RNAs relay genetic information from DNA to proteins or function by themself. Live-cell imaging of RNAs at a single-molecule level is crucial to uncovering their roles in gene expression regulation (Buxbaum et al., 2015). Various tools have been developed to visualize RNAs in live cells (Braselmann et al., 2020; Le et al., 2022), including RNA-binding protein–fluorescent protein approaches (Bertrand et al., 1998), CRISPR-based systems (Nelles et al., 2016; Yang et al., 2019), and those utilizing fluorophore–RNA aptamer pairs (Chen et al., 2019; Paige et al., 2011; Sunbul et al., 2021). The MS2-based system is the most widely used and represents the current gold standard for single-molecule RNA imaging in live cells (Braselmann et al., 2020; Bertrand et al., 1998). MS2 is a short RNA stem-loop bound specifically by the bacteriophage MS2 coat protein (MCP). To image RNA, 24xMS2 are placed at the 3′UTR or 5′UTR, and a fluorescent protein is fused to the MCP (MCP-FP). When coexpressed in cells, up to 48 fluorescent proteins (2 × 24, with two MCPs bound to one MS2) will be recruited to the RNA through MS2–MCP binding. This forms a fluorescent spot indicating a single RNA molecule (Braselmann et al., 2020).

The MS2 system has been successfully used to trace the whole mRNA life-cycle from transcription, to nuclear export, subcellular localization, translation, and to final degradation (Braselmann et al., 2020; Le et al., 2022). However, most RNA imaging studies in animal cells have been performed using exogenous mRNAs in cultured cell lines. 24xMS2 (about 1300 nt in length) have to be knocked into a specific genomic locus to image endogenous mRNA. The difficulty and low efficiency of the knock-in of long sequences into the genome represent a significant obstacle toward visualizing endogenous mRNA using the MS2 system. Thus, it is not surprising that less than 10 endogenous mRNAs have been imaged in live animal cells at a single-molecule level, and examples of endogenous mRNAs imaged in live animals remain extremely rare (Halstead et al., 2015; Levo et al., 2022; Dufourt et al., 2021; Park et al., 2014; Das et al., 2018; Zimyanin et al., 2008; Forrest and Gavis, 2003; Lee et al., 2022). Since overexpressed mRNAs may not faithfully recapitulate endogenous mRNA expression and dynamics, the development of more sensitive techniques for endogenous mRNA imaging is of great value.

In this study, we reasoned that combining the MS2 with a signal amplifier may allow the recruitment of more fluorescent proteins to the RNA with fewer MS2 repeats (i.e., 8xMS2 – see Figure 1A). To achieve this, we combined the MS2 and Suntag systems. Suntag is a 19 amino acid protein tag that binds to its specific single-chain variable fragment (scFv) antibody (Tanenbaum et al., 2014). We fused MCP with a 24xSuntag array and linked scFv with sfGFP. When coexpressed in cells, one MS2 interacts with two MCP-24xSuntag molecules, further recruiting 2 × 24 GFP molecules (Figure 1A). As the Suntag serves as a signal amplifier, the combined system was named as MS2-based signal Amplification with Suntag System (MASS). When an 8xMS2 is placed into the 3′UTR, up to 384 (2 × 8 × 24) GFP can then be tethered to a single mRNA through the MCP–Suntag–scFv–sfGFP interaction (Figure 1A). This leads to the formation of an intense GFP spot associated with single mRNA, facilitating live RNA imaging.

Figure 1 with 6 supplements see all

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Figure 1—source data 1

qRT-PCR (Quantitative Reverse Transcription PCR), number of foci detected by smFISH and MASS, signal-to-noise ratio, velocity, and intensity of the foci of mRNA detected in HeLa cells.

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As proof of concept, 8xMS2 V1⁴ was fused to the 3′UTR of β-ACTIN mRNA and transfected into HeLa cells. When all the required elements of the MASS (MS2, MCP, 24xSuntag, and scFv antibody) were present, bright GFP foci were readily detected (Figure 1B). As controls, no GFP foci were detected when omitting any one of these elements (Figure 1B). With MCP-24xSuntag, an MCP molecule could be labeled with up to 24 GFPs. Under our imaging conditions (100–500 ms exposure time), MCP-24xSuntag particles were not detected (Figure 1B), probably because MCP-24xSuntag are diffusing too fast to be imaged as a spot. Thus, the GFP foci clearly represent β-ACTIN RNA molecules.

