Peer review of: Cryo-EM structure of catalytic ribonucleoprotein complex RNase MRP (2020)

REVIEWER COMMENTS
Reviewer #1 (Remarks to the Author):


The manuscript by Perederina et al reports the 3Å cryo-EM structure of yeast RNase 
MRP, a protein-RNA complex that resembles eukaryotic RNase P.  This is an important 
and novel contribution of general interest and is clearly worthy of publication in Nature 
Communications.  RNase MRP is an RNP ribozyme that shares in common the RNA 
catalytic domain of RNase P, but a unique substrate-speciﬁcity domain.  Remarkably, 
most of the proteins associated with RNase P are also associated with MRP, but are 
arrayed diﬀerently and have a unique binding mode with respect to the diﬀering part of 
the RNA.  The manuscript makes extensive references and comparison to the RNase P 
structure, which greatly adds to its description and our understanding of its 
signiﬁcance.  The structural determination appears quite sound, and although I was 
unable to view the coordinates in the PDB, the validation report appears reasonable.


Some minor questions arise:


Are the diﬀerences observed in P15 a consequence of diﬀerent sequences and 
interactions in the two enzymes, or is P15 simply inherently ﬂexible, and the two 
structures simply capturing two diﬀerent conformations available to both enzymes? If 
the diﬀerence is dependent upon the identify of the enzyme, is there any obvious 
mechanistic consequence?


Based on the diﬀerences within the S-domain and similarity of the C-domain, is 
anything diﬀerent regarding the chemical mechanism apparent, or is this simply a very 
interesting example of how evolution has rearranged an enzyme to confer additional 
activity?  Is there any cross-reactivity between MRP and P (i.e., does MRP have any 
residual pre-tRNA processing capacity, or are the binding activities completely 
orthogonal)?


The methodology description states that the (previously existing) RNase P map was 
used to build in the similar parts of the structure.  This potentially raises the spectre of 
model bias.  (In this sense I ﬁnd the Pop3 protein issue more reassuring than concerning, but a bit more description of how bias was tested for and if present, 
eliminated, would be reassuring).


I fully appreciate the tour de force this structure represents, but the most important 
consequence — how substrate discrimination is achieved — remains a bit obscure.  
Can anything more be said about this, or do we need to await the structure of the 
enzyme-substrate complex?



Reviewer #2 (Remarks to the Author): 

RNase MRP is an important ribozyme involved in rRNA maturation and mRNA metabolism. Here, 
Pederina and colleagues present the cryo-EM structure of yeast MRP at 3.0 Å resolution, which comes 
on the heels of archeal, yeast, and human RNAse P cryo-EM structures, which are analogous enzymes 
to RNase MRP but are involved in tRNA maturation. The authors conduct a thorough structural analysis 
of the RNase MRP structure compared to yeast RNase P, revealing structural similarities surrounding 
the active site and striking differences surrounding the substrate binding domain. Of note, they 
observe that while most subunits are shared between RNase MRP and RNase P, they adopt new 
conformations (e.g. Pop1, Pop4) due to the altered RNA sequence/secondary structure. In addition, 
they resolve the two RNase MRP specific subunits Smn1 and Rmp1, and can describe their roles in 
MRP architecture and substrate binding. One major insight comes from the comparison of substrate 
accessibility, revealing that RNase MRP lacks features required for pre-tRNA binding by RNase P. 

Overall, the RNase MRP structure will be of great interest to the RNA ribozyme and metabolism fields, 
and is thus in principle suitable for publication in Nature Communications. From the figures, it's 
apparent that the RNase MRP coordinate model can be significantly improved, and this would be a 
must prior to acceptance. In my view, the paper would benefit from clarifying edits to the figures and 
shortening of the text or refocusing towards the key points, see below. 

Major comments: 
1. Fig 4b shows the RNase MRP coordinate model for Pop1, compared to that in panel c of Pop1 in 
RNase P. The grey part of the protein looks near-identical, yet apparently the MRP model lacks all 
beta-strands, which are visible in the RNase P model. I would strongly suggest the authors improve 
their coordinate model and correct secondary structure features. At the reported nominal resolution of 
3.0 Å, this should be doable. 

2. The authors frequently compare yeast RNase MRP to yeast RNase P. Since the human RNase P 
structure is also known, a comparison to this would be of interest for the expert reader. 

3. The figures and coloring could be improved for clarity throughout. For example, Fig 2 two views 
would suffice on the top, the bottom views could be replaced with those from the top but with all 
proteins transparent and the RNA as solid - this would highlight the RNA path, which with the current 
choice of RNA and protein colors is very difficult to follow. Similarly, in Fig 6, it is very challenging to 
see the pre-tRNA (maybe add thicker lines?) and labels for example for the active site would be 
helpful. 

4. The text is extremely detailed in the description of structural features, however the importance of 
these is unclear. While I appreciate that a basic description of the structure is required, the authors 
could substantially shorten the text without loosing their key points on RNA-driven protein remodeling 
and the RNase MRP substrate binding domain. This will also make the manuscript more accessible to 
non-experts. 

Minor comments: 

1. There are multiple mentions of 'electron density' in the text, this is incorrect and should be replaced 
with cryo-EM density, density, coulomb potential map, or others. 

2. The authors should acknowledge the human RNAse P structure in the introduction more explicitly. 



Reviewer #3 (Remarks to the Author): 

Eukaryotic RNase MRP is a ribozyme that acts as an endoribonuclease. It consists of a catalytic RNA 
core and several protein subunits. The complex is closely to RNase P., with which it shares most 
protein subunits but also has unique components and a divergent RNA-subunit. RNase P and RNase 
MRP process different target RNAs. A cryo-EM structure of RNase P has been published in 2019, but 
similar structural information is still lacking for the related RNase MRP. This is where the manuscript, 
by Perederina et al. comes in. They represent the cryo-EM structure of the eukaryotic RNAse MRP. The 
comparison to RNase P reveals how the unique components act as adaptors and how some of the 
common proteins adapt their conformation to facilitate the contact to the diverging catalytic RNA-
subunit. 
The manuscript is well written and interesting. It appears largely sound. Minor concerns are: 
1) Pixel size “0.886 Å”. The quoted pixel size is unusual for a Falcon III at a Krios at 75000 
magnification. Many others report pixel sizes for these settings around 1.06 Å. The authors should 
check, whether they have used the correct parameters. 
If the pixel size would be wrong, this could explain the relatively low percentage of “favoured” 
positions in the Ramachandran plot. If the pixel size would be wrong, the authors should re-refine 
their model and adapt the resolution claim. However, a wrong pixel size would not have a significant 
impact on the claims. 
If the pixel size is correct, the authors should briefly explain how the pixel size was determined. It is 
best practice to determine the magnification of the microscope relative to a known X-ray structure. 
Other standards such as catalase or cross-grating can be easily a few percent off. 
2) The distribution of orientations shows a strong preference. Could the authors briefly explain, how 
this impacted their map or how they mitigated the problem? In extended figure 2 the map looks 
somewhat distorted, but this is difficult to see. 
3) In the methods it is stated that “the exposure dose for the dose weighting procedure was set to 70% 
of the actual dose”. Please add the rational why, this was done. 
4) The figures and the legends need some touch-up. The labels (“a”, “b”, etc.) are generally too large. 
The colour coding for the subunit changes between figures and even within a composite figure (e.g. 
Figure 4… Pop 1 is red in a and b and green in c. The same green is also used for Pop6 in a and Pop4 
in d (and e)), which make it difficult to follow the representations through the paper. 
The figure legends are also very concise and often do not provide sufficient information to understand 
what is shown in the figures without reading the main text.