The RNA-binding activity of the TRIM-NHL protein NHL-2 is essential for miRNA-mediated gene regulation

Reviewer #1 (Public Review):

Summary:

C. elegans NHL-2 is a member of the conserved TRIM-NHL RNA binding protein family, with known functions in promoting small regulatory RNA function, including the conserved let-7 family microRNAs. Since NHL-2 promotes microRNA function, the authors seek to address if this function is due to direct binding of a mRNA target shared with the miRNA pathway. They successfully solve the crystal structure of NHL-2's NHL domain and discover residues Tyr935/Arg978 are required for RNA binding in vitro. In C. elegans, they establish that Tyr935/Arg978 are required for nhl-2 to promote let-7 microRNA function. Processing body (P body) size is increased in nhl-2 (Y935A R978A) and null mutants. The microRNA Argonautes, ALG-1 and ALG-2, also show increased binding to known let-7 mRNA targets in nhl-2 null mutants. Together these data suggest a lack of mRNA turnover in the absence of functional NHL-2. NHL-2 may function with CGH-1 and IFET-1 to promote let-7 miRISC function.

Strengths:

The authors successfully solve the structure of NHL-2's NHL domain. Although unable to crystalize it bound to RNA they are able to predict residues important for RNA binding based on charge, position and comparison with other known NHL domain structures crystalized with RNA. In vitro RNA binding assays confirm that Tyr935/Arg978 are required for RNA binding in vitro.

Weaknesses:

(1) In vivo, authors use a combination of established let-7 microRNA genetics and a 3' UTR reporter assay to establish that Tyr935/Arg978 are required for nhl-2 to promote let-7 microRNA function. However, they do not demonstrate that full length NHL-2 actually binds RNA directly in vivo in the Tyr935/Arg978 mutated background. While the presented genetic evidence suggests nhl(RBlf) acts much like the nhl-2 null, it is never demonstrated that full length NHL-2(RBlf) is actually RNA binding defective/dead in vivo. Yet several times in the text this is implied or stated. For example,
o page 8, section title. "RNA binding is essential for NHL-2 function in heterochronic pathway"
o page 9 - line 13-14. "Together, these data indicate that the RING and NHL domains are required for the normal function of NHL-2, but that the loss of RNA-binding activity has a more pronounced phenotype, suggesting that RNA-binding is critical for NHL-2 function."
o page 11, line 3-4. "Together these experiments support the conclusion that... RNA binding is essential for its function"
The language should be softened (e.g., page 8: "Residues required for RNA binding in vitro are required for NHL-2 function in heterochronic path") or additional experiments should be performed to support that NHL-2(RBlf) is in fact RNA binding defective/dead, like wild-type NHL-2 vs NHL-2(RBlf) RIP-qPCR for let-7 targets.

(2) Authors report that Processing body (P body) size is dependent on nhl-2 and the Tyr935/Arg978 residues. microRNA Argonautes, ALG-1 and ALG-2, also show increased binding to known let-7 mRNA targets in nhl-2 null mutants (unfortunately requirement of Tyr935/Arg978 is not tested). However total levels of these mRNAs are unchanged. Authors propose these data together support a role for nhl-2 in promoting microRNA target turnover. Unfortunately, it is unclear how increased P body size with no observed increase of microRNA target levels are to be resolved.

(3) The authors propose a model where NHL-2, CGH-1(DDX6) and IFET-1(eIF4E-transporter/4E-T) promote microRNA mediated translational repression and possibly turnover based on nhl-2-dependent IFET-1 interaction with ALG-1, cgh-1's synthetic interaction with both nhl-2 and ifet-1 to enhance let-7-mediated alae development, and conservation of known interactions between Dead Box helicases and eiF4A, which is supplemented by ALPHAFold modelling of IFET-1. The Boag lab previously characterized ifet-1 as a translational repressor required for germline P granule formation (Sengupta 2013 J Cell Sci). The role of NHL-2 RNA binding is unclear in this model as is any more molecular evidence of direct NHL-2, CGH-1 and IFET-1 interaction.

