RNA sequence to structure analysis from comprehensive pairwise mutagenesis of multiple self-cleaving ribozymes
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Evaluation Summary:
The authors provide a summary of single and double mutants in five self-cleaving ribozymes using next-generation sequencing. They dissect their data in terms of epistasis effects, which provides a new angle to understanding ribozyme function. In principle, this allows conclusions to be drawn on bases involved in pairs and in catalysis that have the potential to be of use to the field, although there is also a series of technical weaknesses that should be addressed.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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Abstract
Self-cleaving ribozymes are RNA molecules that catalyze the cleavage of their own phosphodiester backbones. These ribozymes are found in all domains of life and are also a tool for biotechnical and synthetic biology applications. Self-cleaving ribozymes are also an important model of sequence-to-function relationships for RNA because their small size simplifies synthesis of genetic variants and self-cleaving activity is an accessible readout of the functional consequence of the mutation. Here, we used a high-throughput experimental approach to determine the relative activity for every possible single and double mutant of five self-cleaving ribozymes. From this data, we comprehensively identified non-additive effects between pairs of mutations (epistasis) for all five ribozymes. We analyzed how changes in activity and trends in epistasis map to the ribozyme structures. The variety of structures studied provided opportunities to observe several examples of common structural elements, and the data was collected under identical experimental conditions to enable direct comparison. Heatmap-based visualization of the data revealed patterns indicating structural features of the ribozymes including paired regions, unpaired loops, non-canonical structures, and tertiary structural contacts. The data also revealed signatures of functionally critical nucleotides involved in catalysis. The results demonstrate that the data sets provide structural information similar to chemical or enzymatic probing experiments, but with additional quantitative functional information. The large-scale data sets can be used for models predicting structure and function and for efforts to engineer self-cleaving ribozymes.
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Evaluation Summary:
The authors provide a summary of single and double mutants in five self-cleaving ribozymes using next-generation sequencing. They dissect their data in terms of epistasis effects, which provides a new angle to understanding ribozyme function. In principle, this allows conclusions to be drawn on bases involved in pairs and in catalysis that have the potential to be of use to the field, although there is also a series of technical weaknesses that should be addressed.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The authors provide insight into which regions of the ribozymes are involved in pairings including some tertiary interactions. Overall, the data support known structures and give insight into the roles of bases as pairs, catalytic residues, and extensions. The epistasis analysis is novel and gives deeper insight than previous mutational analyses of ribozymes. However, more can be extracted from this data. This study will impact the field by helping classify the roles of possible bases. There are also numerous technical issues that must be addressed. The authors should consider why short and long pairings show different epistasis and discuss the robustness of pairings from an evolutionary point of view. The effect of the primer binding site on ribozyme activity needs to be discussed.
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Reviewer #2 (Public Review):
In this work, the authors have constructed a library of mutants of 5 different self-cleaving ribozymes starting from synthetic DNA presenting 97% of the wild-type nucleotide at each position mixed with 1% of the three other sequence possibilities.
The catalytic activity of each single and double mutant was then monitored by deep-sequencing analysis. The double mutants allow for assessing positive or negative epistasis, meaning whether the double mutations increase or decrease the catalytic capacities of the ribozyme as compared to the individual mutants. These data were reported on heatmaps for direct and straightforward visualisation.
This high-throughput approach generated very strong data, identifying systematically the residues critical for catalytic activity and the structural elements on which it …
Reviewer #2 (Public Review):
In this work, the authors have constructed a library of mutants of 5 different self-cleaving ribozymes starting from synthetic DNA presenting 97% of the wild-type nucleotide at each position mixed with 1% of the three other sequence possibilities.
The catalytic activity of each single and double mutant was then monitored by deep-sequencing analysis. The double mutants allow for assessing positive or negative epistasis, meaning whether the double mutations increase or decrease the catalytic capacities of the ribozyme as compared to the individual mutants. These data were reported on heatmaps for direct and straightforward visualisation.
This high-throughput approach generated very strong data, identifying systematically the residues critical for catalytic activity and the structural elements on which it relies. These extensive data present a thorough activity landscape of all ribozymes double mutants that could be in the past only obtained for a few positions due to time-consuming mutants construction. This method can be potentially adapted to any RNA, provided that a biological test can be designed. I reckon that this method will have a major impact in the field of RNA structure-activity relationship studies.
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Reviewer #3 (Public Review):
This article by Roberts, Hayden and colleagues expands on an interesting high-throughput experimental approach developed by Kobori and Yokobayashi (2016; Angew Chem) by determining the relative activity for every possible single and double mutant of five known self-cleaving ribozymes. While this approach is not in itself new, the fact that the authors analyze their data by looking at epistasis (non-additive effects between pairs of mutations) provides an additional opportunity for extracting meaningful structural information that is proposed to be similar to chemical or enzymatic probing experiments obtained on these self-cleaving ribozymes. In fact, this type of high throughput mutagenesis analysis might provide data closer to comparative sequence analysis and as such, might provide even more reliable …
Reviewer #3 (Public Review):
This article by Roberts, Hayden and colleagues expands on an interesting high-throughput experimental approach developed by Kobori and Yokobayashi (2016; Angew Chem) by determining the relative activity for every possible single and double mutant of five known self-cleaving ribozymes. While this approach is not in itself new, the fact that the authors analyze their data by looking at epistasis (non-additive effects between pairs of mutations) provides an additional opportunity for extracting meaningful structural information that is proposed to be similar to chemical or enzymatic probing experiments obtained on these self-cleaving ribozymes. In fact, this type of high throughput mutagenesis analysis might provide data closer to comparative sequence analysis and as such, might provide even more reliable structural information than structural probing experiments, especially when a relative activity can be properly assessed for the studied RNAs.
(1) Overall, the experiments have been carefully performed and the data seem to be highly reliable.
(2) The strength of this article is that it demonstrates the generality of the approach initially developed by Kobori and Yokobayashi (2016; Angew Chem) by validating its usefulness in identifying most (if not all) the structural features of the studied ribozymes. The determination of positive and negative epistasis is very useful as it can facilitate the identification of base pairs covariations that are indicative of RNA structural elements.
(3) At the present time, the authors have not really discussed how their data analysis compares to comparative sequence analysis. This aspect is important.
(4) It is necessary to mention more clearly that this article builds on the method of Kobori and Yokobayashi (2016). Overall, with the exception of a few experimental details, the experimental method described herein is almost identical to the one of Kobori and Yokobayashi (2016) and this should be better emphasized.
(5) Most importantly, this article provides an analysis of self-cleaving ribozymes for which the three-dimensional structures are known. Considering the scope of this article, instead of mostly focusing on the 2D structural aspect, it would be absolutely necessary to provide more 3D structural information.
(6) When a self-modifying enzymatic activity is associated with the studied RNA, a relative activity could potentially be derived from high throughput sequencing. Could the authors expand on the generality and requirement of their high throughput approach for the study of RNA? -
