DNA circles promote yeast ageing in part through stimulating the reorganization of nuclear pore complexes
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Evaluation Summary:
This interesting study examines a potential relationship between the tethering of extrachromosomal DNA (ecDNA) to the nuclear pore complex (NPC) and its role in aging; a model is proposed whereby the nuclear basket is displaced by ecDNA anchoring, which leads to a broader remodeling of the NPC that is distinct from NPC damage. This idea is conceptually novel and will represent an important advance, although some more support for the conclusions is still needed.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Abstract
The nuclear pore complex (NPC) mediates nearly all exchanges between nucleus and cytoplasm, and in many species, it changes composition as the organism ages. However, how these changes arise and whether they contribute themselves to ageing is poorly understood. We show that SAGA-dependent attachment of DNA circles to NPCs in replicatively ageing yeast cells causes NPCs to lose their nuclear basket and cytoplasmic complexes. These NPCs were not recognized as defective by the NPC quality control machinery (SINC) and not targeted by ESCRTs. They interacted normally or more effectively with protein import and export factors but specifically lost mRNA export factors. Acetylation of Nup60 drove the displacement of basket and cytoplasmic complexes from circle-bound NPCs. Mutations preventing this remodeling extended the replicative lifespan of the cells. Thus, our data suggest that the anchorage of accumulating circles locks NPCs in a specialized state and that this process is intrinsically linked to the mechanisms by which ERCs promote ageing.
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Author Response:
Thank you for the reviews on our manuscript “Specialization of chromatin-bound nuclear pore complexes promotes yeast aging”. We were pleased to see the overall positive response on our work. Following the reviewers’ advice, we have been able to substantially improve our manuscript.
In the original submission we wrote that the composition of the NPC alters upon the attachment of extrachromosomal DNA circles in old yeast cells. Specifically, the interaction with DNA circles displaces the peripheral subunits from the core of the NPC, leaving the pores without basket and cytoplasmic complexes. We proposed that displacement was not the result of damage, but rather a regulated remodeling of the NPCs. These modifications affected the interaction with mRNA export factors specifically, without changing the residence of import …
Author Response:
Thank you for the reviews on our manuscript “Specialization of chromatin-bound nuclear pore complexes promotes yeast aging”. We were pleased to see the overall positive response on our work. Following the reviewers’ advice, we have been able to substantially improve our manuscript.
In the original submission we wrote that the composition of the NPC alters upon the attachment of extrachromosomal DNA circles in old yeast cells. Specifically, the interaction with DNA circles displaces the peripheral subunits from the core of the NPC, leaving the pores without basket and cytoplasmic complexes. We proposed that displacement was not the result of damage, but rather a regulated remodeling of the NPCs. These modifications affected the interaction with mRNA export factors specifically, without changing the residence of import factors. Mutations preventing the remodeling of the NPCs extended the lifespan of the cells. We concluded that DNA circle accumulation during aging in mother cell drive aging, at least in part, via NPC modulation.
Although the overall positive feedback, some reviewers raised concerns about the the conclusion we had drawn from it. We have addressed all these issues. In particular we have done the following:
We have additionally studied the dynamics of transport factors in the circle- bound NPCs, which are accumulated in the caps at the DNA cluster. Reviewer 2 pointed rightfully out that the displacement of certain Nups might affect the integrity of the NPC’s permeability barrier strongly, leading to a potential collapsing of the RanGTP gradient and preventing transport across the central channel of the circle-associated NPCs. Without a RanGTP gradient, transport factors will not be dissociated from the FG-Nups and ultimately getting stuck in the pores. This would lead to an accumulation of transport factors in these pores. Although we did not observe this for the majority of the transport factors in our studies, two import factors accumulated in circle-bound NPCs (Kap60 and Kap123). To investigate directly whether transport factors got immobilized in these NPC, we measured the dynamicity of these two NPC accumulated transport factors by FLIP. We observed no significant difference for the dynamics of both transport factor in pores localized in the cap at the DNA clusters compared to the NPCs in the rest of the nuclear envelop (new figure 6A-B). This data shows that the transport factor exchange in circle-bound NPCs is comparable to the ones without the association of DNA circles, located in the rest of the nuclear envelop. Thus, we assume that, although the displacement of several important Nups, the RanGTP gradient is not affected in these pores.
