|Type||Kitchen Table Talk|
Professor Dominique Weil from the Sorbonne’s Institut de Biologie Paris-Seine delivered a tour de force lecture for Dewpoint on April 15th as part of our series of Kitchen Table Talks. Dominique studies the roles played by various types of RNA in the behavior of membrane-less ribonucleoprotein granules which form by liquid-liquid phase separation.
Dominique discussed her recent work on the role of mRNA storage in human P-bodies. We enjoyed her talk immensely and hope you do too.
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Mark Murcko (00:00:00):
Dominique, we’re very happy to have you coming to us from Paris this morning, and for those who don’t already know, which is probably nobody on the Dewpoint team, because we’ve already been talking about you, your work in P-bodies especially, but more broadly the role of RNA in condensates is something that we’ve been watching and are very excited in hearing about. And today, you’ll be talking about mRNA storage in P-bodies.
Dominique has also published on some helicases, which are also topics that come up pretty frequently these days in the condensate community. So, that might be another topic of discussion, as well. So, we thank you again for giving this lecture and you can take it away…
Dominique Weil (00:00:57):
Okay, thank you for the invitation, particularly in this very special moment. So, I’m going to share my screen. Okay, just can you confirm you see it? No? Can you confirm you see my screen or…
Dominique Weil (00:01:23):
Can you see my screen?
Yep. We can see your screen now.
Dominique Weil (00:01:36):
Okay, that’s fine. So, my lab is located in Paris, close to the Seine. It’s in the Institute of Biology Paris Seine. So, I would be here. My lab would be here. So, today, I’m going to talk about the GC content that shapes mRNA storage in human P-bodies, or rather, how we came to that idea and to have evidence for that. So, you are a particular audience, so you know about all that, but post-transcriptional regulations of mRNA, it’s a story of RNA binding proteins that are going to control if these RNA are going to be translated, stored without translation, or decayed in the cytoplast. But it’s also a story of cytoplasmic RNP granule where the RNA and the proteins responsible for this regulation are accumulating.
Dominique Weil (00:02:31):
And we are particularly interested in this aspect of post-transcriptional regulations. In general, these granules, these RNP granules, they have very different names, germ granules, neuronal granules, P-bodies, stress granules, now even translation factories. Depending on the cell type where they have been found, like germ cells or in neurons or in all cell types for P-bodies.
Dominique Weil (00:02:58):
But it’s not only a matter of name or of cell lines, of cell type. These granules, they also differ by their composition, their morphology, and maybe their function. But globally, overall, the questions, at least the way we see these granules, the questions are more or less the same for all of them. How do they assemble? What do they contain in terms of proteins and RNAs? What is their function in RNA metabolism? And further than that, what’s their function in cell physiology?
Dominique Weil (00:03:38):
So, in our case, in the recent years at least, we have been particularly interested in P-bodies in human cells. So, these P-bodies, they are here in epithelial cell, human cell. These red dots, red because they are labeled through the expression of DDX6 helicase fused to RFP. In the absence of this protein, they would not be visible in contrast to these other gray granules here, which are visible in phase contrast, because they are surrounded by membrane.
Dominique Weil (00:04:16):
These P-bodies, they have been observed in animals and vegetals, from yeast to human. Since the beginning, we know that they contain a set of proteins related to mRNA metabolism function, mRNA decapping, mRNA decay, specialized pathways for mRNA decay such as NMD, nonsense-mediated decay. RNA interference proteins which act also on RNA decay and mRNA translation. And factors involving translation repression.
Dominique Weil (00:04:52):
So, from this first list of components, everyone postulated that they should be important for mRNA decay, translation regulation, and RNA interference. However, how do they function with respect to these different regulation pathway has been a mystery, and is probably still a mystery.
Dominique Weil (00:05:16):
So, at least we know that they can’t translate these P-bodies. First of all, nobody has seen any signal using antibodies directed against ribosomal proteins. But then, we also confirm that by combining electron microscopy with in situ hybridization using 28S probes or 18S probes. So, here, we have these DDX6 helicase I was talking about fused to RFP. This time it is the endogenous DDX6 protein. And the antibody is coupled to large gold particles 15 nanometer diameter. So, we know that this is a P-body, and the in situ 28S probes or 18S probes are coupled to small gold particles, 5 nanometers. And as you see, all the small particles are in the surrounding cytosol, but they are not located in P-bodies. It’s true for the two ribosomal probes.
Dominique Weil (00:06:20):
And when we quantified that, we found that indeed in granules, the granules are very devoid of 28S and 18S signal compared to the surrounding cytosol. Some groups or for some other granules have proposed that maybe they would be a crown of ribosomes at the surface of the granules. We do not see that at all in the periphery cell concentration. The density of gold particles is very similar in the surrounding cytosol. This is very much in contrast with stress granules, another type of granules which is induced by stress and contains also untranslated mRNA. The same approach that we did a few years ago led to the evidence of accumulation of 18S ribosomal RNA, but not 28S, confirming since observation by immunofluorescence that the stress granules contain the small ribosomal subunit, but not the large ones.
Dominique Weil (00:07:28):
So, just to compare these two ones, which form in the same cell type, most granules contain silent RNAs, but the P-body RNA are totally out of translation, while the stress granules contain mRNA which are pre-initiated for translation. Just to extend a little bit this comparison between P-bodies and stress granules, it has been observed by many labs, including ours, that the P-bodies here in green are very often found in close contact to stress granules when these cells are stressed, here in red. We also visualize that electron microscopy here is a stress granule, and here, the neighboring P-body. Nevertheless, the two types of granules, we never found them intermingling, and in fact, they have different structure. Stress granules have granular ultrastructure, probably related to the presence of the small ribosomal subunit, while P-bodies, it is enlarged here. They have fibrillar ultrastructure.
Dominique Weil (00:08:38):
We also found that this contact, they do not form after the two types of granules form, but in fact, many stress granules, not all of them but majority of them assemble at the contact of pre-existing P-bodies. We don’t know why. It’s like a [inaudible 00:08:56] mechanism that would be worth to study, but so far, we have no clue of what is origin of these.
Dominique Weil (00:09:04):
Now, to come back to P-bodies, as you know, they are formed following liquid-liquid phase separation. So, in electron microscopy, they clearly have no membranes. Here, it is the antigen on DDX6 protein, which is detected through immuno-staining. We have conducted several studies, showing that three proteins are required in human cells for P-body assembly. It’s DDX6 helicase, the DEAD-box family, 4E-T, and LSM14A, which are RNA binding proteins, and also, which interact together and with DDX6. We don’t know how they interplay together, but we know that there’s three of them. If you silence even one of the three, P-bodies disappear, and cannot be reassembled by any trigger we tested so far.
