In this review, we highlight major recent advances in the field, with special emphasis on the potential link between m6A modifications and RNA structure. We will also discuss the role of RNA methylation of neuronal transcripts, and the emerging evidence of a potential role in RNA transport and local translation in dendrites and axons of transcripts involved in synaptic functions and axon growth.Reversible N6-methyladenosine (m Instances in which data are still not available will also be mentioned. Finally, we will detail evidence supporting the hypothesis that RNA modifications play a pivotal role in regulating mRNA transport, translation, and degradation in the nervous system.In this review, we highlight the latest research that provides insights into how m26A is regulated by three groups of proteins. Writers, which add the modification to mRNA, are found in the nucleus and therefore fall outside of the scope of this review. For more information on writers, we direct the reader to two recent reviews by He and Meyer , and ribosome profiling, indicated that YTHDF1 and YTHDF3 both bind to ribosomal subunit proteins with high efficiency (>70% of 40 S and 60 S proteins). Knockdown of either protein severely reduces the translational efficiency of their common targets, but knockdown of YTHDF3 only does not affect the translation of its unique binding targets, perhaps suggesting a different role of YTHDF3 in the absence of functional interaction with other readers  of mRNAs can be translated independently of the 5′ cap , mass spectrometry, and electron microscopy suggested that 5′ cap-independent translation is mediated by an alternative function of the m6A writer protein METTL3. During m6A addition to the mRNA, METTL3 binds to the eIF3h subunit and circularises the mRNA molecule to bring eIF3 closer to the translation initiation site, allowing the complex to recruit both YTHDF proteins and ribosomes  is a biological phenomenon by which components of similar hydrophobic characteristics accumulate and form highly concentrated dynamic condensates, such as granules and membrane-less intracellular structures. Stress granules, ribonucleoprotein complexes, and processing bodies in neurons are all found within LLPS , and in 6A on phase separation has been investigated. In cell lines, multi-modified, but not singly-modified mRNAs promote phase separation when bound to YTHDF proteins 6A reader6A methylation plays a key role in activity-dependent, temporal control of localised mRNA in neurons, although further studies are needed to reveal the additional mechanisms involved.Collectively, these data suggest that mRNA methylation may influence the binding, translation, and decay of localised transcripts. It is also clear that m46A modifications, it is not surprising that changes of mRNA methylation have been involved in several human diseases. Alterations of m6A levels have been observed in a variety of human disorders, from obesity  , 4-thiouridine (4SU) is incorporated into the mRNA  is an adaptation of the iCLIP methodology . When cells are transfected with APOBEC1-YTH C to U deamination is induced in sites adjacent to m6A residues. The most important innovation of this technique is the low amount of input RNA (10 ng) that can be used to map RNA modifications. All current methods of m6A mapping analyse data from cell populations. The low amount of RNA required for DART-seq allowed the development of a more advanced method for single-cell sequencing named the scDART-seq CA motif intact  with the allyl  modifications instead of m6A. The mRNA is isolated and fragmented from the treated with Se allyl- L-selenohomocysteine cells followed by antibody immunoprecipitation of a6A containing mRNA  , it willnd axons . We now Braulio Martinez De La Cruz: Conceptualization, Writing – original draft, Writing – review & editing. Marousa Darsinou: Conceptualization, Writing – original draft, Writing – review & editing. Antonella Riccio: Conceptualization, Supervision, Funding acquisition, Writing – original draft, Writing – review & editing.


