A comprehensive survey of C. elegans argonaute proteins reveals organism-wide gene regulatory networks and functions
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eLife assessment
This impressive study presents the most comprehensive analysis of the Argonautes, their small RNA partners, their targets, and their biological functions in any species to date. The work provides new insights into Argonaute-based pathways, it includes extensive validation of existing models, and describes overall a treasure-trove of reagents and datasets for future exploration of the vast Argonaute world in C. elegans.
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Abstract
Argonaute (AGO) proteins associate with small RNAs to direct their effector function on complementary transcripts. The nematode Caenorhabditis elegans contains an expanded family of 19 functional AGO proteins, many of which have not been fully characterized. In this work, we systematically analyzed every C. elegans AGO using CRISPR-Cas9 genome editing to introduce GFP::3xFLAG tags. We have characterized the expression patterns of each AGO throughout development, identified small RNA binding complements, and determined the effects of ago loss on small RNA populations and developmental phenotypes. Our analysis indicates stratification of subsets of AGOs into distinct regulatory modules, and integration of our data led us to uncover novel stress-induced fertility and pathogen response phenotypes due to ago loss.
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eLife assessment
This impressive study presents the most comprehensive analysis of the Argonautes, their small RNA partners, their targets, and their biological functions in any species to date. The work provides new insights into Argonaute-based pathways, it includes extensive validation of existing models, and describes overall a treasure-trove of reagents and datasets for future exploration of the vast Argonaute world in C. elegans.
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Reviewer #1 (Public Review):
The manuscript by Seroussi et al. presents a comprehensive analysis of the expression and function of the entire, extensive family of Argonaute (AGO) proteins in C. elegans. Using genome editing methods, the authors fused tags to 19 endogenous argonaute genes to allow for visualization of their expression patterns in live worms and immunoprecipitation assays to detect RNA partners. Furthermore, they analyzed how the loss of specific AGOs impacts smRNA populations as well as fertility, developmental, and pathogen susceptibility phenotypes. The methods are rigorous and care was taken to maintain the functionality of tagged proteins. This study offers an extremely valuable resource in its comprehensive evaluation of all AGOs in an organism and the resulting datasets and reagents. Furthermore, the authors …
Reviewer #1 (Public Review):
The manuscript by Seroussi et al. presents a comprehensive analysis of the expression and function of the entire, extensive family of Argonaute (AGO) proteins in C. elegans. Using genome editing methods, the authors fused tags to 19 endogenous argonaute genes to allow for visualization of their expression patterns in live worms and immunoprecipitation assays to detect RNA partners. Furthermore, they analyzed how the loss of specific AGOs impacts smRNA populations as well as fertility, developmental, and pathogen susceptibility phenotypes. The methods are rigorous and care was taken to maintain the functionality of tagged proteins. This study offers an extremely valuable resource in its comprehensive evaluation of all AGOs in an organism and the resulting datasets and reagents. Furthermore, the authors provide thoughtful analyses of their own data pointing out surprises and follow-up experiments to support their interpretations. A good example is the isolation of miRNAs in the ERGO-1 IP, which provide compelling evidence to indicate that this result is likely due to co-IP of ALG-1/2 on transcripts also bound by ERGO-1 rather than a new role for ERGO-1 in directly binding miRNAs. The authors are also to be commended on the clear and engaging figures, which are often difficult to produce from largely genomics data. Overall, this is a highly significant body of work that provides extensive datasets and reagents that will propel the smRNA field forward faster.
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Reviewer #2 (Public Review):
In this manuscript by Seroussi et al, the authors describe a global analysis of Argonaute (AGO) protein biology in the model organism C. elegans. Small RNAs regulate most facets of gene expression and, therefore, play important roles in all aspects of biology, including development and health. C. elegans has been at the forefront of attempts by biologists to understand the biological roles played by AGOs and small RNAs in animals. Here, the authors make a significant contribution to this effort by epitope tagging all 21 of the elegans AGO genes. The reagents they generate allow the authors to explore at a global level the where, when, and why of C. elegans AGOs. Whenever possible the authors confirm that their tagged proteins are functional. The authors then analyze the expression patterns of elegans AGOs, …
Reviewer #2 (Public Review):
In this manuscript by Seroussi et al, the authors describe a global analysis of Argonaute (AGO) protein biology in the model organism C. elegans. Small RNAs regulate most facets of gene expression and, therefore, play important roles in all aspects of biology, including development and health. C. elegans has been at the forefront of attempts by biologists to understand the biological roles played by AGOs and small RNAs in animals. Here, the authors make a significant contribution to this effort by epitope tagging all 21 of the elegans AGO genes. The reagents they generate allow the authors to explore at a global level the where, when, and why of C. elegans AGOs. Whenever possible the authors confirm that their tagged proteins are functional. The authors then analyze the expression patterns of elegans AGOs, sequence small RNAs that associate with these AGOs, and identify small RNA populations that change in animals lacking the AGOs.
In many cases the data presented are consistent with previously published studies, establishing that the author's approach was likely successful. In other cases, the data allow the authors to make novel observations. For instance, the authors categorize the 21 AGOs into four major categories- based on their small RNA binding profiles. They define the types of genomic loci targeted by each group of AGOs and show that these AGO groups regulate distinct classes of transposable elements, such as DNA cut-and-paste transposons, LTR retrotransposons, and Rolling transposons. This differential targeting suggests that sequence features intrinsic to each target transcript may help direct different RNAs into different silencing pathways. A metagene analysis defines differences in the 3' and 5' distribution of small RNAs across target genes for each AGO. These patterns are likely to have important implications for understanding RdRP function. The authors identify an AGO, which they name VSRA-1, which binds broadly to most classes of small RNAs. This data is likely to be an important step towards understanding how different AGOs bind different small RNAs. The authors present evidence that they have identified ten new miRNAs. The low expression levels of these miRNAs suggest that additional work is needed to ensure that these miRNAs have a biological function. The authors show that miRNAs with higher precursor duplex complementarity are preferentially loaded into the AGO RDE-1. This observation has important implications for miRNA evolution. The authors identify several hundred new candidate piRNAs as well as reclassify many mis-annotated miRNAs as piRNAs. Finally, the authors link several AGOs to interesting phenotypes, such as germ cell immortality and innate immunity.
The paper is very well written. No small accomplishment given the huge amount of data presented. As far as I can tell, experimental approaches and statistical analyses follow best practices. Alternative explanations for data are usually acknowledged. In summary, the paper provides novel insights into small RNA biology (described above), establishes high-quality reagents that will empower future studies into the myriad of ways that C. elegans use small RNAs to regulate gene expression, and demonstrate quite clearly that the C. elegans small RNA systems are intertwined and remarkably complex.
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