Condensation of LINE-1 is critical for retrotransposition
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Abstract
LINE-1 (L1) is the only autonomously active retrotransposon in the human genome, and accounts for 17% of the human genome. The L1 mRNA encodes two proteins, ORF1p and ORF2p, both essential for retrotransposition. ORF2p has reverse transcriptase and endonuclease activities, while ORF1p is a homotrimeric RNA-binding protein with poorly understood function. Here, we show that condensation of ORF1p is critical for L1 retrotransposition. Using a combination of biochemical reconstitution and live-cell imaging, we demonstrate that electrostatic interactions and trimer conformational dynamics together tune the properties of ORF1p assemblies to allow for efficient L1 ribonucleoprotein (RNP) complex formation in cells. Furthermore, we relate the dynamics of ORF1p assembly and RNP condensate material properties to the ability to complete the entire retrotransposon life-cycle. Mutations that prevented ORF1p condensation led to loss of retrotransposition activity, while orthogonal restoration of coiled-coil conformational flexibility rescued both condensation and retrotransposition. Based on these observations, we propose that dynamic ORF1p oligomerization on L1 RNA drives the formation of an L1 RNP condensate that is essential for retrotransposition.
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Referee #3
Evidence, reproducibility and clarity
In this work, Sil et al. use fluorescent microscopy and biochemical reconstitution to study the ribonucleoparticles formed by one of the two L1 transposon proteins: ORF1p. Authors show that fluorescently tagged ORF1p forms puncta in HeLa cells within several hours of induced expression. The authors use this system to test how various mutations in the ORF1p affect its ability to form puncta in cells. They then correlate this property to the ability to induce transposition, which is quantified using a reporter system. Mutants that fail to form puncta are also unable to induce transposition. This leads the authors to conclude that …
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Referee #3
Evidence, reproducibility and clarity
In this work, Sil et al. use fluorescent microscopy and biochemical reconstitution to study the ribonucleoparticles formed by one of the two L1 transposon proteins: ORF1p. Authors show that fluorescently tagged ORF1p forms puncta in HeLa cells within several hours of induced expression. The authors use this system to test how various mutations in the ORF1p affect its ability to form puncta in cells. They then correlate this property to the ability to induce transposition, which is quantified using a reporter system. Mutants that fail to form puncta are also unable to induce transposition. This leads the authors to conclude that condensation of ORF1p is required for L1 retrotransposition. A two-color colocalization assay demonstrates that ORF1p is immobile within the observed puncta, showing no evidence of exchange and mixing with a co-expressed ORF1p labeled with a different fluorescent protein. In addition, the authors purify the ORF1p protein, and various mutant variants, and test their ability to undergo phase separation in vitro in various conditions where they vary the concentrations of ORF1p, the salt, and RNA. The simple phase separation assays are complemented with a biophysical characterization of the condensates, where the post-fusion relaxation into a circular shape of the droplet is quantified and used to determine the inverse capillary velocity, which reflects the condensate viscosity and surface tension. These properties and then correlated with the ability of the variants to form puncta and facilitate retrotransposition.
It is an interesting and well-written article. The figures are neat and well documented. The experimental methods are described in sufficient detail. However, I believe that the conclusions made are not sufficiently supported by the presented evidence. The authors show correlation, not causation, of the ORF1p condensation and transposition. The evidence that the ORF1p particles form co-translationally, that they are condensates, and that they directly mediate transposition is insufficient. The in vitro work is interesting, but too preliminary and needs a more careful quantification. I encourage the authors to address my comments experimentally as much as they can. Where not possible, they could tone down the language and address the comments in writing and point out the limitations in the article text.
Major comments:
- What is the evidence that ORF1p forms condensates in an endogenous situation? A more thorough discussion of the evidence, based on the literature is needed. Alternatively, authors could use antibodies (if available) to demonstrate that such structures indeed exist in cell culture of tissues.
- The model that the observed puncta form co-translationally through co-condensation of ORF1p and its encoding mRNA is intriguing and would indeed provide an elegant biophysical explanation for the discussed cis preference of transposition. In my opinion, this idea is the strongest part of the paper. I would advise the authors to provide more compelling evidence for this idea, as currently, it is not well-supported by the data. At the least, the authors need to show that the L1 mRNA is actually present in the studied condensates (for example, using smFISH on fixed cells). This will also allow the determination of the number of L1 mRNAs present in each condensate.
