An adapted MS2-MCP system to visualize endogenous cytoplasmic mRNA with live imaging in Caenorhabditis elegans

  1. Cristina Tocchini
  2. Susan E. Mango

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Abstract

Live imaging of RNA molecules constitutes an invaluable means to track the dynamics of mRNAs, but live imaging in Caenorhabditis elegans has been difficult to achieve. Endogenous transcripts have been observed in nuclei, but endogenous mRNAs have not been detected in the cytoplasm, and functional mRNAs have not been generated. Here, we have adapted live imaging methods to visualize mRNA in embryonic epithelial cells. We have tagged endogenous transcripts with MS2 hairpins in the 3’ Untranslated Region (UTR) and visualized them after adjusting MS2 Coat Protein (MCP) expression. A reduced number of these transcripts accumulate in the cytoplasm, leading to loss-of-function phenotypes. In addition, mRNAs for dlg-1 fail to associate with the adherens junction, as observed for the endogenous mRNA. These defects are reversed by inactivating the nonsense-mediated decay pathway. RNA accumulates in the cytoplasm, dlg-1 associates with the adherens junction, and mutant phenotypes are rescued. These data suggest that MS2 repeats can induce the degradation of endogenous targets and alter the cytoplasmic distribution. Although our focus is RNAs expressed in epithelial cells during morphogenesis, this method can likely be applied to other cell types and stages.

Summary statement

An adapted MS2-MCP method to tag endogenous transcripts in C. elegans embryos for live imaging without affecting mRNA stability.

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  1. Review Commons

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    Reply to the reviewers

    We thank the reviewers for their thoughtful comments. We were delighted that the reviewers found our manuscript and results “solid”, “important”, “well-written”, “thoughtful”, “critical addition to the literature”, that the “design of (these) experiments is high in quality” and “conclusions are convincing and the experiments are well executed”.

    We were thrilled the reviewers appreciated that “this manuscript provides solutions to technical limitations to observe mRNA in vivo” by approaching such limitations “in a thoughtful study where many of the salient features of MS2 epitope tagging are systematically measured” and “it provides a greatly improved tool to track mRNA by live imaging” that “also alerts of experimental noise that can be found and can be specific for each gene/transcript”

    We will address all the concerns raised by the reviewers. Most of the comments concern text edits. In addition, we will add the following to the Results section:

    1. Quantitation of observed phenotypes in Figures 1C-D and 2C-D;

    2. Quantitation of cytoplasmic transcripts in Figure 1G-L.

    Quantitation will be performed as previously done in Tocchini et al., 2021.

  5. Review Commons

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    Reply to the reviewers

    We thank the reviewers for their thoughtful comments. We were delighted that the reviewers found our manuscript and results “solid”, “important”, “well-written”, “thoughtful”, “critical addition to the literature”, that the “design of (these) experiments is high in quality” and “conclusions are convincing and the experiments are well executed”.

    We were thrilled the reviewers appreciated that “this manuscript provides solutions to technical limitations to observe mRNA in vivo” by approaching such limitations “in a thoughtful study where many of the salient features of MS2 epitope tagging are systematically measured” and “it provides a greatly improved tool to track mRNA by live imaging” that “also alerts of experimental noise that can be found and can be specific for each gene/transcript”

    We will address all the concerns raised by the reviewers. Most of the comments concern text edits. In addition, we will add the following to the Results section:

    1. Quantitation of observed phenotypes in Figures 1C-D and 2C-D;

    2. Quantitation of cytoplasmic transcripts in Figure 1G-L.

    Quantitation will be performed as previously done in Tocchini et al., 2021.

  6. Review Commons

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    Referee #3

    Evidence, reproducibility and clarity

    Review of: "An adapted MS2-MCP system to visualize endogenous cytoplasmic mRNA with live imaging in Caenorhabditis elegans"
    Authors: Cristina Tocchini and Susan Mango

    The MS2-MCP imaging platform is an essential imaging system that enables dynamic quantification of mRNA transcription, abundance, location, and turnover in living biological systems. In the last ten or so years, this approach has been used in extremely successful ways in Drosophila embryos to dissect both the regulatory logic underpinning early transcriptional organization and activation with unprecedented resolution and, furthermore, how active mRNA localization outside of the nucleus impacts pattern formation. The authors correctly point out that full implementation of this tool has been suspiciously lacking in the C. elegans community for some time (aside from a few noted implementations).

