also bind to LSU rRNA domain 1.

23) Line 367: “Since the major features of pathways for ribosomal assembly evolutionarily conservation, …”

?

24) Line 373: “The difference between the ratio of co-transcriptional and post-transcriptional is evident from ..”

Insert here “processing”?

25) Line 351 and following: “We therefore posit that the simplest explanation for the buildup of extra-ribosomal uL18 during inhibition of 40S assembly is that the folding of the 60S part of the early rRNA transcript is influenced by 40S r-proteins that bind to rRNA prior to separation of the subunit moieties of the emerging rRNA transcript.”

I am not sure whether this is really the simplest explanation. The effects described here were observed after rather long r-protein depletion times (6 -21 hours according to Materials and Methods). Effects on small subunit synthesis start to be detectable much earlier (after one to two hours in RNA pulse analyses) and after 6 hours and more depletion times quite a massive subunit imbalance can be established in small subunit r-protein expression mutants (see for examples the authors own publication in Life Science Alliance, 2019). That, together with the clear reduction in cellular amounts of functional ribosomes can have various consequences on the balanced production of each of the other ribosomal proteins, including the ones of the large subunit. Did the authors also test effects on extra-ribosomal uL18 accumulation after shorter incubation times in glucose medium at which effects on 40S production just start to be established (1-2 hours)? That might help to distinguish if the observed effects on uL18 are primary consequences of an altered early SSU maturation state or due to secondary effects on the translation status of the cell. Related to this point, see also point 21 in regard to the authors choice of a conditional eS31 expression mutant to control for effects on uL18 by a late SSU assembly defect.

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Reviewer #1: Yes: John Woolford

Reviewer #2: No

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Interaction between the assembly of the ribosomal subunits: Disruption of 40S ribosomal assembly causes accumulation of extra-ribosomal 60S ribosomal protein uL18/L5

PONE-D-19-24366R1

Dear Lasse,

I hope you had a fabulous Christmas and a fantastic start of 2020. Apologies for the delay in processing the manuscript but I had made a promise to my wife that I would not be doing any work during the Christmas period and therefore only started reading the revised version a few days ago. I have have now had a chance to properly read the revised manuscript and I am happy to recommend the paper for publication.

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Academic Editor

PLOS ONE

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