- If authors have access to a microscope that can perform FRAP measurements, I would strongly suggest such an assay, where the individual cytoplasmic and nuclear ORF1p puncta can be examined for their material properties as a function of time (compare 6 hours post-induction and 72 hours post-induction).
- Please provide a more detailed analysis of the formation of nuclear ORF1p condensates. How much later do they appear? The nucleus is the place where transposition occurs. Do the authors suggest that the co-translationally formed condensates enter the nucleus? Or do they form there de-novo? Is there also no colocalization in the nuclear foci? This could be addressed by a quantitative time-course.
- The in vitro assays only use the L1 mRNA fragment. Do other RNAs (for example total RNA, rRNA, mRNA) similarly affect ORF1p condensates? Other studies showed that the presence of specific RNA could nucleate the formation of condensates in vitro, particularly where non-specific RNA is also present, mimicking the cellular environment (Maharana et al. Science 2018, PMID: 29650702; Elguindy and Mendell, Nature 2021, PMID: 34108682). The authors should test if the observed effect of L1 mRNA fragment is sequence-specific. Length dependence should also be addressed, as it may be the key parameter for the "co-translational assembly and gelation" model.
Minor comments:
- The K3/K4 and R261 variants don't form puncta and do not promote transposition, yet phase separate at a similar concentration in vitro. The stammer mutants phase separate less efficiently in vitro, yet form puncta and promote transposition. This suggests that the in vitro phase separation assay is not very informative of the protein's behavior in cells. To me, it suggests that the puncta observed in cells might not be formed through phase separation. Other mechanisms of puncta formation should be explored.
- Based on the fluorescent images, can the authors estimate what percent of the ORF1p protein is actually present in distinct condensates and how much is diffuse in the cytoplasm or nucleoplasm? How does the outside (diffuse) concentration change upon increased expression or ORF1p? Is there any evidence of a saturation concentration?
- Does the ORF1-Halo and ORF1-mNG2 colocalization change at longer time-points where larger condensates are observed?
- The authors often refer to the "the total area of condensed phase". This parameter is not very useful, as it highly depends on the experimental condition. Instead, authors should determine the apparent saturation concentration for each studied mutant in the presence and absence of RNA at a relevant RNA concentration. This requires increasing the resolution at the protein concentration axis and an unbiased analysis pipeline.
- It is shown that decreasing ORF1p protein concentration at a fixed salt concentration decreased the total condensed phase area but increased the protein partition coefficient. The DNA/RNA binding mutant R261A does not show this trend. Moreover, it is the only mutant that shows a change in the phase diagram upon the addition of RNA. One explanation is that there are nucleic acid contaminants present in the protein prep. In fact, the R261A mutant seems to also have a lower 260 nm peak relative to 280nm peak at the chromatogram. That the enrichment of the ORF-1p protein changes with increasing concentration strongly suggests that we are already looking at a multi-component system here, where the contaminant would be a second component. The authors do include an extra step in the purification protocol to reduce nucleic acid contamination. However, they could also run an ion-exchange chromatography to improve the purity. Alternatively, they could test if adding benzonase, RNAse or DNAse changes the phase diagram of the ORF1p alone.
- It would be great to see how the StammerDel behaves in vitro. The authors could at least try the purification with their current protocol. Full-length proteins often behave very differently than the fragments alone.
Significance
The model that the proteins encoded by the L1 transposon form condensates co-translationally and that these assemblies are functional units of the transposon that explain the cis-preference is a significant, important and interesting concept. In my opinion, this idea is the strongest part of the paper. However, unless supported by more evidence (such as experiments and analysis suggested above), it remains just an idea. This work would be of interest of the phase separation community as well as general cell biology and genetics field.
My field of expertise is: biomolecular phase separation, quantitative microscopy, cell biology, protein biochemistry, developmental biology and genetics.