In addition, we performed MASS combined with single-molecule RNA fluorescence in situ hybridization (smFISH) using a probe against the MS2 stem-loop and a probe against the linker region between the MS2 stem-loops (Figure 1C, Figure 1—figure supplement 1A, B). We found that MASS detected a similar number of GFP foci compared to the spots detected by smFISH (Figure 1—figure supplement 1C, Figure 1—source data 1). Moreover, the majority of GFP foci (72%) colocalized with the smFISH spots of β-ACTIN-3′UTR-8xMS2 mRNAs (Figure 1C, D, Figure 1—figure supplement 1B, Figure 1—source data 1). It is reported that not all MS2 stem-loop will be bound by the MCP (Wu et al., 2012). As only 8xMS2 was used in MASS, it is likely that some mRNAs were not fully bound by MCP and were not detected. On the other hand, in theory, only up to 16 probes will be hybridized with the 8xMS2 stem-loops and the linker regions in the smFISH experiment, and it is possible that some mRNAs were miss labeled by smFISH. Therefore, 100% colocalization of MASS foci with the smFISH spots was hard to achieve. Taken together, our data indicated that MASS is able to detect single mRNA molecules and label the majority of mRNAs from a specific gene in live cells.

It was reported that tagging mRNAs with MS2 stem-loops might affect mRNA stability, which could be counteracted by using improved versions of MS2 repeats (Li et al., 2022; Vera et al., 2019). We found GFP foci of β-ACTIN-3′UTR-8xMS2 mRNAs were detected regardless of using MS2-V1⁴ or MS2-V7²¹ with 2× tandem repeats of monomer MCPs (tdMCP) (Figure 1—figure supplement 2A). Therefore, MASS could be performed using different versions of MS2 stem-loops and MCPs. To directly test whether mRNA stability was affected while imaging by MASS, we examined the stability of three mRNAs: MYC, HSPA1A, and KIF18B, which were reported as medium stable mRNAs (Vera et al., 2019; Sharova et al., 2009). We found that the stability of those mRNAs was not significantly affected either by tagging of 8xMS2 V1 or by coexpression of the MASS imaging system (Figure 1—figure supplement 2B, C, Figure 1—source data 1). It is worth noting that we only examined three mRNAs in this study. The stability of specific mRNAs might be affected by MASS. If so, an improved version of MS2 should be used for the imaging experiment.

It has been reported that β-ACTIN mRNAs with 3′UTR can localize to the lamellipodia (Katz et al., 2012). In support of this, we observed that the GFP foci of β-ACTIN-3′UTR-8XMS2 mRNAs were indeed localized to the lamellipodia in HeLa cells (Figure 1—figure supplement 3A and Video 1). To further test whether mRNA localization was affected by MASS, we imaged β-ACTIN-3′UTR mRNA with MASS or with the conventional 24xMS2 system in NIH/3T3 cells, which is a mouse fibroblast cell line. We found that GFP foci of β-ACTIN-3′UTR mRNAs detected by MASS or 24xMS2 system showed similar localization (Figure 1—figure supplement 3B). Thus, these data suggested that MASS did not affect RNA subcellular localization.

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Haven established that MASS with MCP-24xSuntag could image single RNA molecules; we next sought to test whether MASS could be performed with shorter repeats of Suntag arrays (MCP-12xSuntag, MCP-6xSuntag) and compare MASS to the conventional 24xMS2 image system.

With the conventional 24xMS2 mRNA imaging system, a nuclear localization signal (NLS) was usually fused to MCP to localize NLS-MCP-GFP into GFP. NLS-MCP-GFP will be exported into the cytoplasm with mRNAs when binding to mRNAs. This strategy allows the detection of β-ACTIN-3′UTR mRNAs with a signal-to-noise ratio of 1.21 (Figure 1—figure supplement 4A, B, Figure 1—source data 1). MASS with MCP-24xSuntag showed the highest signal-to-ratio of 1.79. MCP-12xSuntag and MCP-6xSuntag labeled β-ACTIN-3′UTR-8xMS2 mRNAs with a similar signal-to-noise ratio of 1.42 and 1.48, which are better than the conventional 24xMS2 system (Figure 1—figure supplement 4C–I, Figure 1—source data 1). Thus, MASS is flexible regarding the length of Suntag repeats used for imaging. MASS with short repeats of Suntag (MCP-6xSuntag) is sufficient for RNA labeling.