    In this manuscript, Tocchini and Mango directly approach this deficit in a thoughtful study where many of the salient features of MS2 epitope tagging are systematically measured. Specifically, the authors use CRISPR genomic engineering to tag two separate dosage-sensitive, developmental genes and study the expression and function of these genes within the context of the MS2/MCP-GFP system. The authors demonstrate that the location of the MS2 epitope insertion within the endogenous 3'UTR is an important design consideration for functional, downstream implementation of the imaging system. In both cases, insertion of the MS2 hairpins near the end of the open reading frame of either gene results in overt and specific developmental phenotypes that phenocopy previously characterized loss of function alleles of each gene. The design of these experiments is high in quality in that they measure both the levels of cytoplasmic abundance of the various epitope-tagged mRNAs as well as the protein expression levels for these transgenes (by monitoring the levels of GFP expression (each MS2-tagged gene encodes a functional GFP-tagged allele). In two clear transgene examples, they demonstrate that the loss of function phenotypes of the proximally-tagged (closest to the ORF) transgenes disrupt mRNA levels and expression and reduce the proper localization of these mRNAs. This may be why previous attempts at implementing this important imaging system have failed.

    The authors then characterize the cellular systems that cause the differential expression of MS2-tagged transgenes. The authors note that previous studies on simpler systems and in C. elegans have suggested that the nonsense-mediated mRNA decay (NMD) pathway limits the expression of mRNAs with exceptionally long 3'UTRs. Tocchini and Mango then use C. elegans NMD mutants to demonstrate that ablation of this natural RNA degradation system corrects the developmental and gene expression defects associated with the reduction of function MS2 insertion alleles. These experiments are complete and compelling as they are validated at all levels (GFP expression (via quantification of GFP expression) and mRNA expression, and mRNA localization levels (via in situ hybridization).

    The authors then make the case that the type and expression levels of the MCP-GFP fusion protein are also essential features that need to be optimized for an effective imaging system. The authors suggest that optimal visualization of endogenous genes requires the surprisingly low-level expression of the MCP-GFP fusion protein. The authors use a novel transgene that differs from the conventional system. Specifically, the Tocchini system employs a 2xMCP ORF fused to 2xmCherry ORFs fusion. This transgene lacks the NLS typically used to localize exported mRNAs in the cytoplasm and also encodes two MCPs that may or may not facilitate dimerization on the MS2 hairpins. They demonstrate that endogenous, epitope-tagged transgenes can be visualized in developing embryos and that tethering this 2xMCP fusion to the reporter transcript does not alter RNA expression levels. While the authors demonstrate that visualization is possible with this system, it is hard to tell if this fusion protein dramatically improves over other available systems without a direct comparison. For instance, measuring the signal-to-noise (S/N) ratio of localized 2xMCP-2xmCherry would be a good addition and support the author's claims. If it were an exceptional system, these calculations should exceed the well-characterized and quantified MCP-GFP system described in Lee et al. 2019 ((Lee et al., 2019). It is just too hard to know if this is a dramatic element that should now be included in future RNA localization experiments.

    Minor critiques:

    1. The authors should provide more details in the experimental description of the MS2-tagged alleles (or in the figure images). It needs to be clarified in the main text how many MS2 hairpins there are, though this can be found in the materials and methods. In addition, it would be nice to know if these were any of the variations of MS2 hairpins that have already been optimized in some other way to increase or decrease structure or RNA metabolism defects in other systems. Specifically, are these hairpins the newest versions, V6 or V7, described in manuscripts from the Singer laboratory (e.g., (Tutucci et al., 2018))? For aficionados of this imaging system, it would be important to qualify each of the potential new features that make the results in this manuscript so clear and important.
    2. For people that are colorblind (or have reduced ability to distinguish some colors from others (like me, a reviewer)), it would be nice to have the MS2 illustrations in Figures 1A and B not have that color within the black, normal UTR. It's picky, but I had to ask someone what color that was.