Dominique Weil (00:10:05):
So, we got particularly interested in this DDX6 helicase for several reason, but maybe mainly because it’s a helicase which is very highly expressed in human cells. Concentration, we measured that the concentration of the protein is of the order of 3.3 micromolar in these cells, and by counting the gold particle in this immuno-staining, we could calculate that the concentration inside the P-bodies reaches 0.5 millimolar, which is extremely high of protein. We don’t know if that would enhance the enzymatic activity of the DDX6, or maybe contact with some of its protein partners, or maybe this very high concentration would do the opposite, like decrease the activity of the protein, because it’s too viscous. But it should somehow change the property of the complex, the activity of the complexes, which are contained in P-bodies.
Dominique Weil (00:11:11):
Okay. One of the reasons it was very difficult to find the function for P-bodies is that by silencing any of these proteins, we can make disappear the P-bodies, dissolve the P-bodies. But that doesn’t lead to major defect, visible defects in the cell. Everything in terms of translation, in terms of decay, in terms of RNA interference continues to happen, and it has been difficult to prove that these P-bodies were required by any way.
Dominique Weil (00:11:50):
One missing information probably to understand the function of P-body, is which RNA are in there. In fact, maybe there would be naturally only a couple, a few RNA that would accumulate there, explaining why when we suppress the P-body, we don’t see major defect, and we need to image in some particular test to evidence the function of P-bodies. So, to know which RNA are in P-bodies, they only way probably to make it systematically is to profile the P-bodies. This has been a major challenge for all labs which try, first reason being that nobody could find any protocol of differential centrifugation that would be efficient at purifying P-bodies, without co-purifying membranous organelles. So, mitochondria, in particular, come with, but many other organelles. All these membranous organelles are the same size.
Dominique Weil (00:12:51):
Another challenge is that they are very small in size. There is a low number per cell. They have no membranes. But maybe, at least to my opinion, one of the major difficulty is how much of any marker we have is really in P-bodies. So, take this DDX6 protein. It’s really hugely concentrated in P-bodies, but nevertheless, if you calculate how many molecules are in P-bodies versus outside of P-bodies, you find that not more than 20% of total that exist is located in P-bodies at any time, despite the fact that it is 170 fold concentrated in P-bodies. But they are too small and very scarce. So, it’s probably true for any P-body marker, so we have to consider that P-bodies contain a high concentration of all the protein that we find in it, but this proteins, they also exist outside. So, any approach using immuno-precipitation is going to co-precipitate both co-purify together the complex, which are in P-bodies and the complex which are outside of P-bodies.
Dominique Weil (00:14:08):
So, we turn to a different approach, which was to engineer cells to get fluorescent P-bodies through the expression of one of the components that is in LSM14A fused to GFP here. We lyse the cells, and we pass this lysate in FAPS in flow cytometry. We could observe a fluorescent signal that was specific of the P-bodies by comparison with cells which express a fluorescent protein as well, but not contained in P-bodies. So, this signal here is specific from P-bodies. Using the FAPS, we sorted these fluorescent granules. After sorting, we spin them down. We analyze them on the FAPS, and then kept size and same fluorescence. So, obviously, they could resist the process that we call fluorescence activated particle sorting. Before sorting, it’s very dirty lysate, with in green, the P-bodies, and in red, it’s just unspecific staining of all membranous contaminants. After sorting, it looks rather clean. And the difficulty was to purify enough material to be able to identify proteins by mass spec and RNA by RNA-seq.
Dominique Weil (00:15:34):
So, we did quite an extensive purification. Six full day of sorting to purify 450 nanogram of protein, that we could analyze by mass spectrometry. Here, we compare the mass spec score before sorting to after sorting, so we reason in terms of enrichment in P-bodies. In yellow, about 125 proteins which are enriched in P-bodies. In red are all the ones which we are already known to be P-body components, so they are mostly enriched. In black are a few proteins that we verified by immuno-fluorescence. They are indeed in P-bodies, but they are also many proteins which are significantly excluded.
Dominique Weil (00:16:26):
If we look at them, looking at the distribution of the proteins by function, in the enriched protein compared to depleted protein, we got three of most mitochondrial protein in violet here, cytoskeleton protein in black. We get some of those, but we think it’s relevant. It’s related probably to the known trafficking of P-body in the cells. And even in terms of proteins related, so three quarters of the proteins are obviously related to nucleic acid metabolism, even these have a specificity, like we got rid of all translation initiation factors in light blue, as expected from the granule which is not translating. We got rid of all stress granules markers in light blue as well, here. So, I don’t want to insist too much on this protein content, because we had enough material to do one mass spec analysis, so it’s not really substantive or verbose, but it was enough to claim that this purification was to a sufficient extent to analyze RNA.
Diana Mitrea (00:17:38):
Can I ask a question?
Dominique Weil (00:17:39):
Yeah, of course. Of course.
This is Diana. That’s really beautiful. I was wondering, how many cells did you need to extract for-
Dominique Weil (00:17:50):
It was 300 millions. 300 millions.
Okay. Great. Thanks.
Dominique Weil (00:17:54):
Yeah, so probably the yield is not very good, because by calculation, starting with 300 million cells, we should have got more P-bodies than what we really got, so in particular here, maybe there are still P-bodies, small P-bodies here that we missed. We don’t know. Or maybe some still dissolved during the FAPS. We don’t know. But at least, the material we get at the end is enough to do the mass spec. But the yield is probably not very good.
Dominique Weil (00:18:28):
So, in terms of RNAs, this time we had enough material, 90 nanogram of RNA, so this was enough to do a triplicate RNA-seq, from independent sorting and independent libraries. So, in red is a profile in size, a size profile of the RNA from the purified P-bodies, compared to the profile before sorting in green. So, it’s not a few RNA species obviously. It’s a large range of size. The RNA are very much depleted in the two peaks for the ribosomal RNA as expected. And in fact, it’s a very, very large number of RNA which are significantly enriched. It’s more than 6,000 of them, but they are also basal. So, a very large number of RNA which are significantly excluded, more than 7,000.