One critical concern about MASS is that intense tagging of mRNAs may affect the dynamics of mRNAs. To address this, we performed live-cell imaging of β-ACTIN mRNA using the conventional 24xMS2 system or MASS with different lengths of Suntag arrays (MCP-24xSuntag, MCP-12xSuntag, and MCP-6xSuntag). The velocity of mRNA movement in each imaging condition was measured. We found that compared to the conventional 24xMS2 system, mRNA labeled by MASS with MCP-24xSuntag or MCP-12xSuntag showed a smaller velocity (Figure 1—figure supplement 5A, B, Figure 1—source data 1), indicating that heavier labeling affected mRNA movement speed. In contrast, mRNAs labeled by MASS with MCP-6xSuntag showed a similar velocity to that labeled with the conventional 24xMS2 system (Figure 1—figure supplement 5A, B, Figure 1—source data 1). Those data pointed out that when MASS is used to measure the speed of mRNA movement, a short Suntag array (MCP-6xSuntag) should be used. We next measured the average intensity of each GFP foci of β-ACTIN mRNA labeled using the conventional 24xMS2 system or MASS with different lengths of Suntag arrays (MCP-24xSuntag, MCP-12xSuntag, and MCP-6xSuntag). We found GFP foci detected by MASS showed higher intensity than those detected by the 24xMS2 system (Figure 1—figure supplement 6, Figure 1—source data 1). These data support that with MASS, a single mRNA molecule could be tethered with more GFP molecules, forming a GFP spot of higher fluorescence intensity. Such an application, retains the ability to image mRNA using low-power lasers, thus lowering any unwanted phototoxicity and photobleaching and still allowing the tracking of mRNA dynamics in high temporal resolution.

We then performed time-lapse imaging of the β-ACTIN-3′UTR-8XMS2 mRNA with a time interval of 1 s in HeLa cells (Video 2). We found that foci of mRNAs showed various dynamics: (1) Fusion. GFP spots fused into a more prominent spot (Figure 1E and Video 3); (2) Fission. Large GFP foci split into smaller spots (Figure 1F and Video 4); (3) Transient interaction. GFP foci touched each other briefly, then moved away (Figure 1G and Video 5), suggesting there are dynamic RNA–RNA interactions in cells; (4) Dynamic movement or anchoring. Despite most foci of β-ACTIN-3′UTR-8XMS2 mRNAs showing dynamic movement in cells, other foci were far more static, showing little movement (Figure 1H and Video 6), suggesting that these latter mRNAs may be anchored to subcellular structures.

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Our ultimate goal was to develop tools for endogenous mRNA imaging in live animals. It has been previously reported that the knock-in of short sequences into the genome is far more efficient than those of longer sequences (Wang et al., 2022; Paix et al., 2017). The MASS exploits this advantage as only 8xMS2 (350 nt) needs to be inserted into a genomic locus, thus overcoming the previous obstacle of the requirement of inserting a long 1300 nt 24xMS2 into the genome for live-cell imaging of endogenous mRNA.

We then used the nematode C. elegans to specifically examine whether the MASS could be used for RNA imaging in live animals. An 8xMS2 was placed into the 3′UTR of cdc-42 mRNA (Figure 2—figure supplement 1). cdc-42-8xMS2, MCP-24xSuntag, and scFv-sfGFP were also expressed in the epidermis of live C. elegans. Consistent with the observations in HeLa cells, bright GFP foci could only be detected when all the required elements were present (Figure 2—figure supplement 1). Similarly, excluding any essential elements resulted in a complete failure of foci formation (Figure 2—figure supplement 1). Thus, the MASS was efficient in imaging exogenous RNAs in live animals.

Next, we set out to visualize gene expression activation and the dynamics of endogenous mRNAs in live animals. We used the skin of C. elegans as a model, which is composed of an epidermal epithelium with multiple nuclei. Upon wounding the epithelium via laser or needle, specific gene expressions and downstream signaling cascades for wound repair are then triggered and activated (Xu and Chisholm, 2014; Figure 2A). To this end, 8xMS2 was knocked into the 3′UTR region of two endogenous genes, C42D4.3 and mai-1 (Figure 2B and Figure 2—figure supplement 2A), the expression levels of which were reported to increase significantly after wounding (Fu et al., 2020). MCP-24xSuntag and scFv-sfGFP were expressed in the epidermis with the tissue-specific promoter semo-1 and col-19. We then used a UV laser to injure an area of the epidermis. Prior to wounding, few sfGFP foci were detected in either wild-type (WT) or in 8xMS2 knock-in animals (Figure 2B and Figure 2—figure supplement 2A). In contrast, numerous GFP foci were formed 15 min after wounding in the C42D4.3 and mai-1 8xMS2 knock-in animals but not in the WT animals (Figure 2B and Figure 2—figure supplement 2A, Videos 7–9). This result suggests that the wounding had activated C42D4.3 and mai-1 mRNA expression. In agreement with this, qRT-PCR showed that C42D4.3 and mai-1 mRNA levels were upregulated more than eightfold 15 min after injury (Figure 2C and Figure 2—figure supplement 2B, Figure 2—source data 1), confirming that GFP foci were able to detect endogenous C42D4.3–8xMS2 and mai-1-8xMS2 mRNAs. In addition, the expression level (Figure 2—figure supplement 2C, Figure 2—source data 1) and stability of C42D4.3 mRNA (Figure 2—figure supplement 2D, Figure 2—source data 1) were similar in WT, C42D4.3 8xMS2 knock-in animals and animals expressing the MASS imaging system, indicating MASS did not affect the expression level and stability of endogenous C42D4.3 mRNAs.