We have furthermore expanded our studies on whether the circle-bound NPCs are defective and recognized by the mechanism to remove damaged or misassembled NPCs or are rather remodeled via posttranslational modifications. As indicated by reviewer 3, this is indeed a challenging idea, and we do not want to stretch our claims here. We have adjusted the manuscript to explain better that we assume that the remodeling of the NPC indeed might have subsequent damaging consequences for the NPCs and the physiology of the cell. The displacement of the mRNA export factors from these NPCs are indeed indicative for a malfunction of these pores and might have drastic impact on protein synthesis rates. However, what we propose is that the displacement of the peripheral subunits itself is a regulated modulation of the NPC, important for its function to retain DNA circles in the mother cell. We have adapted the text to clarify our conclusion better and we added additional experiments to test the idea that circle-bound NPC are indeed not damaged. These new data indeed support our conclusion:
a) First, we studied the localization of additional components of the storage of improper assembled NPCs compartment (SINC) in respect to DNA circles in old mother cells. The SINC represents a quality control system that recruits the ESCRT III machinery to detect and remove defective NPCs in the nuclear envelope. These SINCs were shown to accumulate in mother cells. However, when we studied the location of the SINC components in old cells on the microfluidics chip, we did not see the SINC proteins colocalizing with DNA circle clusters (Fig 4A-C). Although damaged NPCs accumulate in old cells, our data showed no enrichment for these damaged NPCs at the DNA circle clusters. We interpreted this data that DNA circle-loaded NPCs thus are not recognized by the ESCRT III machinery as being defective.
b) We next investigated the possibility that circle-bound NPCs of old cells are recognized and targeted by the SINC. Thus, we used the fact that ERCs bind the protein Net1 to ask whether the SINC accumulates to the vicinity of ERC-bound NPCs. However, this was not the case either. These data are now shown in figure 4D-E.
c) The new data showing that the dynamics of Kap60 and Kap123 is not affected in circle-bound NPCS (see above) support as well the notion that these NPCs are dramatically defective.
d) We have added a new experiment to confirm that the NPCs of old cells are not leaky, as already observed by others (see Morlot et al., 2019; Rempel et al., 2019).
Together, the additional data did not indicate that DNA-bound NPCs in old cells show a sign of any immediate defect. This supports our initial idea that these NPCs are specialized for DNA circle retention in the mother cell. However, we acknowledge that the progressive accumulation of many of these modified NPCs can be considered to be aging-induced defect in the cell.
Finally, we discussed and studied in more detail the cause and consequence relation between DNA circle attachment and basket displacement. This concern was raised by reviewer 3, asking whether altered (i.e. defective) NPCs are more present in the old cells and that they could attract DNA circles, rather than DNA circles displace the peripheral structures from the NPC. We now discuss this point more in depth and make several points further supporting our initial conclusions. First, we have noticed in an earlier study (Denoth-Lippuner et al., Elife, 2014) that acetylation of Nup60 is required for DNA circle binding to the NPC. This speaks for a specific regulated posttranslational modification of Nup60 for DNA circle binding, induced by the circle association with the pore, considering that the circle is bound with SAGA’s acetyltransferase Gcn5. A random aging-induced alterations is unlikely to bind DNA circles in such a regulated fashion. Second, as we previously showed, DNA circles no-longer colocalize with NPCs in mlp1∆ mlp2∆ double mutant cells and DNA circle are no-longer confined to these mutant mother cells (Shcheprova Z. et all, Nature 454:728–734). This implies that NPCs without the basket cannot attach to DNA circles anymore. How this mechanistically works is up for further investigation, but it at least indicates that the basket is involved in the interaction of circles at the NPCs. Thus, our observation that the basket is displaced from circle-bound NPCs indicate that this displacement is subsequent to circle-binding. Likewise, the Nups Nup82 and Nup159 have been recently shown to require Nup116 for their recruitment to the NPC. The fact that these Nups are not displaced from circle-bound NPCs but Nup116 is argue for Nup116 being displaced upon circle binding rather than being absent in the first place. Accordingly, we show that preventing Nup60 acetylation, which our data identify as a target of SAGA upon circle binding, restores Nup116 localization. Thus, Nup116 displacement seems to be a consequence of Nup60 acetylation upon circle anchorage to NPCs. Thus, the most parsimonious hypothesis for explaining these different observations is that DNA circle anchoring to the NPC core drives the displacement of peripheral subunits, starting by the nuclear basket.