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Referee #2
Evidence, reproducibility and clarity
This paper revisits aggregate formation by ORF1p, a nucleic acid (NA) binding protein encoded by the L1 retrotransposon. This topic dates to 1996 (Hohjoh and Singer - 1996 Embo J 15: 630) and was extensively examined again in 2012 using highly purified ORF1p by Callahan et al (Callahan et al - 2012 Nucleic Acids Res., 40, 813), to determine the effect of salt and nucleic acid on this process. The earlier studies employed chemical cross linking and gel electrophoresis to examine ORF1p aggregates in the presence and absence of NA and neither were cited in the present study. As ORF1p contains several intrinsically disordered regions …
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Referee #2
Evidence, reproducibility and clarity
This paper revisits aggregate formation by ORF1p, a nucleic acid (NA) binding protein encoded by the L1 retrotransposon. This topic dates to 1996 (Hohjoh and Singer - 1996 Embo J 15: 630) and was extensively examined again in 2012 using highly purified ORF1p by Callahan et al (Callahan et al - 2012 Nucleic Acids Res., 40, 813), to determine the effect of salt and nucleic acid on this process. The earlier studies employed chemical cross linking and gel electrophoresis to examine ORF1p aggregates in the presence and absence of NA and neither were cited in the present study. As ORF1p contains several intrinsically disordered regions (IDRs) ORF1p aggregates can form phase separated condensates (droplets) which were characterized microscopically in the present study, and the authors assume that condensate formation is intrinsic to the function of ORF1p in retrotransposition, or as they state on page 2: "...we hypothesized that ORF1p undergoes condensation to carry out its roles in L1 RNP formation...". The authors attempt to correlate the ability of L1 encoded ORF1p complexed or not with RNA to form phase separated condensates in parallel with retrotransposition assays. They couple these observations with in vitro studies on condensate formation by the purified protein.
I have the following major comments:
- (A) The functional relevance of condensate formation by IDR-containing proteins has been questioned (Martin, E. W. and A. S. Holehouse - 2020; Emerging Topics in Life Sciences 4: 307). These authors conclude their review as follows: "In summary, IDRs are ubiquitous and play a wide range of functional roles across the full spectrum of biology, and in a large number (likely the majority) of cases their biological function has nothing to do with the ability to form large macroscopic liquid droplets. The notion that the presence of an IDR means a protein has evolved to phase separate is an inaccurate inference that has unfortunately been used to justify questionable lines of inquiry and questionable experimental design." And in terms of ORF1p this admonition is exemplified by the findings of Newton et al (2021, Biophys J 120;2181) cited by the present authors. This study showed that phase separated condensates readily form by just the N-terminal 152 amino acids (NTD + coiled coil). As this region of ORF1p cannot bind NA, condensate formation is indifferent to RNA binding, an obviously critical function of ORF1p.
- (B) Earlier studies (Ostertag et al - 2000; NAR 28:1418) showed that sufficient retrotransposition events have occurred by 48 hours after introduction of an L1 retrotransposition reporter to be readily detectable by whole cell staining for the retrotransposition-generated reporter gene product. The 48-hour lag presumably reflects the time to accumulate sufficient L1RNPs or their retrotransposed products to be detectable. Does this mean that the puncta (Fig 1F) accumulating during the first 24 hours after introduction of their full-length L1 retrotransposition reporter (Fig 1C) are the L1RNPs generated by the reporter? If not, what are they? If they are L1RNPs, are they thought to be or expected to exhibit the properties of phase separated condensates or are such properties just a feature of disembodied ORF1p that the authors posit could form an active L1RNP? The Ostertag paper should be cited here given its relevance to this issue.
- (C) Four of the IDRs in ORF1p harbor or are juxtaposed to phosphorylation sites essential for retrotransposition (their citation - Cook et al, 2015). As the authors expressed their purified proteins in E. coli, it is not phosphorylated and would not only be inactive for retrotransposition and given the structural effects of phosphorylation (e.g., Bah, A., et al.;2015; Nature, 510, 106) it would differ significantly from the structure of the active protein. As variables they introduce into ORF1p several not too subtle mutations particularly regarding the ORF1 coiled coil. They thereby aim to assess the role or particulars of ORF1p condensate formation for L1 retrotransposition. In their Abstract they state (p.1, l. 11) "...we propose that ORF1p oligomerization on L1 RNA drives the formation of a dynamic L1 condensate that is essential for retrotransposition."