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Figure 2 with 5 supplements see all

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Figure 2—source data 1

qRT-PCR (Quantitative Reverse Transcription PCR), number, and size of foci of mRNAs detected by MASS in C. elegans.

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As the mRNA expression level of C42D4.3 and mai-1 significantly increased after wounding, we expected a boost in transcription. GFP will form extensive foci in the nuclei with active transcription. However, we failed to detect the appearance of bigger GFP foci in the nucleus. The epidermis of C. elegans is a syncytium with 139 nuclei located in different focal planes. With our microscopy, we could image only one focal plane, in which there are usually 4–10 nuclei. Therefore, it is likely that the nuclei with active transcription were out of focus, and therefore the GFP foci formed at the transcription site were not detected.

Next, we tracked the dynamics of endogenous C42D4.3–8xMS2 mRNA in the C. elegans epidermis after wounding. We found that in proximity to the injury site GFP foci were detected as early as 1 min after wounding (Figure 2D, Figure 2—figure supplement 3, and Videos 7 and 8). As a control, we pretreated the C. elegans with Actinomycin D, which potently inhibits gene transcription. In such cases, no GFP foci could be detected after wounding (Figure 2—figure supplement 4). This indicated that GFP foci are newly synthesized C42D4.3–8xMS2 mRNAs. Our data demonstrated that gene expression activation and transcription occur extremely fast (in this case, within 1 min after the stimulation). In addition, the appearance of GFP foci gradually spreads from the area around the injury site to distal regions. The total foci number steadily increased in the epidermis (Figure 2D, E, Figure 2—figure supplement 3, and Videos 7 and 8). These data suggested that wounding generated a signal to activate downstream gene expression around the injury site. The signal was then diffused to distal regions and able to induce gene expression there. In addition, GFP foci preferentially underwent fusion leading to increased foci size (Figure 2F, G, and Video 10, Figure 2—source data 1). In contrast, when exogenous BFP-8xMS2 was expressed and imaged with MASS in the epidermis (Figure 2—figure supplement 5A, Figure 2—source data 1), such fusion events were not frequently observed (Video 11), and the puncta size of BFP-8xMS2 mRNAs kept constant in 5 min (Figure 2—figure supplement 5A–C, Figure 2—source data 1). In addition, the puncta size of C42D4.3-8xMS2 mRNAs was significantly larger than that of BFP-8xMS2 mRNAs (Figure 2—figure supplement 5C, Figure 2—source data 1). These data suggested that C42D4.3 mRNAs undergo clustering after wounding and form RNA granules in vivo. In agreement with our observation, it has been previously reported that mRNAs formed large clusters and are co-translated in Drosophila embryos (Dufourt et al., 2021).

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It has been recently reported that the SunRISER and MoonRISER system by combination of PP7 and Suntag or Moontag enables imaging of single exogenous mRNAs in living cells. Through computational and experimental approaches, the authors further optimized the SunRISER system and showed that SunRISER provided an excellent approach for long-term imaging of overexpressed mRNA in living cells (Guo and Lee, 2022). The principle of the SunRISER system and MASS is similar, which uses a protein signal amplifier to generate a higher fluorescence signal for RNA imaging. In this study, we compared MASS to the conventional 24xMS2 mRNA imaging system and characterized the effect of MASS on mRNA stability and dynamics. In addition, we primarily explored the application of MASS to image endogenous RNA in live animals, which has not been tested in the previous study. Here, we showed such signal amplification strategy is valuable for imaging endogenous mRNAs that utilizing only 8xMS2 in comparison to the 24xMS2 used in the classic MS2-based live-cell RNA imaging. The advantage of a short MS2 is of prime benefits for imaging endogenous mRNA as it reduced difficulties involved in inserting a long 1300 nt 24xMS2 into a genomic locus. We expect this tool will help promote studies of RNA transcription, nuclear export, subcellular localization, translation, RNA sensing, and degradation, at the endogenous level in culture cells and live animals.