Acropora palmata, if successful, could mitigate the most extreme impacts of coastal flooding by reversing projected trajectories of reef erosion and allowing reefs to keep pace with the ~0.5 m of sea-level rise expected by 2100 with moderate carbon-emissions reductions. This highlights the potential long-term benefits of pursuing coral-reef restoration alongside climate-change mitigation to support the persistence of essential coral-reef ecosystem services.The ability of reefs to protect coastlines from storm-driven flooding hinges on their capacity to keep pace with sea-level rise. Here, we show how and whether coral restoration could achieve the often-cited goal of reversing the impacts of coral-reef degradation to preserve this essential function. We combined coral-growth measurements and carbonate-budget assessments of reef-accretion potential at Buck Island Reef, U.S. Virgin Islands, with hydrodynamic modeling to quantify future coastal flooding under various coral-restoration, sea-level rise, and storm scenarios. Our results provide guidance on how restoration of  The capacity of coral reefs to keep pace with sea-level rise is central to their ability to continue to provide shoreline protection to vulnerable coastal communities. Here, the study shows that whereas restoration has the potential to minimize climate-change impacts, doing nothing will amplify them. Widespread losses of reef-building corals as a result of climate change and other anthropogenic impacts13 have decreased reef elevation14, reduced topographic complexity15, and diminshed the capacity for reef accretion to keep pace with sea-level rise16. Reefs whose vertical accretion lags rising sea level may no longer maintain their role as protective barriers, because dissipation of wave energy decreases as water depths increase7.Coral reefs provide critical barriers that can mitigate shoreline erosion and flooding for tropical coastal communities around the world9. The role of the reef crest is especially important in the western Atlantic, where the broad, shallow reef flats that help dissipate waves in the Indo-Pacific are generally absent17. Historically, reef crests in the western Atlantic were dominated by Acropora palmata, a species whose robust morphology and rapid growth made it the most important reef-builder in the region for hundreds of thousands of years19. Over the last half century, however, the combined impacts of coral disease and thermal stress have decimated A. palmata populations20. The unprecedented loss of this ecosystem engineer has fundamentally changed the structure and function of many western Atlantic reefs18; reef-crest habitats that once reached sea level are now significantly flatter and deeper in many locations15.The shallow reef-crest zone is responsible for the majority of wave dissipation over coral reefs and is, therefore, the most important habitat for shoreline protection3 and for developing effective coral-reef management strategies22. Many coral-restoration efforts are now reaching a scale at which they are expected to have positive impacts on reef function and ecosystem services such as shoreline protection23; however, the ability of coral restoration to achieve those broader goals has yet to be demonstrated.For coral-reef-lined coasts, understanding the changing nature of the reef-building process, particularly in reef-crest habitats, is essential for estimating future risks from coastal hazards under sea-level rise21. Carbonate budgets can also be used to evaluate how management activities such as coral restoration could help reverse declines in reef building and support ecosystem function22; however, because of the difficulty of surveying the high-energy reef crest, few carbonate-budget studies have included this critical habitat16. Similarly, whereas hydrodynamic modeling studies have shown how increasing water depth over reefs can amplify coastal flooding in theory8, including one recent analysis at our study area, Buck Island Reef National Monument (BIRNM)24, to date no studies have combined these approaches to explore how ongoing changes in reef-accretion capacity could affect reefs’ ability to provide coastal protection with and without management intervention.Carbonate-budget studies provide a simplified model of the complex processes controlling the modern accretion-erosion balance and a means for estimating how reef accretion and, therefore, water depth, could change in response to the combined impacts of reef degradation and sea-level rise in the future25. Buck Island is surrounded by a narrow, emergent barrier reef approximately 10-m wide, enclosing a 1- to 4-m deep lagoon. Since the mid-1950s, shoreline erosion on the northwest side of Buck Island has reduced beach habitat26 that has cultural and historical significance, supports the island’s active tourism industry27, and provides nesting habitat for endangered sea turtles28. Shoreline change coincided with dramatic degradation of reef ecosystems surrounding the island29, but the potential contribution of reef degradation to shoreline erosion has yet to be evaluated.We present a real-world example of the impacts of reef degradation on coastal flooding using data collected at BIRNM, located off the northeastern coast of St. Croix, U.S. Virgin Islands Fig. , in the A. palmata