Dominique Weil (00:19:30):
If we look at what they are, close to 90%, 89% of the RNA which are significantly enriched correspond to messenger RNA, while the long encoding here in light blue, or the anti-sense in blue, they are mostly excluded from the P-bodies. This is most coding and non-coding. If we consider the coding transcriptome, it’s one third of the coding transcriptomes which is found enriched in P-bodies. So, a very, very large amount, proportion of the coding transcriptome. I have no time to describe that here, but it’s published. We couldn’t find any sign of 5′ decay from the analysis of the [inaudible 00:20:18] of the enriched RNA.
Dominique Weil (00:20:21):
So, now we compare this dataset to what happens when we disrupt P-bodies through silencing of one of the [inaudible 00:20:29], otherwise the DDX6 protein, proteins completely required for assembling P-bodies. So, we are going to compare cells, the transcriptome of cells which have P-bodies, and cells which do not have P-bodies. And we did the polysome profiling, so when looking at the total mRNA, this should reveal any function in mRNA stability. Here, we compared in the Y axis what happens in terms of abundance of total mRNA, and on the X axis, it’s enrichment in P-body. The RNA which are found in P-bodies, they are not increased or decreased when we dissolve P-bodies. So, they were not degraded in P-bodies. They don’t mind the fact, in terms of abundance, the alternative to the presence or absence of P-bodies.
Dominique Weil (00:21:20):
However, when we look at the polysomal RNA, and here, it’s the polysomal to total ratio that should reflect any regulation of translation. We find that the RNA which are in P-bodies, they are more translated when we disrupt P-bodies than before, which is an argument, I think, that P-bodies are a site of storage that prevent translation. So, no increased stability, because it has been postulated before that P-bodies would be for decay. This was mostly coming from yeast studies, but they were also a few studies in human cells. We could not find any argument for the role in stability of mRNA. This is pointing to translation, a function in translation repression.
Dominique Weil (00:22:10):
Now, we could combine all these data. We could cross them with many existing CLIP experiments. Again, RNA binding proteins, which identifies the targets of these RNA binding proteins. So, I will make it short, because it’s a complex slide, but here, you have the set of proteins, RNA binding proteins, against which CLIP were performed. We rank them depending on the accumulation on the Y axis of the target in P-bodies. So, for instance, on the left, FXR1, its targets identified by CLIP are massively enriched in P-bodies. On the other side, ATXN2, its targets are massively excluded from P-bodies. And then, it’s just ranked depending on the accumulation.
Dominique Weil (00:23:04):
In red, you have here all the proteins which we’ve found in our mass spec for all the groups found, been present in P-bodies. And underlined in green, it’s one which were shown to be absent in P-bodies, particularly ATXN2. It’s particularly true for ATXN2. So, as you see, the proteins which accumulate in P-bodies, they tend to accumulate with a target RNA. These red proteins, many of them, they are known as translation regulators. So, this is a certain argument to think that the P-bodies are a place of storage for mRNA, which are repressed at the level of translation.
Speaker 2 (00:23:56):
Excuse me, I have a quick question.
Dominique Weil (00:23:58):
Speaker 2 (00:24:02):
So, you mentioned DDX6 was 170-fold increase in P-body, but the total number of protein is still below 20% in total.
Dominique Weil (00:24:16):
Speaker 2 (00:24:16):
Right? It’s enriched. So, is this mRNA enrichment kind of expressed the same way? There are a lot in the cytosol as well, or it was just-
Dominique Weil (00:24:28):
Yeah, we can’t really… This is a calculation we cannot do, because for the first 170-fold enrichment of the DDX6, this is a conservative analysis. We just look at section of cells, and in this section, we count the density of gold particles inside and outside. So, we are sure not to miss anything. But here, we are going to compare a sample before FAPS and after FAPS, and we have no idea of the yield. So, we can’t really come back to the same type of… We can discuss in terms of enrichment, but we can’t discuss in terms of absolute ratio.
Speaker 2 (00:25:14):
Right, right. Thank you. Thank you.
Dominique Weil (00:25:16):
Yeah. So, our conclusion from these data and other I didn’t detail is that human P-bodies are primarily involved in mRNA storage, and not so much in mRNA decay, as has been proposed earlier. Then, we also considered what is the protein yield of the mRNA depending on the localization in or out of P-bodies.
Dominique Weil (00:25:43):
So, I assume that you know that the protein yield. So, what I call protein yield is what is the abundance of a protein with respect to the abundance of its cognate mRNA. Everyone knows, I think, that it’s not really very much correlated. There are obvious reason, like if a protein is very unstable. Of course, it will give protein yield which is lower than if the protein is particularly stable. But another source of protein yield variation can also be translation regulations.
Dominique Weil (00:26:16):
So, here, I plotted the protein yield for all the transcriptome, and this is taken from literature for the protein, quantitative protein levels and mRNA levels from our own data. And we just color-coded depending on the localization of the mRNA in P-bodies, in yellow, or outside of P-bodies, in blue. And as you see, the protein yield is lower for the RNA which are in P-bodies compared to the one which are excluded from P-bodies. But even more, the correlation coefficient between the amount of protein and the amount of RNA is also… It’s more correlated for the one which I excluded than for the one which are enriched in P-bodies, which is very much suggesting a regulation at the level of translation. So, I ask you to remember this protein yield story, because I’m going to skip to something else, but I will come back to it later on in the seminar.
Dominique Weil (00:27:25):
So, now, which type of mRNA are stored in P-bodies? When we look at the gene ontology categories, which are in, which are out? So, it’s a large, large variety, because as I said, it’s one third of the coding transcriptome, so it contains all types of… Very large panel of function. But still, there seem to be one constant, which is that the RNA which are in P-bodies, they tend to encode regulating function, at the level of chromatin, transcription, RBP, PTM, but also many other ones, even for mitochondria, or other type of function. When the RNA which are significantly excluded, they tend to encode basal functions. To be more explicit on that, I am going to drive you through a few examples.
Dominique Weil (00:28:18):
For instance, if I take protein degradation, it’s operated by the proteasome. Here is the enrichment on the Y axis in P-bodies of the mRNA encoding. Here is the different subunits of the proteasomes. Most of these mRNA, they are excluded from P-bodies. However, the RNA which encode protein which will regulate the targeting of proteins to the proteasome, mainly the E3 ligase, or the ubiquitinase. The mRNA tend to be enriched in P-bodies. It’s tendencies, not systematic, but it is tendency that we found in many different processes.