Videos 1–11 contain source data for figures. Figure 1—source data 1 contains source data for statistical analysis in cells. Figure 2—source data 1 contains source data for statistical analysis in C. elegans.

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Decision letter after peer review:

Thank you for submitting your article "Enhanced Single RNA Imaging Reveals Dynamic Gene Expression in Live Animals" for consideration by eLife. Your article has been reviewed by 4 peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by James Manley as the Senior Editor. The reviewers have opted to remain anonymous.

The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission. All reviewers are positive about the concept of labeling by MASS but are not completely convinced that it is working without any effect on the mRNA. They all request further data that rigorously report on the mRNA tagged compared to non-tagged mRNA, or singly tagged mRNA.

Essential revisions:

The authors have not compared their MASS system to a standard in the field, the MS2 system. There are questions on the relevance of this approach for single molecule imaging, and the effect of the tagging on the physiology of the mRNA. There needs to be a more rigorous approach showing that the mRNA tagged in this way behaves as if it was not tagged. Suggestions also include comparison to FISH images in fixed cells, and measurement of velocities with and without the extensive tagging. It may be that the massive amount of protein on the mRNA may render it inert.

The referencing does not include similar work in the field (eg Guo et al) that needs to be discussed and compared more thoroughly.

Provide quantitative evaluation of signal and increase in signal to noise. Refer to the reviewers' many comments and suggestions.

Reviewer #2 (Recommendations for the authors):

1. Line 44: There are more previous works reporting endogenous mRNA imaging in live animals.

Forrest and Gavis, "Live imaging of endogenous RNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila", Current Biology, 13, 1159 (2003)

Lee et al., "Real-time visualization of mRNA synthesis during memory formation in live mice" PNAS, 119, e2117076119 (2022)

2. Line 410: More details need to be described for live imaging in C. elegans. For example, spinning disk confocal microscope model, objective maker, Z-stack interval, exposure time or pixel dwell time, time-lapse interval and duration. And did the authors show z max-projection image in Figure 2? Any image processing also needs to be described.

Reviewer #3 (Recommendations for the authors):

– Doublecheck grammar and spelling (e.g., "themself" in line 25).

– Consider rewriting some parts to avoid ambiguity (e.g., 10 mRNAs or mRNAs from 10 genes in the line 43?).

– A bit more expanded introduction would help readers to appreciate the importance of the authors' work.

– In the Methods, the live imaging section needs more details such as the C. elegans immobilization method.

– The authors need to be clear on what version of MS2 stem loops they used as a different version can make different results. For example, the version 5 can induce clustering of MCP signals even after the target mRNAs are degraded due to its nature of being stable and aggregated in the cell.

Reviewer #4 (Recommendations for the authors):

1. While the manuscript provides ample details on constructs, the information on C. elegans imaging is minimal. The authors should provide z-stack information (how many planes and step size), temporal resolution, camera used and settings, laser settings, etc.

2. Analysis of foci sizes. Naively, one would expect quantile sizes of the foci with one, two, three, … mRNAs in a dot, although the apparent/measured size increase may not be linear. Figure 2F, which shows foci sizes over time, looks puzzling in this context.

a. How should one interpret the initial ramp up (first 2 minutes) before hitting an apparent plateau? Could it be an artifact of measurement?

b. Given the assumption of quantal sizes, the plotted mean +/- sem can be misleading. A scatter plot of individual size would make more sense. Alternatively, the authors could provide the size distribution at some representative time points (eg, the two plateaus 3-6 min and 10-15).

c. Related, to what extent could one infer the unit size/single mRNA signal? Some of the zoomed in images leave the impression that there are a large number of foci with comparable and smallest size, which could be single mRNAs?

d. What is the y-axis? I assume it's diameter in um? Since the sizes are quantal in volume, would it make more sense to make some of the plots in d³?

3. Some missed opportunities on the extent of usefulness of the current reagents. For example:

a. Is it possible to see signals in nuclei to capture transcription and early posttranscriptional regulation?

b. What's the practical depth penetrance of imaging? Could one get reasonable signal at the deep side (basal side) of the epidermal cells? Or the far side of the worm (different larval stages provide different distances for imaging).