We thank the reviewers for their valuable comments on our manuscript. We believe that we have covered all the concerns raised.
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Evaluation Summary:
This interesting study examines a potential relationship between the tethering of extrachromosomal DNA (ecDNA) to the nuclear pore complex (NPC) and its role in aging; a model is proposed whereby the nuclear basket is displaced by ecDNA anchoring, which leads to a broader remodeling of the NPC that is distinct from NPC damage. This idea is conceptually novel and will represent an important advance, although some more support for the conclusions is still needed.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Nuclear Pore Complexes (NPCs) controlling the trafficking between nucleus and cytoplasm are known to under changes during aging. However, their underlying cause and association with aging factors were unclear. In this manuscript, the authors investigated the link between a well-known aging factor in yeast, rDNA circles, and age-related NPC changes. They found that 1) NPC-bound DNA circles displace cytoplasmic components and nuclear baskets of the NPC in aged cells; 2) these remodels NPCs are not recognized by defective and not specifically limited to old NPCs; 3) NPC remodeling is caused by acetylation of NPC basket component; 4) remodeled NPCs are a cause of aging; 5) remodeled NPCs specifically affect the mRNA export process.
The experimental systems were well designed to address their questions. The data …
Reviewer #1 (Public Review):
Nuclear Pore Complexes (NPCs) controlling the trafficking between nucleus and cytoplasm are known to under changes during aging. However, their underlying cause and association with aging factors were unclear. In this manuscript, the authors investigated the link between a well-known aging factor in yeast, rDNA circles, and age-related NPC changes. They found that 1) NPC-bound DNA circles displace cytoplasmic components and nuclear baskets of the NPC in aged cells; 2) these remodels NPCs are not recognized by defective and not specifically limited to old NPCs; 3) NPC remodeling is caused by acetylation of NPC basket component; 4) remodeled NPCs are a cause of aging; 5) remodeled NPCs specifically affect the mRNA export process.
The experimental systems were well designed to address their questions. The data presented are of high quality in general and support their major conclusions. This manuscript clearly links NPC remodeling and dysfunction during aging to DNA circles known to accumulate during yeast aging. Importantly, they also demonstrated some of the mechanistic details of this process: specific NPC components being displaced due to DNA circle binding; regulation of this process by protein acetylation; and the specific functions of NPCs being affected. Hence, this manuscript has made a significant progress toward our understanding of mechanism of action by a well-known aging factor and should be of interest to researchers studying yeast and cellular aging, nuclear pore function and aging, as well as those interested in the functions of protein acetyltransferase SAGA.
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Reviewer #2 (Public Review):
Meinema et al. investigated the poorly understood mechanism of how extrachromosomal rDNA circles (ERCs) promote aging in yeast cells. ERCs are well-established aging factors that are derived from ribosomal DNA (rDNA) repeats cut out of the genome during aging. Notably, ERCs are known to associate with nuclear pore complexes (NPCs) via the nuclear basket, yet how this interaction affects NPC architecture and function remains unclear.