Dominique Weil (00:29:02):
If you think to RNA processing, so the splicing is done by the snRNPs, the messenger are excluded. But the splicing regulators here, they tend to be enriched. LSM128 is also constant, an heptamer that control splicing. And it’s also excluded.
Dominique Weil (00:29:25):
If I looked at mRNA translations, the ribosomal proteins, their messenger are massively excluded. Whether belonging to the cytoplasmic, cytosolic ribosomes or mitochondrial ribosome, they are excluded from P-bodies. But regulator of translation, like proteins of the CPB complex or proteins of the RISC complex, their messenger RNA are enriched. And there would be many further example. I took the ones which are the most familiar to you. So, it suggests that this P-body related regulation may be involved in producing the right protein at the right moment, the right regulators when required by the cells.
Dominique Weil (00:30:15):
And even more in detail, it seems that proteins that are going to interact together, their messenger are co-regulated. So, either co-excluded or co-enriched in P-bodies. And so, this is the case of the proteasome, the big complex, or the snRNP, or the ribosome, or this LMS128 heptamer. But it’s also the CPB complex or RISC complex, whose messenger RNA are enriched in P-bodies. This raise the possibility that the regulation associated to P-body localization could also contribute to the protein stoichiometry in complex. It’s a speculation, but we’ve found it interesting.
Dominique Weil (00:31:07):
So, now, if I just summarize what I presented to you. So, in P-bodies, we find one third of the coding transcriptome. These mRNA are rather evident, I can say, but it’s rather whether they’re well-expressed. However, they have a very low protein yield. This storage is reversible. This is from our polysome profiling experiment, but it has also been shown by others. One third of the coding transcriptome is significantly excluded. These mRNA can be also very abundant or not, more or less, but they have a high protein yield. And all these RNA are decayed outside of P-bodies. Just of note, decay has also not been seen in P-bodies, using RNA decay reporters by a series of group. So, I think in human cells, at least became consensus that decay is occurring outside, and P-bodies are for storage. And in terms of function, storage is rather for mRNA encoding regulatory proteins, while the mRNA encoding basic function are rather excluded.
Dominique Weil (00:32:25):
So, now, I reconsider the story about protein yield. At first glance, it could seem very logical that a granule which is for storage contains mRNA, inefficiently translated, have a low protein yield. But the main question is, which fraction of a given mRNA can really be stored in P-bodies? Because I also insisted that the P-body are very small in size, and they are very few per cell. For this, we address this question using the single molecule FISH. So, here, I show the data for the SPEN mRNA, an mRNA which is 10-fold enriched in our purified P-bodies, which is quite high level of enrichment. When we do the single molecule FISH, so here in red, each dot is a single molecule of these RNA, yes, we see if we look at that enlargement, we see clusters of these mRNA in P-bodies in green, but we see also many other molecules outside of P-bodies. So, when we quantified that on the cell basis… So, we counted the number of molecules in and out P-body for 40 cells here. We found that it was very heterogeneous between cells, but on average, it was not more than 15% of the molecules of SPEN mRNA that were located in P-bodies, which was much less than what I would have expected.
Dominique Weil (00:34:07):
So, we have to consider that exactly like the proteins, the mRNA, they can be enriched in P-bodies, but nevertheless, they always exist outside of P-bodies. And maybe for people interested in LLPS, it’s just very logical, because the phase transition is just a new balance between the condensed and soluble phase, which means that it could have been expected that both the protein and mRNA component would be present in the dilute phase, even if they are very much concentrated in the condensed phase.
Dominique Weil (00:34:48):
Nevertheless, we have a problem in terms of amount because 15% of SPEN mRNA molecule in P-body is never going to explain how these mRNA have 100-fold lower protein yield than most P-body excluded mRNA. So, in other words, it’s not the sequestration of this mRNA in P-bodies that can explain this very low protein yield, and we had to conclude that this low protein yield is an interesting property of the P-body mRNA. It’s not related. It’s not due to the sequestration in P-bodies.
Dominique Weil (00:35:32):
So, our next task was trying to understand what could be the reason for this very low protein yield. We first looked at mRNA length, because Parker’s group on stress granules had noted that the RNA which accumulate in stress granules are longer than average, so here, we divided the human transcriptome in six classes, depending on the mRNA lengths, from less than 1.5 kB to more than 10 kB. And like in stress granules, the longer RNA accumulate more in P-bodies than the shorter ones. The correlation coefficient is 0.39, so it’s relatively high or moderately high. However, what was striking, the most striking feature of this RNA is the GC content. Here, it’s divided depending on the GC content. Transcriptome is divided based on GC content, then from less than 40% GC to more than 60% GC. And obviously, the RNA which have a low GC content accumulate much more in P-bodies than the one which have a high GC content. This time, the correlation coefficient is -0.63, so very high. Can be represented a different way. Here, it’s a distribution of GC content in gray for the whole transcriptome, from 35% to 70% for the human transcriptome. It’s like a bimodal distribution, and only the enriched RNA accumulate in P-bodies, here in red. The ones which are GC rich, they are completely excluded. So, it’s a very striking effect of the GC content.
Dominique Weil (00:37:29):
So, here is a representation of the GC content of the lengths of the whole RNA, but if we try to distinguish contribution of the GC content of the 5′ UTR, CDS, and 3′ UTR, we find that it is related to the GC content of the CDS and the 3′ UTR, not so much of the 5′ cap. So, if there is a very strong bias of the GC content in the CDS, it’s expected to have a strong effect on the coding property of these mRNA.
Dominique Weil (00:38:12):
So, we therefore looked at the amino acid usage in CDS of mRNA which are in, in red, or out, in blue, P-bodies. For the 20 amino acids here, as you see if I pick up a few example. Taking alanine. Alanine is much less frequent in the protein encoded by P-body mRNA than encoded by the mRNA which are excluded from P-bodies. It’s the same for glycine. And reversely, the lysine or asparagine, they are much more frequently encoded by the mRNA which are in P-bodies than the one which are outside. So, these very strong GC content bias has consequences on the amino acid frequency in the proteins encoded by P-body mRNA. I have no idea which type of property. I could not make a coherent picture of what type of property it would lead to, but it’s an observation.