- (D) Although the authors provide no direct experimental evidence for the above statement and whatever the authors mean by "dynamic L1 condensate" how does this conclusion materially differ from the conclusions published by Naufer et al, in 2016 (NAR; 44,281), which also was not cited by the authors. Naufer et al used single molecule studies and highly purified ORF1p that had been expressed in insect cells (and thus was fully phosphorylated, Cook et al, 2015). They showed that oligomerization of nucleic acid (NA)-ORF1p complexes to a compacted stably bound polymer was positively correlated with retrotransposition. Both properties could be eliminated by coiled coil mutations that had no effect on biochemical assays of ORF1p activity - high affinity NA binding and NA chaperone activity. As both properties map to the carboxy terminal-half of ORF1p, the inactivating coiled coil mutations are an example of the numerous instances of strong epistasis exerted by amino acid substitutions in the coiled coil on the retrotransposition activity of ORF1p. In some cases epistasis is exerted at the single residue level (e.g., Martin,et al - 2008, Nucleic Acids Res., 36, 5845; Furano, et al. - 2020, PLOS Genetics 16 e1008991.)
While the authors are apparently also not mindful of the PLOS Genetics paper examining the effect of a single inactivating coiled coil substitution at the level of microscopically observed condensates could have provided compelling evidence linking their formation and retrotransposition. On the other hand, lack of a condensate-based readout for single amino acid inactivating coiled coil mutations would question the validity of equating ORF1p condensates with retrotransposition competence.
- (E) The afore mentioned Callahan et al study (2012, NAR, 40, 813) in addition to producing results partly recapitulated in Fig. 2 of the present paper, showed that ORF1p polymerization was mediated by interactions between the highly conserved RRM-containing region of ORF1p. This observation is consistent with previous studies showing RRM-mediated protein interactions of other proteins (Clery, et al 2008, Curr. Opin. Struct. Biol., 18, 290; Kielkopf, et al Genes Dev., 18, 1513)
As well as including the missing citations of the L1 literature, implications of the above considerations need to be addressed before publication.
I have the following additional comments and issues:
- p.2. l. 8, the citation to TPRT should include Luan,et al.- 1993, Cell 72: 595
- p. 5, middle of 2nd para - what does "different diffusivity" mean? - what are "stereotyped puncta"?
Any invocation of cis preference should cite the foundational study by Kaplan, N., et al. (1985). "Evolution and extinction of transposable elements in Mendelian populations." Genetics 109 459.
- p.10 middle paragraph, the authors state: "The decreased phase separation of the R261A mutant was unexpected, as we predicted that mutating a core RNA-binding residue would only affect condensation in the presence of RNA. We also noted that the protein partition coefficients of the R261A condensed phases were higher than their counterparts for WT and K3A/K4A. Taken together, these experiments showed that K3/K4 and R261 are not essential for protein condensation in vitro."
these findings would have been predicted by the afore mentioned findings of Newton et al, which should be cited here.
- p. 14, first paragraph "we predicted that stammer-deleted ORF1p would maintain an elongated coiled coil conformation that might disfavor trimer- trimer interactions that are mediated by the N terminal half of the protein (Figure 4A, left two cartoons)."
It seems that the authors are stating that different fully formed trimers can form larger complexes mediated by interactions between their coiled coils, an idea apparently based on results published by Khazina and Weichenreider (2018). This paper states that "Additional biophysical characterizations suggest that L1ORF1p trimers form a semi-stable structure that can partially open up, indicating how trimers could form larger assemblies of L1ORF1p on LINE-1 RNA." However, the cited Khazina structural data ((PDB) entry 6FIA)) were derived from coiled coils that had been solubilized to monomers in guanidinium HCl from inclusion bodies (insoluble aggregates) that had accumulated during their synthesis in E. coli...a common condition for highly expressed proteins. Fully denatured ORF1p coiled coils such as these, which also lack the entire NTD are an in vitro artifact and never exist in "nature". It is almost certain that ORF1p monomers trimerize while being synthesized on adjacent ribosomes (e.g., Bertolini et al.- 2021; Science 371: 57). I am not aware of any biochemical evidence from the Martin laboratory on mouse ORF1p or the Weichenrieder or Furano laboratories on human ORF1p indicating that the coiled coils of fully formed trimers synthesized in vivo can unravel to mediate interactions between different trimers. In fact, the authors' results in Fig 1F supports this contention.