    References:

    Lee, C., Shin, H., and Kimble, J. (2019). Dynamics of Notch-Dependent Transcriptional Bursting in Its Native Context. Dev Cell 50, 426-435 e424.

    Tutucci, E., Vera, M., Biswas, J., Garcia, J., Parker, R., and Singer, R.H. (2018). An improved MS2 system for accurate reporting of the mRNA life cycle. Nat Methods 15, 81-89.

    Significance

    In summary, this is a well-written and critical addition to the literature that will hopefully increase the implementation of this system in C. elegans research. The systematic approach to getting a new experimental platform up and running certainly has a place in the canon. Aside from the missing elements regarding the putative improvements and/or direct comparisons between different MCP fusion proteins, the manuscript is solid, important, and nearly ready to go.

    It is an advance and will, as noted above, likely serve to help implement this system by other C. elegans reserachers.

  7. Review Commons

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    Referee #2

    Evidence, reproducibility and clarity

    The transparency of C. elegans invites us to push the limit of live imaging. In this context, observation of endogenous mRNA using the high affinity between MS2 RNA hairpins (from a bacteriophage) and a protein (MCP) that can be fluorescently labeled. In the time of CRISPR-Cas, the editing of endogenous genes is feasible and authors use it to insert 24 MS sequences (about 650 bp in total) at the UTRs of a couple of genes. Once they found a way to insert the MS2 sequences at the UTRs, although with phenotypic consequences that are solved in mutants defective in Non-mediated decay of transcript, they tune the expression of the MCP using a heat-shock promoter with a leaking expression at 25C and its location in the cytoplasm avoiding nuclear location signal (NLS) of the protein.

    Major comments:

    They present solutions for live imaging endogenous mRNA that would be useful for colleagues interested in this technique but also show experimental noises that would be specific to each gene/transcript/UTR. In the end, the best value of this technique is to observe "real" or physiological levels but to reach this point they need to use a mutant background (NMD mutants), which may alter the "real" scenario. They found a smart way to title the article using "An adapted..." but it would be more realistic/honest to mention in the title that this is happening only in NMD mutant backgrounds. I also have doubt ion the use of the acronym MS2-MCP in the title. What about something like "Visualization of endogenous cytoplasmic mRNA with live imaging in C. elegans embryos requires an inactive Non-mediated decay"?

    In any case, the conclusions are convincing and the experiments are well executed. I do not find the need for any essential experiments if they are clearer through the manuscript (from title and abstract to discussion) that this technique may need to be optimized and (and maybe validated with FISH) for each specific transcript, and developmental stage cell type where NMD and polyadenylation may work differently. Another source of experimental noise may come from the use of mcherry, which is known for forming aggregates in some cells/stages.(would this artefactual aggregation occur in figure 1L?)
    The only experiment that I missed, not essential but easy to perform, is a better description of the slight developmental delay of dlg-1 MS2 v2 animals. Size measures? Time until they lay embryos?

    In this sense, although is not the main purpose of the article, they could highlight the fact that this is an additional option to produce hippomorphic alleles of essential genes.

    Regarding methods, I miss information about the CRISPR-Cas efficiency of inserting the MS2 sequences at the UTRs. Sizes are "small" and can facilitate the insertion of dsDNA repair template, but it would be useful to know what efficiency would be expected. It would be good to mention somewhere how frequent are GG PAM sequences at UTRs sequences (probably less common than in other regions). In this sense, the use of minimal PAM Cas9 variants (Vicencio et al, Nat Comm 2022) would be necessary.

    Minor comments:

    In the abstract, line 33, remove epithelial? I do not think this is relevant in this sentence.
    In figure 4, panels B and C, add the two different embryonic stages on the left side. Then, it wouldn't be necessary to read the legend to understand the figure.

    Referees cross-commenting

    I find useful and reasonable the comments of my colleagues

    Significance

    I work in C. elegans on diverse topics, with an interest in RNA, and I have used FISH in C. elegans in the past. I find this study useful to expand the C. elegans toolbox in C. elegans. This manuscript provides solutions to technical limitations to observe mRNA in vivo in the cytoplasm, but also alerts of experimental noise that can be found and can be specific for each gene/transcript.