Dominique Weil (00:39:27):
Beyond these amino acid usage, it should also have an effect on codon usage. So, here, I represented for the 20 amino acids. It’s a ratio of usage between the mRNA which are in P-bodies and the one which are out. So, take leucine for instance. It is encoded by 6 codons. One codon here is two-fold… It’s not two, sorry. So, four-fold more used in P-body mRNA than in P-body excluded mRNA. Now, of course, some are less used. In every single case, it’s completely related to the percentage of GC in the codons. It’s color coded in gray here. So, this codon which is more used for leucine is completely devoid. It’s only composed of A and U, and it’s devoid of G and C. And the ones which are less used, they have more GC. So, the GC content bias leads to a codon usage bias in P-body mRNA. And this is very interesting, because in human, the codons which are less frequently used most often they end with an A or a U. So, these letters to think that maybe it was related to the low protein yield that we observed earlier.
Dominique Weil (00:40:50):
So, we plotted the frequency of these low usage codons with the protein yield. As expected, we see that the RNA which are in P-bodies in red, they have a much higher frequency of low usage codons, because they are just much more AU-rich than the ones which are outside of P-bodies. But the correlation with the protein yield is moderate, 0.2. However, I also tell you that the mRNA which are P-bodies, they tend to be longer, and this is also true of the CDS. So, if we combine the two, and this time plot the absolute number of low usage codons per CDS, this is still very correlated to localization in or out of P-bodies, and this time, it has a good correlation with the protein yield, with a correlation coefficient of -0.46. So, we think that we have found what was the interesting properties of these P-body mRNA to have low protein yield. The reason is the GC content, which is very poor.
Dominique Weil (00:41:57):
Okay, now, to extend that, we thought, “Okay, GC content.” And we were very surprised by that, so we did check and recheck several times, but there’s no problem. It’s true. So, we were wondering if it’s very important for P-body localization, maybe it’s also important for other aspect of post-transcriptional control. So, we address for that question by considering very general factors involving mRNA decay or translation. So, I told you we have the polysome profiling after silencing the DDX6. The DDX6 is known as both partners of the decapping complex that stimulate the decapping in yeast, but also as a translation repressor.
Dominique Weil (00:42:45):
We generated transcriptomes after silencing XRN1, the exonuclease which is responsible for the 5′ decay. And after silencing PAT1B, another factor that is both known in yeast as a co=factor of decapping and in all species, has a co-factor of translation repression. And I just give you a flavor of it through this heat map. So, here, represented the whole transcriptome, and we clustered it depending on the GC content, P-body localization, and behavior of the mRNA after silencing the DDX6, the XRN1, or PAT1B. And we also have the dataset of polysome RNA, polysomal RNA after silencing the DDX6. And as you see, just hierarchical clustering of these genes, leads to the separation of two main group of RNA, which are more or less the AU-rich RNA and the GC-rich RNA. The GC-rich RNA, they decay or the abundance, so they decay, is sensitive to silencing of the exonuclease XRN1 and the helicase, the DDX6, but not of the silencing of PAT1B, which was not expected at all. We thought that PAT1B and DDX6 had exactly the same targets. And these RNA, they are excluded from P-bodies.
Dominique Weil (00:44:22):
And conversely, the RNA which are AU-rich, the one which are in P-bodies, they are very sensitive to silencing of XRN1 and DDX6 in terms of decay. However, the decay is controlled by the level of PAT1B and the translation by the level of the DDX6. So, now, if it’s important for this… First, sorry, this general factors that we thought were general at the beginning of the study, they are not so general. They have a large number of targets, but they have preferences for either GC-rich or AU-rich target.
Dominique Weil (00:45:03):
If it’s true for these general factors, it is also true for the sequence specific factors, sequence specific RBPs. So, we took a set of factors and used either clear data or search for identification of mRNA binding motif. And we observed that, in fact, targets of all these RBPs, they have bias in terms of GC content. Some of them, here is median in red. Some of them have targets which are more GC-rich. Some of them have targets which are more AU-rich. And here, in this heat map, it’s just ranked depending on the GC content. As you see at the top are the two only factors we could find that were clearly decay regulators and only decay regulators. YTHDF2 is related to m6a-dependent decay, and SMG6 is the nonsense mediated decay. And the other one, here at the bottom, so liking AU-rich RNA, most of them are translation regulators.
Dominique Weil (00:46:14):
And if we look at our other datasets, the behavior in the different datasets, including the P-body localization, is mostly expectable, can be predicted from the GC content. I would have thought that being the target of PUM [inaudible 00:46:34] for instance, would make a huge difference with being a target of HuR. But it’s not decay. They tend to follow exactly the behavior of RNA of similar GC content. There are a few exceptions. I have no time to detail, but in general.
Dominique Weil (00:46:53):
So, we wondered what was the situation for miRNA targets, and we took the targets of the 22 miRNA which are the most expressed in our cells. And it’s very similar. In most cases, the targets of these micro RNA are AU-rich, except for these two ones. In most cases, they are AU-rich, and their behavior mostly follows the GC content, maybe with small variation. But particularly, the accumulation in P-bodies is completely related to the GC content. There’s very few exceptions. So, it seems that the RNA sequence specific factors, the decay regulators, the targets are GC-rich. The translation regulators, they target are AU-rich, including the miRNA target.
Dominique Weil (00:47:53):
So, now, this is for the CDS. So, our next question was… I told you that the GC content was important, both at the level of the CDS and at the level of 3′ UTR, or at least in terms of correlation coefficient. We could not make the difference in our first analysis. So, our question was okay, the CDS, it has an impact on mRNA regulation, mRNA control, but what is the function of the 3′ UTR on the top? Does it have function or just because of the GC content of the CDS and the GC content of the 3′ UTR? They are very highly correlated together, so can we distinguish the function of the two. It’s what we tried to do in this type of analysis, where we fixed… We consider mRNA depending on the GC content of the 3′ UTR, and for each bin, we look if the GC content of the CDS makes a difference.
Dominique Weil (00:49:02):
So, in all cases, whatever is the GC content of the 3′ UTR, in every case the GC content of the CDS is lower if the RNA are in than if the RNA are out. So, this is a primary factor that the GC content of the CDS is much lower for mRNA which are in P-bodies. And we think it’s related to translation efficiency. In fact, we can also look at lncRNA which cannot be translated. They are mostly excluded from P-bodies, and the correlation is very poor with the GC content. It’s -0.2, -0.63 for coding messenger RNA.
