Comprehensive re-analysis of hairpin small RNAs in fungi reveals loci with conserved links
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Curated by eLife
eLife assessment
This paper is of interest to scientists within the field of RNA silencing and evolution. The data analysis is rigorous, and the conclusions are justified by the data. The key claims of the manuscript provide a compelling approach to identifying and annotating microRNAs in fungi although there is a limitation in the functional validation of the identified miRNAs.
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Abstract
RNA interference is an ancient mechanism with many regulatory roles in eukaryotic genomes, with small RNAs acting as their functional element. While there is a wide array of classes of small-RNA-producing loci, those resulting from stem-loop structures (hairpins) have received profuse attention. Such is the case of microRNAs (miRNAs), which have distinct roles in plants and animals. Fungi also produce small RNAs, and several publications have identified miRNAs and miRNA-like (mi/milRNA) hairpin RNAs in diverse fungal species using deep sequencing technologies. Despite this relevant source of information, relatively little is known about mi/milRNA features in fungi, mostly due to a lack of established criteria for their annotation. To systematically assess mi/milRNA characteristics and annotation confidence, we searched for publications describing mi/milRNA loci and re-assessed the annotations for 41 fungal species. We extracted and normalized the annotation data for 1727 reported mi/milRNA loci and determined their abundance profiles, concluding that less than half of the reported loci passed basic standards used for hairpin RNA discovery. We found that fungal mi/milRNA are generally more similar in size to animal miRNAs and were frequently associated with protein-coding genes. The compiled genomic analyses identified 25 mi/milRNA loci conserved in multiple species. Our pipeline allowed us to build a general hierarchy of locus quality, identifying more than 150 loci with high-quality annotations. We provide a centralized annotation of identified mi/milRNA hairpin RNAs in fungi which will serve as a resource for future research and advance in understanding the characteristics and functions of mi/milRNAs in fungal organisms.
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**Author Response""
Reviewer #2 (Public Review):
The work systematically reassesses fungal mi/miRNA-like characteristics and annotation confidence and identifies that many of the loci fail to meet the key points of the methods developed for animal or plant miRNAs. Therefore, the authors establish a set of criteria suitable for the annotation of fungal miRNAs and provide a centralized annotation of identified mi/milRNA hairpin RNAs in fungi based on their established rules.
Here are some comments and suggestions for the manuscript to be improved:
- The title mentions "ancestral links", however, the main context of this paper does not include the evolution of fungal mi/milRNAs or show the origins of conserved mi/milRNAs in fungi. The authors are suggested to consider a more appropriate title for this work.
Agreed, we have modified …
**Author Response""
Reviewer #2 (Public Review):
The work systematically reassesses fungal mi/miRNA-like characteristics and annotation confidence and identifies that many of the loci fail to meet the key points of the methods developed for animal or plant miRNAs. Therefore, the authors establish a set of criteria suitable for the annotation of fungal miRNAs and provide a centralized annotation of identified mi/milRNA hairpin RNAs in fungi based on their established rules.
Here are some comments and suggestions for the manuscript to be improved:
- The title mentions "ancestral links", however, the main context of this paper does not include the evolution of fungal mi/milRNAs or show the origins of conserved mi/milRNAs in fungi. The authors are suggested to consider a more appropriate title for this work.
Agreed, we have modified our title to include a more fitting description of the outcome of the study:
“Comprehensive re-analysis of hairpin small RNAs in fungi reveals loci with conserved links”
- The work proposes a fungal mi/milRNAs hairpin precursor recovery pipeline with three minimal criteria to annotate fungal mi/milRNA loci, which allows nearly half of the loci to pass these rules. To highlight the innovation of this annotation, it is strongly suggested that the authors compare their established pipeline and criteria for fungi with those used in animal or plant miRNAs in detail, and emphasize the advantages of the established pipeline. A figure showing the established pipeline and detailed parameters is needed.
We have now included a clear workflow diagram for establishing miRNA annotation records and confidence tiers (Figure 1-supplemental 3). As for the comparison with rules in plants and animals, this is stated in Table S6, where it shows some rules employed by other tools/papers/species. We believe these combined supplementals give a strong overview of our approach and how it differs from rules in other approaches.
- The established "standard rules" for fungal mi/milRNA annotation still require more evaluation. It would be better if there is experimental validation to improve confidence.
Sequencing evidence is generally regarded as the gold-standard of experimental support for identifying and annotating miRNAs (Axtell and Meyers, 2018) though the rules are not clear yet in fungi. We agree that developing a standard-rule-set is a high-priority for identifying complete annotation standards. We had a statement (~ line 290) affirming this need, and have now modified this sentence to highlight the need for a sufficient standard.