    It is focused on the C. elegans embryo, which is the system of interest for the authors. I miss a bit of discussion at least on the use of this methodology in other stages. One of the interesting aspects of observing mRNA in vivo is the capacity to manipulate the environment. Such capacity is very limited in embryos but feasible in larvae or adults with the use of microfluidics.

  8. Review Commons

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    The authors of this manuscript have adapted the MS2-MCP system to visualize endogenous cytoplasmic mRNA with live imaging in Caenorhabditis elegans. They have identified some of the issues that might have prevented the MS2-MCP system's adaptation to C. elegans. Specifically, they have identified that the length of the 3'UTR, which is significantly increased upon the insertion of the MS2 sequences, impacts the mRNAs' stability. They have also shown that removing the nonsense-mediated decay pathway can prevent the destabilization of the MS2 transcripts. Moreover, they have also optimized the MCP expression to avoid nuclear retention of the MS2 transcripts and mislocalization of the mRNAs.

    Major comments:

    • The authors show that the insertion of 24xMS2 in two endogenous genes, spc-1 and dlg-1, causes some phenotypes such as slow growth, lack of coordination (Unc), small body size (Sma), and reduced brood sizes. However, only an image example is provided in Fig. 1 C, D, and quantifying all these phenotypes would be nice. Same in Fig. 2C, D.
    • Similarly, the reduction in mRNA spots from smFISH in Fig. 1 G-L is difficult to visualize by eyes, and proper smFISH quantification will help interpret the results.
    • The authors also claim a reduction in cytoplasmic RNAs and increased signal in nuclear RNAs in Fig. 1J, L. A proper quantification of nuclear and cytoplasmic smFISH will help interpret the results.
    • In Fig. 3D-F, the authors quantified the signal of nuclear smFISH. However, it is unclear to me in what samples or conditions the statistical test is performed. For example, do the three stars in Fig. 3D refer to the significant decrease or increase of NMD strain compared to the WT? What about the stars in Fig. 3E? The authors should indicate what samples they compare in the statistical test.

    Minor comments:

    • on line 195, the authors reference Fig. 3A. However, it should be Fig. 4A.
    • In Fig. 4B, C the authors can add close to the image of the embryos the developmental stages. This will help the reader identify the embryo's developmental stage in the figure's upper and lower parts.
    • The authors can expand a bit the discussion on how their method differs (advantages and disadvantages) from the MASS system by Hu et al., 2023.

    Significance

    This manuscript will help the C. elegans community to adapt and use the MS2-MCP system to visualize endogenous mRNAs by live imaging. Their finding could also be adapted to other animal model systems. At the moment, only one published report has described the usage of the MS2-MCP system in C. elegans (by Hu et al., 2023), which combined the MS2 and Suntag systems. In this way, Hu et al., 2023 could shorten the length of MS2 insertion. I am unsure if this is why they do not observe any impact on endogenous mRNA tagged with MS2. However, they only track one gene, and it is possible that different 3'UTR will react differently to the insertion of MS2 repeats. Another manuscript (Kinney et al., BioRxiv 2023) showed the usage of the MS2-MCP-GFP system to track miRNA transcription. In this case, the insertion of the MS2 repeats in the transgenic lin-4 miRNA precursor rescued lin-4 mutation. In this manuscript, Tocchini and Mango identified possible issues in inserting MS2 repeats in endogenous 3'UTR. They have overcome this potential issue by changing the position of the insert in the 3'UTR and by removing the nonsense-mediated decay pathway to prevent destabilization of the mRNA-MS2 transcripts. One possible limitation is that possible system users need to work in a mutant background for the nonsense-mediated decay pathway, which is not ideal. However, it provides a greatly improved tool to track mRNA by live imaging. Therefore, their improved methodology will certainly contribute to expanding the use of MS2-MCP system in C. elegans.

    I have expertise in C. elegans biology and transcription, but I do not have expertise in the imaging system, and therefore, I cannot fully judge the methodology they have used and the quality of the imaging system.

  9. Version published to 10.1101/2023.06.13.544769v1 on bioRxiv