growth in combination with the carbonate-budget models to examine whether and how restoration of A. palmata onto the shallow reef crests at BIRNM could help close the gap between the reef and rising sea level to mitigate coastal flooding impacts in the future. Finally, we use hydrodynamic modeling to determine how projected changes in bathymetry from both reef erosion and restoration, in combination with sea-level rise, could impact coastal flooding potential during storms by 2100. We demonstrate that although sea-level rise will be the primary driver of coastal flooding, reef erosion will result in a small, but measurable increase in flooding potential by the end of the century. Our study also provides insight into how successful coral-reef restoration efforts initiated within the present decade could, in combination with larger-scale climate-change mitigation, reduce the impacts of sea-level rise and wave-driven water levels on coral-reef-lined coasts like BIRNM.Here, we first quantify spatial variability in reef-accretion potential—a measure of the maximum capacity for the vertical accretion of a reef—using census-based carbonate-budget models. We then use in situ measurements of −1 and averaged -1.56 mm y−1 (±0.27 standard error [SE]) across 54 sites within shallow fore-reef, reef-crest, and back-reef habitats  around BIRNM . As a result, reef-accretion potential in the southern sector was significantly higher than in the northern sector . Average projected elevation change of the southern reef crest by 2100 was negligible at +1 cm (±10 cm). In contrast, bioerosion was the dominant process at 91% of sites, with estimated decreases in elevation due to reef erosion averaging -13.09 (±0.02) cm by 2100 . The relatively higher reef-accretion potential on the southern reef crest was instead a result of significantly higher coral cover . Pseudodiploria spp., A. palmata, Orbicella spp., Millepora spp., and Porites astreoides were the most significant contributors to carbonate production at BIRNM , with higher production by A. palmata, Pseudodiploria spp., and Millepora spp. on the southern reef crest . The average rate of change in the height of A. palmata colonies was 6.99 cm y−1 , with average planar surface area of the colonies increasing by 196.21 cm2 y−1 . Although 14 of the 30 A. palmata colonies included in the experiment died within days of transplantation due to the occurrence of an early thermal-stress event at BIRNM during the experimental set-up, only one colony died in the subsequent two years of the experiment.During the coral-growth experiment we conducted at BIRMN from 2019 to 2021, calcification rates of Ps. strigosa colonies in our experiment ranged from 7.96 to 21.31 kg m−2 y−1, with an average of 14.30 kg m−2 y−1 (±0.86). The average annual change in height of Ps. strigosa was 0.95 cm y−1  and its change in planar surface area averaged 12.88 cm2 y−1 . Survival of Ps. strigosa was high throughout the experiment with only four colonies experiencing complete mortality and an additional four experiencing partial mortality. Stony coral tissue-loss disease (SCTLD) was just starting to impact some colonies during the last time interval (see photographs in30). Overall, although there was some variability in coral growth across space and time  based on average, planar growth rates of A. palmata from our study and literature-derived mortality and fragmentation rates to project potential changes in A. palmata cover on the BIRNM reef crest  following outplanting of 100,000 (0.5 m−2), 250,000 (1.5 m−2), and 500,000 (3 m−2) colonies, respectively ; however, in more optimistic scenarios , A. palmata cover is projected to remain elevated by more than 10% to 2100. The best outcome in our model was for the reduced-mortality scenario with 500,000 initial outplants, for which a >30% increase in A. palmata cover was projected to persist to 2100.To evaluate how coral restoration could harness the rapid growth of est Fig.  under vaely Fig. . After 2A. palmata cover above the 2016 baseline coral cover of 6.14% ± 1.40 and 14.86% ± 4.03  in the northern and southern sectors of the BIRMN reef crest, respectively, would bring total coral cover in those zones to ~36 and 45%. Under that scenario, our carbonate-budget models indicate that restoration could increase reef-accretion potential on the BIRNM reef crest to 7.38 mm y−1 (±0.60) on average . At that rate, the reef crest could be capable of as much as 0.25 m (±0.02) of vertical accretion on average by 2050 and 0.62 m (±0.05) of vertical accretion by 2100 , which would be sufficient to allow the reef crest throughout BIRNM to keep pace with Low to Intermediate-Low predictions of