- p.10, Legend to Figure 1G The cells were stained simultaneously with two Halo ligand dyes (Halo-JF549 and Halo- JF646), giving a positive control for colocalization.
Why is staining the same ligand (Halo) with two different dyes a colocalization control?
- The authors conclude their paper with the statement "The L1 system characterized in this work employs a uniquely powerful combination of biochemical reconstitution, live-cell imaging, and functional phenotyping in cells. In vitro reconstitution allows us to study the biophysical properties of condensates in a minimal and controllable system."
However, there are several instances where the in vitro biochemical properties of ORF1p variants are somewhat discordant with their in vivo results. In the case of their coiled coil mutants. replacement of the coiled coil stammer, MEL (uniquely invariant for more than 50 Myr of primate coiled coil evolution) with AAA or AEA exhibited reduced retrotransposition that was not accompanied by a corresponding reduction in condensate formation (Fig 4). In another instance, while mutation of the highly conserved residue (R261) necessary for RNA binding eliminated retrotransposition it did not have a corresponding effect on condensate formation even in the presence of RNA (Fig 3).
General comments on the Figures - Although I rather liked the cartoon version of ORF1p (Fig 1B) and when used to show the location of mutated site, versions that purport to show the effect of mutations on structure (Fig 4A) are misleading and should be eliminated.
Closing Comment:
Overall, I enjoyed reading this paper, and feel that when the issues I raised are appropriately addressed and the relevant missing citations are included it would make a useful contribution. However, it seems that the authors could make a more compelling case that dissociates condensate formation of ORF1p and its activity in retrotransposition, consistent with the Martin and Holehouse review cited above. So, I urge them to reconsider their conclusions. I did not find the highly speculative discussion about the relevance of phase separation / condensate formation to cis preference at all convincing as it is just as it is just as likely (maybe more so) to be enforced at the level of selection - evolutionary failures, by definition, are not propagated.
Significance
Although this paper addresses a long-studied topic in L1 biology, namely how the L1 encoded proteins assemble into an L1 RNP (the retrotransposition intermediate), the authors posit that the formation of phase/separated protein condensates (visible as microscopic droplets) are involved. Such droplets are a currently popular biochemical feature exhibited by some proteins, but their functional relevance is a currently a contentious topic in protein biochemistry. I do not think that the authors make a convincing case that condensate formation is involved, rather I think that their evidence provides reasonable evidence that condensation has no role. I urge the authors to consider this possibility, but whatever which conclusion proves to be correct, their study would make a useful contribution to the field.
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Referee #1
Evidence, reproducibility and clarity
This study shows that the ORF1 protein of the LINE-1 retroelement forms puncta in vivo that they define as cytoplasmic biomolecular condensates based on the characterization of the biophysical properties of ORF1p condensates in vitro.
Defective retrotransposition of some ORF1p mutants correlates with defects in puncta formation in vivo and alteration of biophysical properties of in vitro condensates leading the authors to conclude that condensation of ORF1p is required for retrotransposition.
The study combines biochemical reconstitution, biophysic analysis and live-cell imaging. In particular, the authors take advantage of a new …
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Referee #1
Evidence, reproducibility and clarity
This study shows that the ORF1 protein of the LINE-1 retroelement forms puncta in vivo that they define as cytoplasmic biomolecular condensates based on the characterization of the biophysical properties of ORF1p condensates in vitro.
Defective retrotransposition of some ORF1p mutants correlates with defects in puncta formation in vivo and alteration of biophysical properties of in vitro condensates leading the authors to conclude that condensation of ORF1p is required for retrotransposition.
The study combines biochemical reconstitution, biophysic analysis and live-cell imaging. In particular, the authors take advantage of a new powerful tool they have developed based on the tagging of ORF1 within a functional L1 reporter element. The fluorescent tag allows following the dynamics of ORF1p by live-cell imaging.