Dominique Weil (00:49:46):
Can we show it directly in the cell experimentally? So, we use reporter plasmids, so luciferase reporter plasmid, that we transfected in HEK cells, except that we just switched the coding sequence, GC-rich coding sequence to AU-rich coding sequence, so they encode exactly the same protein. It’s just the codon usage which is different, because we change the GC content. We transfect them in HEK cells, and then in the cells, we visualize P-bodies in green, and we visualize the messenger RNA by single molecule FISH in red. As you see, if we have a GC-rich CDS, it does not accumulate in P-bodies. There are very few molecules. There are no clusters of RNA molecule in P-bodies. However, with our AU-rich CDS reporter, then we observe clusters of RNA molecules in P-bodies. So, changing just the CDS GC content is enough to change the localization with respect to P-bodies, and this was quantified here.
Dominique Weil (00:51:00):
So, in two independent experiments, we have much more clusters of molecules in P-bodies if the CDS is AU-rich than if the CDS is GC-rich. Of course, that has also an impact on protein yield. AU-rich CDS provides more protein than GC-rich CDS, despite the same amount of RNA.
Dominique Weil (00:51:32):
Now, ok for CDS, there is an impact on the GC content. If we want to show if there is also an impact on the 3′ UTR, we have to do exactly the reverse analysis so we fix the GC content in the CDS, and look if the GC content in the 3′ UTR is dependent in and out of P-bodies. If the CDS is AU-rich, we can’t visualize any particular contribution of the GC content in the 3′ UTR. However, if the CDS is GC-rich, then the RNA which accumulate in P-bodies, they have a GC content in the CDS… In the 3′ UTR, sorry, which is much lower than if they are excluded. So, there is clearly also a contribution of the GC content of the 3′ UTR, at least when the CDS is GC rich. And we postulate that it could be through the capacity of this 3′ UTR to bind factors that like AU rich sequence and favor condensation in P-bodies.
Dominique Weil (00:52:44):
Again, can we give some experimental argument? For that, we set up an in vitro constitution assay on ice, so we have our cells with fluorescent P-bodies. We lyse the cells, eliminate the nuclei, eliminate the P-bodies. The pelleted P-bodies here under microscopy, they look like that. And then, you have the cytosol, the supernatant, which is devoid of P-body, but still contains some soluble LSM14A which is fused to GFP. And we add to the cytosol purified recombinant DDX6 protein. That reforms granules that look like that under microscope. So, they are very similar to the pelleted P-bodies. We can count them by FACS. The formation is DDX6 dose dependent, and then, on the top, we can add RNA. So, either RNA sensitized in vitro from the GC-rich reporter or from the AU-rich reporter. When we add on the top GC-rich RNA, then we tend to form less granules here, that we call P-body-like granules. Just, we can’t claim that they are P-bodies, but that is, they contain LSM14A and DDX6. So, if we add GC-rich, that prevents the formation of granules. However, if we add AU-rich RNA, that favors the formation of granules. In that case, it’s on ice. The RNA, they are not poly-Adenylated. They are not capped, so we think this reflects the contribution of the RNA as such, independently of translation to favor condensation of P-body-like granule.
Dominique Weil (00:54:43):
So, if I summarize all that, in human, there are large variation of GC content in the transcriptome. The RNA which are GC-rich, they are very highly translated, very efficiently translated. They bind a set of specific factors, mostly related to mRNA decay pathways. They are decayed, possibly involving the 5′ exonuclease XRN1 with the help of the DDX6 helicase. It’s a helicase, so that would be useful to unfold a GC rich RNA. The ones which are AU-rich, they are poorly translated. They bind a bunch of other factors that like AU-rich motif. The decay is depending on PAT1B. We speculate that it’s from the 3′, but we have no direct evidence of that. The translation repression depends on DDX6, and they are the ones that accumulate in P-bodies. This effect of the GC content is due to the GC content of the CDS, probably through translation efficiency. But also, to some extent, the GC content of the 3′ UTR may be through its factor binding capacity.
Dominique Weil (00:56:07):
And lastly, I just want to add this comment. So, I was thinking that this type of model could be particularly relevant for haplo-insufficient genes, which are genes which, by definition, have limited capacity of producing proteins. So, there are some papers that I gave of a genome-widely. Some probability of being haplo-insufficient. So, I used that and here is a presentation that the RNA in P-bodies, in red, that they tend to have much higher proportion of genes with high probability to be haplo-insufficient than the ones which are excluded from P-bodies. So, probably that many of these regulations apply to the RNA which come from haplo-insufficient genes.
Dominique Weil (00:57:04):
And so, to conclude, just the people I work with, who have been involved with this work. So, these are the people of my group, particularly Maite Courel, who was involved in the polysome profiling experiment. Michele Ernoult-Lange, Marianne Benard, in the immunofluorescence data. Michel Kress in some bioinformatic analysis. Arnaud Hubstenberger, who was the first actor of the P-body purification, who now has is own group in Nice. Racha Chousaib, who initiated the single FISH molecule experiment, and then we have plenty of collaboration with platform but also for electron microscopy, Gerard Pierron’s group. All the values datasets of the silencing from Nancy Standart’s group, Patrick Brest’s group, Antonin Morillon’s group. And for codon usage analysis, the Hugues Roest Crollius group. And I thank you for your attention.
Thank you very much. You’ve covered so much. Very interesting.
Dominique Weil (00:58:09):
Maybe too much. Maybe too much.
No, no, no. It’s great. It’s phenomenal. I mean, I could ask a lot of questions, but I’m sure many other people have questions. I’ll just ask one. I’m really interested in the idea that different types of condensates may use different helicases.
Dominique Weil (00:58:27):
So, your particular work on DDX6, how much is that sort of teaching us a more generalizable sort of lesson about-
Dominique Weil (00:58:43):
My first answer would be that… There are opinions that say it [can/cannot? inaudible 00:58:43] be done. First of all, in our mass spec analysis of the profiled P-bodies, we noted that there is another representation of helicases. So, we didn’t test all of them. We tested DDX3… No, DDX3. DDX3, we silenced it. This is not required for making P-bodies, so there is another representation of helicase in there, but it’s very particular to DDX6 to be required for assembling P-bodies. This is one thing. There may be some others we didn’t try. I mean, there are many, many different DDX in the transcriptome, and we haven’t had time or money or energy or hands to test all of them.
Dominique Weil (00:59:30):
But second, it cannot be completely universal. Like even for the DDX6 in yeast, DDX6 is not completely required for making P-bodies. Yeast P-bodies, they seem to be different and you have to silence both DDX6 and PAT1B to observe the disappearance of P-bodies. So, even the function of DDX6 for P-bodies, I always insist that it is in human cells, because in yeast, it seems to be different.