sea-level rise for the mid- and end-of-century31  and 4.94 m (±0.02) under the 50-year storm scenario . Maximum total water levels vary along the shoreline due to variability in nearshore bathymetry and its subsequent effects on waves and wave-driven water levels; however, the alongshore variation declines as sea level increases, because greater water depth results in relatively lower bathymetric variability on the reef that, in turn, causes lower frictional wave dissipation. The overall trends were similar when projected changes in reef elevation were incorporated into the model  nearshore. Whereas the mean infragravity wave  heights are less affected by sea-level rise, maximum values are more sensitive Fig. . TogetheA. palmata cover to ≥36% total cover) could promote reef accretion and help narrow the gap between reef elevation and sea level by more than 0.5 m on average by 2100  kg CaCO3 m−2 y−1. Bythell et al.29 hypothesized that the loss of A. palmata may have been sufficient to halt reef accretion at BIRNM, and our study supports that conclusion.The capacity of coral reefs to keep pace with sea-level rise is central to their ability to continue to provide shoreline protection to coastal communities in the future−1  and the contemporary western Atlantic reef-accretion potential average of 1.87 mm y−1 (ref. 16), erosion had become the dominant process on most of the reef, with reef-accretion potential averaging -1.56 mm y−1. This rate is only slightly lower than the erosion rate of -2.7 mm y−1 that Yates et al.14 estimated from landscape-scale changes in seafloor elevation around Buck Island from 1981–2014, and is comparable to the highest rates of net erosion estimated for other reefs in the western Atlantic40. The few sites in our study that had positive carbonate budgets all had coral cover >16% and were dominated by A. palmata and/or the massive reef-building corals Ps. strigosa and Orbicella spp. . This difference is primarily due to the relatively high rate of parrotfish bioerosion estimated in our study, as well as the higher macrobioerosion rates incorporated in our budgets based on in situ measurements of macrobioerosion by Whitcher43 (cf.37). It is possible that parrotfish bioerosion was overestimated in our study because our carbonate budgets incorporated the relatively high parrotfish bioerosion rates suggested by the ReefBudget v1 methodology44 (see Methods); however, BIRNM is also one of the oldest no-take marine reserves in the western Atlantic (established in 1961), which has resulted in elevated parrotfish biomass there compared with nearby locations open to fishing45. Because many parrotfish scrape and excavate the reef structure as they graze, protection of these key herbivores could have the unintended consequence of elevating reef bioerosion12.We found that by the time of our surveys in 2016, not only was reef-accretion potential at BIRNM well below the average regional Holocene baseline accretion rate of 3.09 mm ypp. Fig. , highligA. palmata and Ps. strigosa there . This difference would cause alongshore gradients in waves and wave-driven water levels at the east and west ends of the island, likely resulting in net erosion and sediment transport to the south46. Indeed, since 1954, the primary beach habitat on the western side of the island—which supports important cultural and ecological resources such as critical nesting habitat for endangered sea turtles28—has migrated to the south, and nearly 30% (0.88 ha) of the total beach area has been lost due to storm-driven erosion26. For coral-reef-lined coasts fronted with wider shallow-water reefs than the bank-barrier reef at BIRNM, reef erosion would have an even greater impact on the flooding potential47.Overall, we found that only one habitat—the southern reef crest—exhibited marginally positive reef-accretion potential, as a result of the relatively high abundances of ere Fig. . Whereasere Fig. . We foun12. Indeed, just one year after our surveys were completed, Hurricane Maria passed ~60 km west of BIRNM as a Category-5 storm and caused significant damage to southern reef habitats. Many of the large A. palmata colonies that were responsible for the southern reef crest’s rapid rate of carbonate production —in modulating long-term reef accretion48. Most recently, in 2021, SCTLD began to impact the reef at BIRNM30. Acroporids are not susceptible to SCTLD, but the disease has had significant impacts on Pseudodiploria and Orbicella spp. , which were dominant in many reef habitats at the time of our surveys . By increasing reef complexity and minimizing the divergence between the elevation of the reef crest and sea level, restoration of a living barrier