4. Some phenotypic assays on the physiological relevance/potential interference of abelling could greatly strengthen the usefulness of the systems. Some suggestions for the authors to consider:

a. Labeling of cdc-42: Does it affect the actomyosin dynamics in wound repair? Does it affect any developmental process (eg, post-embryonic cell divisions)?

b. Functional rescue by the tagged allele (heterozygous over null/loss of function allele) on any of the three genes tagged?

5. Some analysis and discussions on the range of optimization would be very useful for the field.

a. What is the signal to background ratio? With gross simplification/linear extrapolation, could one consider reducing the abelling (number of GFP recruited) by 10x, 20x or 50x reduction of amplification?

b. How practical is the imaging setting in terms of phototoxicity and longer term imaging? If the authors could provide a powermeter readout of laser after the lens, it would be most objective and transferable to other labs. If not, a more detailed documentation of the laser used and settings would help.

6. I expect the community would respond enthusiastically to the publication and there would be a lot of interest on the reagents. Given the open science spirit of eLife, the authors should affirm the deposition of constructs and worm strains to public platforms, eg, Addgene and CGC.

Essential revisions:

The authors have not compared their MASS system to a standard in the field, the MS2 system. There are questions on the relevance of this approach for single molecule imaging, and the effect of the tagging on the physiology of the mRNA. There needs to be a more rigorous approach showing that the mRNA tagged in this way behaves as if it was not tagged. Suggestions also include comparison to FISH images in fixed cells, and measurement of velocities with and without the extensive tagging. It may be that the massive amount of protein on the mRNA may render it inert.

The referencing does not include similar work in the field (eg Guo et al) that needs to be discussed and compared more thoroughly.

Provide quantitative evaluation of signal and increase in signal to noise. Refer to the reviewers’ many comments and suggestions.

We thank the editors for spending time with our manuscript and encouraging comments. We have done FISH experiments, performed more quantitative evaluations of signal-tonoise, and signal intensity, examined mRNA stability, and measured the velocities with a conventional MS2 system and MASS.

Reviewer #2 (Recommendations for the authors):

1. Line 44: There are more previous works reporting endogenous mRNA imaging in live animals.

Forrest and Gavis, “Live imaging of endogenous RNA reveals a diffusion and entrapment mechanism for nanos mRNA localization in Drosophila”, Current Biology, 13, 1159 (2003)

We thank the reviewer for providing more references. We have added the missing

references in the new version of the manuscript on page 2.

2. Line 410: More details need to be described for live imaging in C. elegans. For example, spinning disk confocal microscope model, objective maker, Z-stack interval, exposure time or pixel dwell time, time-lapse interval and duration. And did the authors show z max-projection image in Figure 2? Any image processing also needs to be described.

We updated the methods section and the missing information on page 25.

Reviewer #3 (Recommendations for the authors):

– Doublecheck grammar and spelling (e.g., “”I" in line 25).

– Consider rewriting some parts to avoid ambiguity (e.g., 10 mRNAs or mRNAs from 10 genes in the line 43?).

We apologize for the ambiguity. We have checked grammar and spelling and c“anged

"less than t”n mR“As" to "mRNAs from less than t”n genes".

– A bit more expanded introduction would help readers to appreciate the importance of the’authors' work.

We noticed there are several beautiful review articles about RNA imaging published recently. We, therefore, cited those papers and wrote a short introduction.

– In the Methods, the live imaging section needs more details such as the C. elegans immobilization method.

We have now updated the methods section, and the missing information was added to page 26.

– The authors need to be clear on what version of MS2 stem loops they used as a different version can make different results. For example, the version 5 can induce clustering of MCP signals even after the target mRNAs are degraded due to its nature of being stable and aggregated in the cell.

We used the V1 version of MS2 stem-loop in our study. We added this information to the manuscript. We also tested the MS2 V7, an optimized version of the MS2 stem-loop, and found that MASS also works with MS2 V7. We added this new data in Figure 1-figure supplement 2.

Reviewer #4 (Recommendations for the authors):

1. While the manuscript provides ample details on constructs, the information on C. elegans imaging is minimal. The authors should provide z-stack information (how many planes and step size), temporal resolution, camera used and settings, laser settings, etc.

We updated the methods section, and the missing information was added to page 26.

2. Analysis of foci sizes. Naively, one would expect quantile sizes of the foci with one, two, three, … mRNAs in a dot, although the apparent/measured size increase may not be linear. Figure 2F, which shows foci sizes over time, looks puzzling in this context.

a. How should one interpret the initial ramp up (first 2 minutes) before hitting an apparent plateau? Could it be an artifact of measurement?