By combining a previously-established ERC reporter assay, a microfluidic yeast aging platform, and quantitative co-localization imaging, Meinema et al. show that tethering of NPCs to ERCs displaces the key nuclear basket component Mlp1, Mlp2, Nup60 and Nup2. This finding is in agreement with previous proteomics data. Notably, the authors convincingly show that tethering to ERCs …
Reviewer #2 (Public Review):
Meinema et al. investigated the poorly understood mechanism of how extrachromosomal rDNA circles (ERCs) promote aging in yeast cells. ERCs are well-established aging factors that are derived from ribosomal DNA (rDNA) repeats cut out of the genome during aging. Notably, ERCs are known to associate with nuclear pore complexes (NPCs) via the nuclear basket, yet how this interaction affects NPC architecture and function remains unclear.
By combining a previously-established ERC reporter assay, a microfluidic yeast aging platform, and quantitative co-localization imaging, Meinema et al. show that tethering of NPCs to ERCs displaces the key nuclear basket component Mlp1, Mlp2, Nup60 and Nup2. This finding is in agreement with previous proteomics data. Notably, the authors convincingly show that tethering to ERCs is necessary for NPC remodeling, as aging alone is not sufficient to cause basket loss. Further arguing a against damage through aging model is the finding that basket-less NPCs are not recognized as defective by the ESCRT III quality control machinery. Yet, NPC remodeling is not without consequences, as the authors show that a number of transport factors involved in mRNA export showed reduced interaction with ERC-tethered, basket-less NPCs. A strength of the paper is the clever use of yeast genetics and mutagenesis to systematically perturb the interaction between ERCs and NPCs. This allowed the authors to identify that acetylation of basket components by the SAGA complex drives their displacement from ERC-bound NPCs.
Taken together, the authors conclude from their findings that ERC binding results in specialized NPCs with reduced mRNA export capacity, and that this NPC specialization promotes aging. However, the authors do not yet test this model rigorously enough to draw such firm conclusions and exclude the possibility of NPC damage-driven aging. In particular, the ERC-induced loss of FG nucleoporins such as Nup116, Nup42 and Nsp1 raises concerns about the integrity of the NPC permeability barrier. This bears the risk of collapsing the RanGTP gradient that fuels all nucleocytoplasmic transport, and thus have far-reaching consequences. Similarly, the paper would benefit from clarifying if the nuclear transport receptors Kap60 and Kap123 (and their cargoes) get stuck in ERC-tethered NPCs or pass more efficiently, as this is central to distinguish between specialized and defective NPCs.
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Reviewer #3 (Public Review):
This study by Meinema et al. explores changes to nuclear pore complex (NPC) composition associated with extrachromosomal DNA (ecDNA) anchoring with aging in budding yeast. The major new concept is that NPC remodeling driven by the displacement of the nuclear basket by ecDNA and concomitant loss of the cytosolic mRNA platform, is an example of NPC specialization, which may also be detrimental to cells and drive aging. This somewhat challenges the idea espoused by others that NPC damage might contribute to aging - an idea that the authors try to rule out. In general, the data are of good quality but the cause and effect relationship between the tethering of DNA circles, SAGA, acetylation of Nup60 and the loss of cytosolic nups remains uncertain. There is little direct data presented, for example, that DNA …
Reviewer #3 (Public Review):
This study by Meinema et al. explores changes to nuclear pore complex (NPC) composition associated with extrachromosomal DNA (ecDNA) anchoring with aging in budding yeast. The major new concept is that NPC remodeling driven by the displacement of the nuclear basket by ecDNA and concomitant loss of the cytosolic mRNA platform, is an example of NPC specialization, which may also be detrimental to cells and drive aging. This somewhat challenges the idea espoused by others that NPC damage might contribute to aging - an idea that the authors try to rule out. In general, the data are of good quality but the cause and effect relationship between the tethering of DNA circles, SAGA, acetylation of Nup60 and the loss of cytosolic nups remains uncertain. There is little direct data presented, for example, that DNA circles "displace the nuclear basket" and alternative explanations are not considered.
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