of A. palmata colonies could have the near-term effect of increasing wave breaking across the reef crest7. This could prevent additional wave-driven increases in water levels, and, therefore, help mitigate coastal flooding49. In the longer-term, the ability of restoration to continue to support coastal protection will depend on its impact not just on coral growth, but on the process of reef accretion.Increasing investments in expanding the scale of coral restoration in recent years23, our study provides important guidance on what it would take for short-term restoration efforts now to have a sustained impact on coastal protection in the long-term. Our models indicate that, if successful, a significant effort to restore A. palmata populations at BIRNM in the near-term  could result in substantial and sustained increases in coral cover and, therefore, reef-accretion potential until the end of the century, even in the absence of sexual reproduction , can be realized, our carbonate-budget models indicate that reef-accretion potential of the reef crest at BIRNM could exceed 5 mm y−1 , which could occur under the highest CO2 emissions scenarios31 . At present, however, the most probable magnitude of end-of-century sea-level rise across all CO2-emission scenarios is between ~0.5 and 1 m 31. Our models indicate that under that scenario significant and successful restoration  could allow the BIRNM reef crest to keep pace with sea-level rise to 2100  and estimated historic reef-accretion potential based on the maximum observed A. palmata cover there in the 1970s of 62%35 (see Methods). That 40% or 3.08 mm y−1 offset is three times higher than what was estimated for reefs in south Florida22, likely because of differences in methods for estimating reef-accretion potential. Although this result supports the conclusion that reef-accretion potential almost certainly overestimates millennial-scale reef accretion48, geological estimates of reef accretion, based on limited dating of reef-core records, also necessarily underestimate reef accretion over the shorter, multi-decadal timescales that are most relevant for coral-reef management. Given both the uncertainties in reef-accretion potential and in the overall efficacy of large-scale restoration amid a regime of ongoing disturbance, it is likely that the most realistic outcome will likely lie somewhere in between these two end members.To estimate how the omission of non-biological erosion could affect our results, we followed the approach of Toth et al.A. palmata  on the BIRNM reef crest, then the reef could have the potential to keep pace with the low-end sea-level projection (~0.36 m) expected with moderate emissions reductions31. Under more pessimistic sea-level-rise scenarios, following impacts of major pulse disturbances like storms, or if the impacts of climate change on coral reefs increase rather than abate over time, then additional restoration efforts would likely be necessary for reefs to provide optimal coastal protection. One possibility in this case would be the adoption of a hybrid restoration approach wherein engineered structures could provide a baseline increase in reef elevation upon which coral restoration can be initiated59.Importantly, in our projections, the possibility of even a 40% reduction in realized reef accretion does not change our overall conclusion that if restoration can produce a sustained increase of 30% cover of 23. Bayraktarov et al.60 found that the median cost of restoration projects globally was ~$400,000 in U.S. dollars per hectare (USD ha−1), which equates to ~$6.6 million USD to restore an area the size of the reef crest at BIRNM . At that rate, increasing A. palmata cover on a reef the size of the one at Buck Island to a level that could significantly impact shoreline protection  would cost ~$12.5 million USD. By bolstering the ability of reefs to provide coastal protection, the high up-front costs of restoration could be balanced by both the direct value of restoration to local communities and economies  and the millions of dollars of damage to coastal infrastructure that could be avoided and the people in vulnerable communities that could be protected from flooding49. With limited resources, local restoration efforts could also be strategically placed to protect the most vulnerable human communities and infrastructure, or critical natural, cultural, and historic resources.Our study demonstrates how restoration could help to mitigate reef degradation and coastal flooding in locations like BIRNM. It also supports the conclusion that for restoration to move beyond short-term enhancement of coral populations and achieve a meaningful impact on reefs’ long-term accretion function, will require a substantial investment of resources in the near-term40 seem trivial; however, realized erosion rates may already be higher than our study suggests and will likely be higher going forward because of projected increases in the frequency of severe thermal-stress events and storms, and the impacts of ocean acidification, as climate change accelerates39. Furthermore, even the relatively low rates of maximum erosion estimated in our study (0.45 m by 2100) could almost double the realized water levels over the reef crest at BIRNM by 2100 under SSP1-2.6 to SSP2-4.5 (~0.49 m) to a level comparable to the highest sea-level rise predicted under SSP5-8.5 (0.96 m31). Based on our models, this would result in a projected ~0.2 m increase in wave-driven water levels during storms , preventing wave-driven increases in coastal flooding during storms, even under pessimistic emissions-reduction scenarios. Additionally, every area of reef restored not only contributes to positive accretion, but it also protects the reef structure with a cap of living coral, staving off erosion13. Restoration can, therefore, work on both sides of the carbonate-budget equation to close the gap between rising sea levels and the reef surface. Put another way, whereas coral restoration has the potential to minimize climate-change impacts, doing nothing to combat erosion would amplify them.The realities of the high cost of coral restoration are often met with pessimism over the prospects for long-term restoration efficacy and return on investment, but the alternative—allowing coral-reef degradation to continue—would also come at a cost. At just ~0.2 m on average, the conservatively low estimates of net erosion predicted by the end of the century in our study and other carbonate-budget assessments from the western Atlanticrms Fig. . On the 3. Coastal communities and decision-makers will be faced with increasingly difficult choices when allocating limited resources to address the growing array of urgent climate-change impacts, and large investments in coral restoration would have to be weighed against other priorities. Our study helps to support that cost-benefit analysis by providing new guidance on the potential scale of restoration necessary to revive reef accretion and maintain reefs’ capacity for coastal protection. The success of restoration will ultimately depend on whether ongoing coral mortality, and its underlying causes, can be mitigated. Nonetheless, our results support the conclusion that if restoration is successful, there is currently a window of opportunity during which substantial restoration efforts in the near term could help combat the worst projected impacts of sea-level rise and resulting coastal-flooding risk by the end of the century, whereas allowing reef erosion to continue unchecked could counteract progress from emissions reductions. We conclude that restoration has the potential to be more than a stop-gap measure: it can provide an important component of the portfolio of solutions that communities and managers can draw from to support the persistence of coral reefs and their function as coastal barriers into the future.Coastal flooding and erosion due to extreme weather events have significant impacts on coastal communities globally, and those impacts are projected to increase as sea-level rises over the coming centuryn = 18 sites in each habitat; Fig. n = 27 sites in each sector) of BIRNM. For each site, carbonate budgets were estimated by analyzing reef-census data collected in July 2016 (ref. 61), following a modification of Hubbard et al.37 and the ReefBudget v1 methodology44.We calculated the carbonate budgets of 54 stratified-random sites within shallow fore-reef, reef-crest, and back-reef habitats  to quantify the percent cover of benthic calcifiers  and total consolidated substratum. Gross carbonate production (kg CaCO3 m−2 y−1) was estimated by calculating the product of percent cover of each calcifying taxon, taxon-specific calcification rates from ReefBudget v144, and, for corals, taxon-specific rugosity correction-factors (Table 62). Carbonate production rates for all calcifying taxa were summed for each transect.To estimate carbonate production, we collected photographs of one 10 × 1 m benthic transect at each site and conducted point-count analysis  using the program CoralNet . Those rates were multiplied by the densities of parrotfish of each species, size class, and life phase observed within each transect, summed within transect, and averaged between the two transects to estimate total parrotfish bioerosion at each site (kg CaCO3 m−2 y−1).To estimate parrotfish bioerosion, we