Interesting. Thank you. Other questions?
Alicia Zamudio (01:00:04):
Hi. Yeah, I had a question about the FAPS purification. So, I’m wondering if in your RNA extraction, you’re able to capture micro RNAs, and whether the micro RNAs are depleted or enriched in your P-bodies?
Dominique Weil (01:00:22):
This is a very interesting issue, but unfortunately, what we purified was just a standard RNA purification protocol, so that we didn’t purify the micro RNA. And the library particularly we prepared, they were for over 200 nucleotides, so we have no idea about micro RNA. Some groups have shown by in situ hybridization that micro RNA can be present in P-bodies one by one, but it would require for us to redo complete purification, and have enough material to do micro RNA preparation and the specific library to answer this issue. So, we don’t know. We can only look at the targets.
Edgar Boczek (01:01:13):
Hey, Dominique. Super interesting talk. Thank you very much. It’s beautiful work. I was wondering, also on the FAPS method you used to purify the P-bodies. I found it very interesting that P-bodies are actually stable in the cell lysate. Have you ever investigated the material properties of the P-bodies in the cell and in the lysate? Does that change? Is it more gel-like? So, how do you explain the stability? It’s very interesting.
Dominique Weil (01:01:52):
So, yeah, the stability. I don’t explain anything. Many people said, “How is it possible to profile them by FAPS?” Because FAPS, our procedure is more or less an extreme dilution. It’s a purification by dilution, so one would expect that they should dissolve, and they didn’t dissolve. All I insist that the yield is low, is poor, so maybe some dissolved, but at least, the material that we have, we can spin it down and then put it back in the FAPS and we still have the material. So, they seem to be very resistant to dilution. I don’t know. In the FAPS procedure, there is no crosslinking. There is no particular treatment, so there is no trick. And they remain more or less intact. I don’t say intact, but all the protein expected to be there, they were there, so they [inaudible 01:02:47]
Dominique Weil (01:02:47):
The only thing I can speculate is from what I discussed with bio physician, the concentration dependent has not to be symmetrical between condensation and dissolution. So, there may be a certain concentration. After a certain threshold, it condensates, but for dissolution, that it would require, for instance, ATP. I say ATP because there has been quite a few papers saying that maybe ATP and helicase are important for condensation. And somehow, we got completely rid of ATP by our extreme dilution as well. So, maybe, and we didn’t try that… If we would add back the ATP, maybe they would dissolve. There has to be something that disappeared in the surrounding cytosol that makes them so resistant, or at least not completely concentration dependent. This is one thing.
That’s super interesting, and it also loops back to Mark’s question, right? So, ATPase driven machines like helicases or maybe chaperones like HSP70 has been shown to be important for the dynamics of stress granules. Maybe it’s conceivable that these machines are also important for keeping up the dynamics of P-bodies, and would be very interesting to study these specifics in these cells.
Dominique Weil (01:04:18):
So, there is Karsten Weis in Switzerland has performed an experiment on DDX6 in yeast, and in yeast, he’s saying that when he’s mutating the helicase function, the ATPase activity of the DDX6, then this stabilized P-bodies. They are more stable. In human, we did this type of experiment long time ago. I think we published that in 2008, so 12 years ago now. We performed several mutation of the DDX6 helicase and the complementation assays. None of them was able to assembles P-bodies, meaning we could not re-silence the endogenous DDX6 protein. And then, we transfect the cells with transcription to express various mutants of DDX6. This was based on the mutation performed for other DDX6 helicase in other systems. None of them was able to re-assemble P-bodies. While these complementation assays was working very well, if we use a wild-type protein, it works very well.
Dominique Weil (01:05:26):
So, in human, the helicase function is required for condensation. We don’t know if it’s for condensation in terms of local condensation or some upstream regulation event, which is required for the RNA to be able to condensate together, but it is required. And it seems to not be the case in yeast. And Karsten Weis has written a long review on the function of the helicase, but his observations are very restricted to yeast, at least for the DDX6. So, this very large enthusiasm about the function of helicase. Yes, there is a function of helicase that’s helicase, but I don’t know how to make generality of it, because most generalized review paper that was published recently does apply to yeast DDX6, but not at all to human DDX6.
Dominique Weil (01:06:24):
And now, just to complete, so you said do you know anything about the properties? This is very small object. It’s not like stress granules. Stress granules can do FRAP experiment very easy on them. P-bodies, they are less than 500 nanometers diameter, and personally, I was not successful in FRAPing them. Most of the case, they would just flow away and I would just lose them from the focus. There are a few papers that described some FRAP experiment in it, and they say it’s very common. Half a minute recovery, sometimes a little bit less, sometimes a little bit more, but it is in the range of that for others, and doing really accurate measurement of fusion time of things like that, I don’t think there is anyone who was successful for that.
Hi, Dominique. This is Violeta.
Dominique Weil (01:07:27):
Violeta Yu (01:07:27):
Thank you so much for being here. I’m just so excited that you gave this seminar, and it’s just fabulous work. I have a few questions. You mentioned about reversible storage in mRNAs and P-bodies. Is there anything known about those mechanisms and what that is in response to, in terms of signaling pathways?
Dominique Weil (01:07:51):
No. Not really. There are no examples of natural situation where, suddenly, a P-body disappear, except in mitosis. In mitosis, like all other condensates, the P-body completely disappear. Nevertheless, we got interested… I mean, I tried to cross our data with data of translation regulation during mitosis to look if there would be some particular increase of translation of the RNA which are found in P-bodies, and it’s not the case. So, it’s not because they are… They are obviously released from P-bodies, because P-bodies dissolve at mitosis, but the translation doesn’t resume, or at least not enough for people to have noted it when they look at mitosis-specific translation.
Dominique Weil (01:08:46):
And beside that, I am not aware of any situation that would drastically change the number of P-bodies. However, what I can imagine is that… What I imagine, in fact, if that’s for regulation, then it’s not because the object seems to be maintained all the time, but its content is maintained all the time. And what I imagine is that the content is renewing all the time, and depending on the trigger, depending on the environment in cell culture, whatever, hormones, but the object as such, the P-body as such, doesn’t look changed. I don’t know if I’m very clear in what I’m saying.