We imaged exogenous BFP-8xMS2 mRNA in the epidermis of C. elegans. We found that the size of GFP foci of BFP-8xMS2 mRNA kept constant over time. We also found that the size of endogenous C42D4.3-8xMS2 mRNAs is bigger than the GFP foci of BFP-8xMS2 mRNAs. Those data indicate that the growing size of the GFP foci of endogenous C42D4.3 mRNA is not an artifact of measurement.

Our data suggested that there is a transcription boost in the first two minutes after wounding, and those mRNAs undergo quick clustering to form RNA granules.

b. Given the assumption of quantal sizes, the plotted mean +/- sem can be misleading. A scatter plot of individual size would make more sense. Alternatively, the authors could provide the size distribution at some representative time points (eg, the two plateaus 3-6 min and 10-15).

We made the scatter plot of the individual size of each GFP foci at five representative time points (1 min, 2 min, 3 min, 4 min, and 5 min after wounding). We added that information to Figure 2—figure supplement 5.

c. Related, to what extent could one infer the unit size/single mRNA signal? Some of the zoomed in images leave the impression that there are a large number of foci with comparable and smallest size, which could be single mRNAs?

There are indeed small and large foci of mRNAs in cells. We imaged exogenous BFP8xMS2 mRNAs in the epidermis of C. elegans and found that the size of the GFP foci of endogenous C42D4.3-8xMS2 mRNAs is larger than that of BFP-8xMS2 mRNAs. Those data suggest that the large GFP foci in C42D4.3-8xMS2 mRNA are mRNA granules. GFP foci with the smallest size should be single mRNAs. We added those new data in Figure 2—figure supplement 5 and the text on page 7.

We also performed MASS combined with single-molecule RNA FISH. We found that MASS detected a similar number of GFP foci compared to the spots detected by smFISH. In addition, the majority (72%) of GFP foci colocalized with the smFISH spots of b-ACTIN8xMS2 mRNAs. Thus These data suggested that MASS could label single mRNA

molecules, and GFP foci with minimal size are likely single mRNAs. We have added the new data in Figure 1, Figure 1—figure supplement 1, and the text on page 3.

d. What is the y-axis? I a’sume it's diameter in um? Since the sizes are quantal in volume, would it make more sense to make some of the plots in d³?

The y-axis is the area (µm²) of the GFP foci. We added this missing information in Figure 2 and Figure 2—figure supplement 5.

3. Some missed opportunities on the extent of usefulness of the current reagents. For example:

a. Is it possible to see signals in nuclei to capture transcription and early posttranscriptional regulation?

We expected a boost of transcription after wounding. However, we failed to detect the appearance of bigger GFP foci in the nucleus. The epidermis of C. elegans is a syncytium with 139 nuclei located in different focal planes. With our microscopy, we were able to image only one focal plane, in which there are usually only four to ten nuclei. Therefore, it is likely that the nuclei with active transcription were out of focus. We discussed this point in the manuscript (page 6).

b. What's the practical depth penetrance of imaging? Could one get reasonable signal at the deep side (basal side) of the epidermal cells? Or the far side of the worm (different larval stages provide different distances for imaging).

The practical depth penetrance of our spinning microscopy is 50 µm. The depth of the epidermis of C. elegans is 60 µm. Therefore we are only able to image the basal side of the epidermal cell.

4. Some phenotypic assays on the physiological relevance/potential interference of labeling could greatly strengthen the usefulness of the systems. Some suggestions for the authors to consider:

a. Labeling of cdc-42: Does it affect the actomyosin dynamics in wound repair? Does it affect any developmental process (eg, post-embryonic cell divisions)?

We tagged 8xMS2 into the 3′UTR of cdc-42 mRNA. The coding sequence of cdc-42 mRNA was not changed. We previously found that epidermal wounding induces rapid clustering of CDC-42 proteins. When cdc-42-8xMS2 mRNAs were overexpressed and labeled by MASS in the epidermis of C. elegans, we found similar clustering of CDC-42 proteins, suggesting MASS did not affect the function of CDC-42. Therefore, the actomycosin dynamics in wound repair were likely not affected. We did not observe any developmental defects in worms expressed with cdc-42-8xMS2 mRNAs.

b. Functional rescue by the tagged allele (heterozygous over null/loss of function allele) on any of the three genes tagged?