The key conclusion is that ORF1p condensation is important for L1 retrotransposition. The correlation is clearly shown but raises several questions: Is the defect in ORF1p condensation the only explanation for the retrotransposition defects of the ORF1p mutants analyzed here? Can we exclude that the mutations in ORF1p affect other functions of the protein such as its binding to RNA (as in the case of the R261 mutant) and cis-preference, or its binding to other factors involved in L1 replication? Could the loss of these functions affect L1 retrotransposition independently of ORF1p condensation?
Major comments:
On several occasions, the authors propose that ORF1p-HALO dynamics in vivo is linked to its co-translational association with L1 RNA. However, they never show the presence of L1 RNAs in ORF1p-HALO puncta in vivo. To strengthen the conclusion that the puncta observed in vivo are L1 RNPs, the authors should add experiments showing the presence of L1 RNA in the cytoplasmic puncta (by RNA FISH) or that the puncta are dependent on the presence of L1 RNA (expressing ORF1p-HALO alone should not be sufficient for puncta formation). These experiments seem to be realistic in few weeks with the tools already available in the laboratory.
Apart from this comment, the authors are cautious in their conclusions. It is clear, as they indicate in the Discussion, that showing that ORF1p condensation is also required for the mobility of other retroelements will strengthen the implication of ORF1p condensation in L1 replication.
The data are well presented and the methods described in detail so that others should be able to use them. The experiments seem to be adequately replicated and the statistical analysis adequate.
Minor comments:
Figure 1F: Having the pictures of cell nuclei (like in Figure 1D) would be nice to know how many cells we are looking at in this panel.
Figure 2E: it is surprising that there is no correlation between the ORF1p:RNA ratio and the number of individual fusion events (i.e. curves of ORF1p+RNA 10000:1 and 1000:1 overlap while 3000:1 is different). Could the authors discuss this point?
Previous studies are appropriately referenced. Text and figures are clear and precise.
Referees cross-commenting
The main critical points shared by all reviewers are: 1) the need to show the presence of LINE1 RNAs in ORF1p condensates in vivo and 2) the lack of evidence for causality between ORF1 condensate formation and L1 transposition efficiency (At this stage, the authors should moderate their conclusions, especially in the abstract). Regarding the other reviews, we noticed the need to cite additional relevant studies in the field (reviewer #2) and the interesting points raised by reviewer #3 to investigate the formation of ORF1 condensates in an endogenous situation, and whether other RNAs do affect ORF1p condensates.
Significance
The study is technically interesting in that it describes a new system for tracking ORF1p puncta formation in vivo. The findings are not unexpected because it comes after the publication of Newton et al. in 2021 (PMID : 33798566), describing that ORF1p does phase separation in vitro. Furthermore, the fact that RNPs form "membrane-less" structures is already established in other situations as the authors point out. Compared to Newton et al., condensates are better-defined biochemically, especially for RNA association features and in vivo dynamics.
The ORF1 protein is widely studied for its role in L1 retrotransposition. The protein forms a homotrimer in vitro, binds to L1 mRNA in a cis-preferential manner, and is required for retrotransposition. On the other hand, RNA-binding proteins are often involved in the formation of membrane-less organelles (stress granules, RNA processing bodies...). These observations suggest that ORF1p may also form RNP condensates required for L1 retrotransposition. A study published in Biophysical Journal in 2021 (Newton et al. PMID: 33798566) has already reported the phase separation of the LINE-1 ORF1p that is mediated by the N-terminus and coiled-coil domain. This former study was based on in vitro microscopy and NMR approaches and is cited in the submitted manuscript. The study submitted to Rev commons goes further by analyzing the biochemical properties of ORF1p condensates in the presence of L1 RNA and by following in vivo condensates of ORF1p (WT or mutants) expressed from a functional L1 reporter element by live-cell imaging. The findings will interest a wide audience investigating the biology of retroelements and more particularly scientists who study the L1 retrotransposon.
I am an expert in retrotransposon biology but I do not work on L1. I am not expert enough to assess the quality and relevance of the biophysical experiments in the paper. In particular, panels 2D, 3B and 3D were difficult to analyze.
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