Yeah, yeah, yeah. Yeah, that the content is…
Dominique Weil (01:09:33):
Like, the RNA have to go out and in, but selectively, or it would not make sense. When I give the landscape where inside it’s mRNA that tend to encode for regulators, the cell doesn’t need all regulators at once. It needs the regulator, one after the other, so I imagine that the content change and from time to time, the cell needs some type of regulator, but not others, but globally, at middle scale, we don’t see any difference in terms of size of P-body or morphology of P-body.
I have two more questions. I think you started to touch upon… when you talked about the haplo-insufficiency, but I was interested to hear about any diseases that were P-body dysregulation is indicated. I guess, it’s too early to know.
Dominique Weil (01:10:35):
Yeah, yeah. It’s just an observation that these haplo-insufficient genes, which are haplo-insufficient, so just one version of the gene is not enough to produce enough protein. It needs two of them, so it means that they have a limited capacity of producing protein, and they correspond to the one which are in P-bodies. I thought it was interesting, even if I can’t apply it to anything particular.
Yeah. And then, the other question [inaudible 01:11:04] was kind of pointing out. So, you covered that the P-bodies contain mRNA targets, micro RNA targets. I guess it’s difficult to look at mRNAs themselves. I’m curious about mRNAs a lot, just because there’s a lot of pre-clinical data, and some clinical data now emerging in regards to using mRNAs as therapies. And so, it makes me curious about its function in the P-body, in particular because a lot of its targets are in there, and whether or not efficiency of these therapies may depend on its ability to get into the P-body. Another question I have is if mRNAs are stored in P-bodies, if they’re enriched in the P-bodies, perhaps there’s a way to manipulate the in and out of the mRNAs. So, the cells become their own source.
Dominique Weil (01:12:12):
Okay. So, manipulating the in and out… This seems very far. I don’t know. I have some difficulty to envision it. However, if you would like an RNA to go into P-bodies, tentatively I would advise first to try an AU-rich one. I would bet that the GC-rich one is not going to work, so I would try to put at least a large part of the RNA AU-rich, to make sure it goes in. Now, to control the in and out… We don’t know enough. We would first need to know why it’s going in, which is it depending on one single protein? So, on the whole dataset, I didn’t show it, but we tried to… At the beginning, we were dreaming, particularly the first author of the paper…
Dominique is frozen.
Yeah, frozen for me too.
Dominique Weil (01:13:15):
[crosstalk 01:13:15] dreaming that maybe… [inaudible 01:13:19] fine?
It’s better now, thanks.
Yeah, I can hear you now.
Dominique Weil (01:13:21):
Sorry. I think it’s my connection. So, yeah, then I repeat. At the beginning, I was hoping, and the first author of the paper was also very much hoping that they would be like a zip code, like some particular motif related to some particular protein that would be enough to drive RNA in P-bodies. So, we did a PCA analysis of the whole dataset, and we found that yes, there are proteins, that protein binding is related to the fact that they can accumulate in P-bodies, but there is no single combination. So, we didn’t go further to analyze it in detail, but there are probably many different factors and many combinations of them that can drive an RNA in P-bodies. So, as long as we don’t master that, I…
Dominique Weil (01:14:08):
I can’t imagine how we could master the in and out trafficking.
John Manteiga (01:14:21):
Dominique Weil (01:14:24):
I have a question around the changes in the translation of mRNAs, based on being in P-bodies. So, I might have missed this, but when you guys dissolved P-bodies by knocking down DDX6, did you measure translation differences there, and is that where you saw the differences?
Dominique Weil (01:14:45):
Yeah, it’s where we did… It was a polysome profiling, so then we measured to see. We quantified the RNA, the total RNA and polysomal RNA. And we say when we dissolve, then we see that the RNA that was before in P-bodies, they tend to be more translated. It’s a very limited extent, but which can… It’s like 30% increase. It’s not a huge increase, but it’s consistent with the amount that can be in P-bodies.
Yeah. I was going to say, it seems like that potentially isn’t super consistent with the fact that… Because you also found only like 15% of the RNA is in the P-body to begin with. So, if that’s the case, you could only ever expect a 15% increase, right? So, I guess, were the increases on that magnitude? I can’t remember.
Dominique Weil (01:15:41):
So, the extent of increase in polysomal RNA was consistent with the amount of RNA that is not sequestered, but in P-bodies at a certain moment.
Dominique Weil (01:15:56):
And this is not enough to make a huge regulation.
Okay. Cool. Thank you.
Dominique Weil (01:16:06):
So, this is possible, but it’s not a factor 10. It’s like a very small factor.
Yeah, so given that, I guess, do you think that’s the primary function of P-bodies then? Very fine tuned translational regulation? Or is there any other kind of [crosstalk 01:16:25]
Dominique Weil (01:16:25):
I would say it’s completely… It’s completely open. Either it’s really fine tuning, and maybe in the past… In the not very old past, but people were saying if it’s not a factor two difference, it’s not biologically relevant. I think this is wrong, so some time 20% difference make a difference. So, then it would have to be things that are very sensitive to very small differences. Or, another possibility is that what we see is only the condensation. Maybe something happened in the condensate, but then the RNA is released, but still has been marked with something that is going to impact its fate then. We still should see it when we do our silencing experiment to look at cells after dissolving the P-bodies, and we don’t see evidence of that, but maybe we don’t look at the right thing. Like, we only look at translation and decay, I mean, the abundance of RNA. Maybe we should look at modifications of RNA, or maybe we should look at… I don’t know, the exact cap, what is exact identity of the cap. Maybe something could change when it goes through the condensate. That’s one thing. Or, alternatively, maybe we don’t ask the right question at all, and what is important is not the amount in the condensate, but rather, the condensate can buffer in differences in the cytosol, in which case we just focus the wrong way.
Okay. Thank you.
That’s a subtle topic. Other questions? Well, if not, I think we’re close to time anyway, so Dominique, we just want to thank you again. Great lecture, and very much enjoyed it.
Dominique Weil (01:18:24):
Okay. We may have some other follow up questions we’ll come back to you with, if that’s okay.
Dominique Weil (01:18:29):
Of course, of course, of course.
You know how it is. Sometimes, things hit you later.
Dominique Weil (01:18:33):
Yeah. No problem. No problem.
Really appreciate it.
Dominique Weil (01:18:37):
It was nice to do my presentation. A little bit strange, but interesting.
It is. It is a little weird, but it’s still great to have the chance to connect like this, so thanks very much.
Dominique Weil (01:18:46):
Dominique Weil (01:18:47):
Take care. Okay. Bye bye.
Dominique Weil (01:18:49):