(4) In Figure 5, adult nhl-2(ok818) worms express the mCherry when the putative NHL-2 binding sites in the lin-28 3'UTR reporter are mutated. Couldn't this be interpreted as suggesting that the observed phenotype is nhl-2 independent? The authors mention this as an "interesting" observation in text, but I find it concerning. The authors should address this issue more directly. The reporter expression data needs to be quantified.

(5) I am frankly confused at the direction the manuscript takes in the Discussion section. The role of NHL-2 RNA binding, which has been the core of the paper, is seemingly disregarded and exchanged for what is mainly speculation about protein-protein level regulation with CGH-1 and IFET-1. This is all based on only a few pieces of data that do not include any analysis using the nhl-2(RBlf): nhl-2-dependent IFET-1 interaction with ALG-1, cgh-1's synthetic interaction with both nhl-2 and ifet-1 to enhance let-7-mediated alae development, and conservation of known interactions between Dead Box helicases and eiF4A, which is supplemented by ALPHAFold modelling of IFET-1. I'd strongly suggest reworking the text to better integrate IFET-1 or skip it and refocus the Discussion around the majority of the data characterizing NHL-2 RNA binding.

Reviewer #2 (Public Review):

Summary:

In this manuscript, the authors provide structural analysis of the NHL domain for C. elegans NHL-2 and provide functional analysis of the NHL RNA binding domain. Their data support a model in which NHL-2 binding to mRNA targets through U rich motifs to promote miRISC regulation of translation and mRNA stability.

Strengths:

The authors present convincing data to describe the structure of the NHL-2 NHL domain along with functional analysis that supports an important role for two amino acids that are required for RNA binding activity. The function of these two amino acids were further studied through phenotypic assays to analyze their contribution to miRNA mediated regulation through the let-7 pathway. These data support an important role for RNA binding activity of NHL-2 in the regulation of miRNA dependent pathways. Genetic interactions support a role for the eIF4E binding protein IFET-1 in the miRISC activity.

Weaknesses:

The use of phenotypic assays to monitor let-7 pathway activity could be better explained so that the reader can more easily follow the significance of changes in alae formation or col-19::gfp expression.

The challenges of comparing expression levels using extrachromosomal arrays should be acknowledged.

The figure legends need to be revised to more clearly and accurately explain what is shown in the figures.

Reviewer #3 (Public Review):

Summary:

The manuscript by Saadat et al., examines the structure and function of the NHL-2 RNA binding domain in miRNA-mediated gene regulation in C. elegans. NHL-2 has previously been shown to function in miRNA and other smRNA pathways in C. elegans but its mechanism of action is unclear. The authors present a crystal structure that revealed candidate RNA binding residues. In vitro binding assays confirmed that these amino acids were required for RNA binding. The authors tested the importance of the RING and NHL domains in NHL-2 by mutating the endogenous gene using CRISPR and analyzing developmental and molecular effects in C. elegans. They concluded that the RNA binding domain of NHL-2 and co-factors, including CGH-1 and IFET-1, are important for the regulation of some miRNA targets in developing C. elegans.

Strengths:

The NHL-2 structural work and in vitro analyses of RNA binding activity are rigorous. The work is important for providing new structural information for an important post-transcriptional regulator.

Weaknesses:

The in vivo studies to better understand the role of NHL and several cofactors require further controls, replicates or better explanations of the methods and analyses in order to support the conclusions. In particular, protein levels of the mutant NHL-2 strains should be analyzed to rule out differences in expression contributing to the results; the reporter strategy would be improved by showing it is dependent on miRNA regulation, including an internal control and adding quantitative data; validation of similar levels of ALG-1 protein in the immunoprecipitation experiments would add confidence for the differences in levels of miRNA targets detected.