recorded the number, size classes (in 15-mm bins), and life phases  for each of seven bioeroding parrotfish species—44 Table  to calcuEchinometra lucunter, Ec. viridis, Diadema antillarum, and Eucidaris tribuloides and recorded their test sizes within 20-mm bins along the same 10 × 1 m transects used for the benthic surveys to estimate size-specific densities of each species. Sea-urchin bioerosion (kg CaCO3 m−2 y−1) was calculated by multiplying the urchin densities by size-specific bioerosion rates derived from the generalized relationship between urchin test size and bioerosion rate described in the ReefBudget v1 methodology44  by multiplying the percent available substrate by the microbioerosion rate of 0.27 kg m−2 y−1 derived by Vogel64 in the Bahamas . Total bioerosion at each site was estimated as the sum of bioerosion by parrotfish, sea urchins, and endolithic macro- and microbioeroders.We estimated site-level endolithic macrobioerosion  by multiplying the available substratum for endolithic bioerosion, defined here as the percent cover of dead coral substratum from our benthic surveys, by the average macrobioerosion rate of 0.4 kg m3 m−2 y−1) was estimated by subtracting bioerosion from gross carbonate production at each site and was converted to estimates of reef-accretion potential using information on local reef-framework density and porosity34. For sites with positive net carbonate production, we estimated reef-framework density as the weighted average of the densities of coral taxa at the site according to the following equation:Di is the weighted mean density of coral framework at site i (kg m−3), xn is the percent cover of coral species n at site i, Xi is the percent cover of total live coral at site i, dn is the density of coral species n (kg m−3). For net erosional sites we used the average Caribbean coral density value of 1670 kg m−3. Estimated vertical reef-framework accretion (or erosion) potential was calculated by dividing net carbonate production by estimated framework density. For sites with positive net carbonate production, we also accounted for the contribution of unconsolidated sediments and void space to total reef accretion using data from Holocene reef cores collected by Hubbard et al.34 at BIRNM according to the following equation:Ai is the reef-accretion potential at site i (mm y−1), Fi is the rate of framework accretion at site i (mm y−1), sed is the mean percentage of linear depth within the cores that was composed of sediment, void is the mean percentage of linear depth within the cores composed of void space, and frame is the mean percentage of linear depth within the cores composed of in-place coral framework and rubble. Based on Hubbard et al.34sed was set at 33%, void was set at 18%, and frame was set at 49%. The assumed rate of sediment reincorporation at our sites was validated by Whitcher66 who independently quantified the rate of sediment production from bioerosion at BIRNM. We emphasize that reef-accretion potential quantified in our study is likely a maximum estimate of realized reef accretion, as carbonate budget models do not incorporate the impacts of physical erosion or chemical dissolution48.Net carbonate production  since this study was conducted, and the updated method would produce some small changes in our estimated budgets; however, because the hydrodynamic models were run using bathymetries created using the budgets we calculated with ReefBudget v1, we did not update the budget calculations here. One of the most substantial changes to the method is that ReefBudget v2 no longer considers bioerosion by juvenile (<10 cm fork length) parrotfish and incorporates substantially lower bioerosion rates for Sp. aurofrenatum, Sp. rubripinne, and Sp. chrysopterum, as these fish may not contribute substantially to bioerosion65. It is possible, therefore, that we overestimated bioerosion by those taxa, but because they were substantially less common in our surveys than the more important bioeroding parrotfish, Sp. viride, their contribution to overall rates of parrotfish bioerosion in our study was relatively minor , those data were collected after the carbonate budget and hydrodynamic modeling for this study was completed, so we were not able to incorporate them into the 2016 carbonate-budget models; however, the range of local calcification rates (see Results) was similar to the regional estimates we derived from ReefBudget v144, so the use of regional rather than local calcification data is likely not a significant source of uncertainty in the models.The ReefBudget v1 methodologyor Table . For cares Table . The fac67). We statistically evaluated spatial variability in coral cover, gross