“While this minimal rule-set is useful for filtering the lowest-confidence loci, it is likely not sufficient to form the basis of an annotation and this analysis further confirms the need for a standardized pipeline and set of criteria for miRNA annotation in fungi.”
To address the question of experimental validation, we have included descriptions of loci with strong-functional support in Table S5, including a section discussing top-tier loci in the discussion, described in the response to reviewer 3.
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eLife assessment
This paper is of interest to scientists within the field of RNA silencing and evolution. The data analysis is rigorous, and the conclusions are justified by the data. The key claims of the manuscript provide a compelling approach to identifying and annotating microRNAs in fungi although there is a limitation in the functional validation of the identified miRNAs.
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Reviewer #1 (Public Review):
This is an elegant article where the authors define valuable criteria to identify and classify high-confidence miRNA in fungi. The data supporting the conclusions are solid, and the results are essential to unify the annotation criteria in these organisms. Interestingly, the paper shows that miRNAs in fungi look and position within the genome more similarly to their animal counterpart but may act like plant miRNAs.
The conclusions of this paper are well supported by data, but some aspects need to be clarified and extended.
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Reviewer #2 (Public Review):
The work systematically reassesses fungal mi/miRNA-like characteristics and annotation confidence and identifies that many of the loci fail to meet the key points of the methods developed for animal or plant miRNAs. Therefore the authors establish a set of criteria suitable for the annotation of fungal miRNAs and provide a centralized annotation of identified mi/milRNA hairpin RNAs in fungi based on their established rules.
Here are some comments and suggestions for the manuscript to be improved:
1. The title mentions "ancestral links", however, the main context of this paper does not include the evolution of fungal mi/milRNAs or show the origins of conserved mi/milRNAs in fungi. The authors are suggested to consider a more appropriate title for this work.
2. The work proposes a fungal mi/milRNAs hairpin …Reviewer #2 (Public Review):
The work systematically reassesses fungal mi/miRNA-like characteristics and annotation confidence and identifies that many of the loci fail to meet the key points of the methods developed for animal or plant miRNAs. Therefore the authors establish a set of criteria suitable for the annotation of fungal miRNAs and provide a centralized annotation of identified mi/milRNA hairpin RNAs in fungi based on their established rules.
Here are some comments and suggestions for the manuscript to be improved:
1. The title mentions "ancestral links", however, the main context of this paper does not include the evolution of fungal mi/milRNAs or show the origins of conserved mi/milRNAs in fungi. The authors are suggested to consider a more appropriate title for this work.
2. The work proposes a fungal mi/milRNAs hairpin precursor recovery pipeline with three minimal criteria to annotate fungal mi/milRNA loci, which allows nearly half of the loci to pass these rules. To highlight the innovation of this annotation, it is strongly suggested that the authors compare their established pipeline and criteria for fungi with those used in animal or plant miRNAs in detail, and emphasize the advantages of the established pipeline. A figure showing the established pipeline and detailed parameters is needed.
3. The established "standard rules" for fungal mi/milRNA annotation still require more evaluation. It would be better if there is experimental validation to improve confidence. -
Reviewer #3 (Public Review):
The authors provide a centralized annotation of miRNA and miRNA-like hairpins in fungi. They aim to develop a standardized pipeline and criteria for miRNA annotation in fungi focusing only on sRNAs derived from hairpin structures, seeking to identify essential characteristics of fungal miRNA and miRNA-like.
Overall this paper will be of interest to readers trying to understand the characteristics and functions of miRNA and miRNA-like hairpins in fungi. The conclusions of this paper are mostly well supported by data, but some aspects of the methodology need to be clarified and extended. The absence of follow-up experiments somewhat limits the impact of this paper. Subsequent work should focus on searching and validating targets of miRNA in fungi. In particular, the strong mi/milRNAs candidates detected in …
Reviewer #3 (Public Review):
The authors provide a centralized annotation of miRNA and miRNA-like hairpins in fungi. They aim to develop a standardized pipeline and criteria for miRNA annotation in fungi focusing only on sRNAs derived from hairpin structures, seeking to identify essential characteristics of fungal miRNA and miRNA-like.
Overall this paper will be of interest to readers trying to understand the characteristics and functions of miRNA and miRNA-like hairpins in fungi. The conclusions of this paper are mostly well supported by data, but some aspects of the methodology need to be clarified and extended. The absence of follow-up experiments somewhat limits the impact of this paper. Subsequent work should focus on searching and validating targets of miRNA in fungi. In particular, the strong mi/milRNAs candidates detected in their work.
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