We tagged 8xMS2 into the 3′UTR of endogenous C42D4.3 and mai-1 mRNAs. The coding sequences of C42D4.3 and mai-1 mRNAs were not changed. We showed that MASS did not affect the expression level and mRNA stability of C42D4.3 gene. We expect the function of C42D4.3 and mai-1 will not be affected, and we did not observe any developmental defects in the tagged worms. Therefore we did not perform functional rescue experiments by the tagged allele.

5. Some analysis and discussions on the range of optimization would be very useful for the field.

a. What is the signal to background ratio? With gross simplification/linear extrapolation, could one consider reducing the labeling (number of GFP recruited) by 10x, 20x or 50x reduction of amplification?

For the signal-to-noise ratio, we did more experiments and analyses. We imaged overexpressed b-ACTIN mRNAs using the conventional 24xMS2 system or MASS with different repeats of Suntag arrays (MCP-24xSuntag, MCP-12xSuntag, MCP-6xSuntag). For the conventional 24xMS2 system, we followed the previous protocol that added a nuclear localization signal (NLS) to MCP, and b-ACTIN mRNAs were nicely detected with a signal-to-noise ratio of 1.21.

We found MASS showed a comparable or better signal-to-noise ratio than the conventional 24xMS2 system. (MASS with MCP-24xSuntag: 1.79, MASS with MCP-12xSuntag: 1.48, MASS with MCP-6xSuntag: 1.42). These data indicate that using Suntag as a signal amplifier did not increase background noise.

b. How practical is the imaging setting in terms of phototoxicity and longer term imaging? If the authors could provide a powermeter readout of laser after the lens, it would be most objective and transferable to other labs. If not, a more detailed documentation of the laser used and settings would help.

We do not have a powermeter readout of laser after the lens. We updated the methods section, and the information on the laser used and settings were added to page 26.

We did not observe significant phototoxicity in the C. elegans after 15 min continuous imaging with a time interval of 2 seconds.

We did not test whether MASS could be used for longer-term imaging in this study. However, it is recently published that SunRISER, which used a similar strategy with MASS, allows long-term (24 h with a 10 min frame rate) live cell mRNA imaging (Guo, Y. and Lee, R.E.C., Cell Reports Methods, 2022). Taking together, Guo and we demonstrated that it is an efficient strategy to combine the MS2 system and the Suntag system as a signal amplifier for long-term and endogenous mRNA imaging in live cells.

6. I expect the community would respond enthusiastically to the publication and there would be a lot of interest on the reagents. Given the open science spirit of eLife, the authors should affirm the deposition of constructs and worm strains to public platforms, eg, Addgene and CGC.

Thanks for the suggestion; yes, we will deposit all the constructs and worm strains to

Addgene and CGC after this paper's publication.

Author details

1. Yucen Hu

   Zhejiang Provincial Key Laboratory of Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   Contributed equally with

   Jingxiu Xu and Erqing Gao

   Competing interests

   No competing interests declared

2. Jingxiu Xu

   International Biomedicine-X research center of the Second Affiliated Hospital, Zhejiang University, Hangzhou, China

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   Contributed equally with

   Yucen Hu and Erqing Gao

   Competing interests

   No competing interests declared

3. Erqing Gao

   International Biomedicine-X research center of the Second Affiliated Hospital, Zhejiang University, Hangzhou, China

   Contribution

   Formal analysis, Validation, Investigation, Visualization, Methodology

   Contributed equally with

   Yucen Hu and Jingxiu Xu

   Competing interests

   No competing interests declared

4. Xueyuan Fan

   Zhejiang Provincial Key Laboratory of Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China

   Contribution

   Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8297-1596

5. Jieli Wei

   Zhejiang Provincial Key Laboratory of Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China

   Contribution

   Supervision, Methodology

   Competing interests

   No competing interests declared

6. Bingcheng Ye

   Zhejiang Provincial Key Laboratory of Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China

   Contribution

   Methodology

   Competing interests

   No competing interests declared

7. Suhong Xu

   1. International Biomedicine-X research center of the Second Affiliated Hospital, Zhejiang University, Hangzhou, China
   2. Center for Stem Cell and Regenerative Medicine and Department of Burn and wound repair of the Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Project administration, Writing - review and editing

   For correspondence

   shxu@zju.edu.cn

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-4079-340X

8. Weirui Ma

   Zhejiang Provincial Key Laboratory of Cancer Molecular Cell Biology, Life Sciences Institute, Zhejiang University, Hangzhou, China

   Contribution

   Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Visualization, Methodology, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   maweirui@zju.edu.cn

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9193-7311

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