carbonate production, bioerosion, and coral reef-accretion potential at BIRNM with linear mixed-effects models (LMEs) using the nlme package68. Reef sector (northern and southern) and habitat  were treated as fixed factors and site was treated as a random intercept in the models. To further evaluate the drivers of differences among zones (sector and habitat), we compared the contributions of calcifying taxa to carbonate production using ANOSIM and SIMPER in the vegan package69. We summarize the results of the LMEs using the anova function and evaluate significant fixed effects using the Tukey method in the emmeans package70.All statistical analyses of the were conducted in R Studio  to delineate the spatial footprint of fore-reef, reef-crest, and back-reef habitats. The 2100 bathymetry based on estimated reef-elevation change was used to estimate the impact of reef degradation on future total water levels at BIRNM (see Hydrodynamic Modeling section).To predict how coral-reef degradation will change the bathymetry of the BIRNM fringing reef in the future, we calculated the estimated change in reef elevation for each site by 2100  based on estimated reef-accretion potential. We modified the 2014 USGS lidar bathymetry for BIRNM (in ArcGIS v.10.5.1) based on the mean projection for each habitat in each sector, using benthic habitat maps created by the National Oceanic and Atmospheric Administration  of A. palmata were selected within BIRNM by Z.H.S. for subsampling for our study based on knowledge of each colony’s history with respect to survival during disease outbreaks and bleaching events, or recruitment to environments of interest to park resource managers. Thirty Ps. strigosa colonies were selected based on target size and depth from the area surrounding each calcification-assessment site  were transplanted onto PVC discs fitted with stainless-steel bolts that slide through a hole in the top of cement blocks secured to the reef71. One specimen of each species was deployed together on each block and the corals were photographed, measured (height and planar dimensions), and weighed using the buoyant-weight technique71 twice per year for two years. Planar area occupied by each colony was calculated using calipers to measure maximum and minimum diameter of the footprint estimated as an ellipse and used to normalize coral calcification rates.To assess the potential for restoring populations of reef-building corals and, therefore, reef-accretion potential at BIRNM, we measured the vertical growth and calcification rates of ite Fig.  except fA. palmata is the primary species responsible for constructing shallow reef-crest habitats in the western Atlantic18 and is, therefore, the species for which restoration could have the most significant impact on coastal-hazards protection, we assessed how the restoration of this species in particular could impact the potential of reefs at BIRNM to keep pace with future sea-level rise. To do this, we first used the mean (±SE) rate of change in A. palmata surface area measured at BIRNM in this study30 to develop a simple matrix model projecting the potential change in percent cover of A. palmata in reef-crest habitats of BIRNM for each decade from 2020 to 2100 following outplanting of 100,000, 250,000, and 500,000 colonies: equivalent to ~0.6, 1.5, and 3.0 individuals m−2 across the 166,407 m2 total area of the BIRNM reef-crest habitat  from 2030–2040.Because tat Fig. . For theA. palmata has only begun in earnest in the last few years and, as a result, few data are available on the long-term survival of outplanted colonies of this species; however, a study by Garrison and Ward74 that tracked the fate of transplanted storm-generated A. palmata fragments in St. John, U.S. Virgin Islands, found that after 12 years (1999–2011), survival was 3%. Although this rate may seem low, it is similar to the benchmark annual survival rate of 70% proposed by Schopmeyer et al.72 for the congener A. cervicornis, when projected over a decadal scale . Some restoration programs have achieved much higher rates of survival ; however, we chose to adopt the relatively conservative estimate of 3% for our model.Restoration of A. palmata in recent decades52, which makes accurately parameterizing reproductive rates problematic. Because the inclusion of the presently low rates of sexual reproduction by A. palmata is not likely to significantly change its population projections56, we did not include sexual reproduction in our model. We did, however, account for the high rate of asexual fragmentation in this species, using the generalized  annual fragmentation rate of 10% and fragment-mortality rate of 40% for A. palmata suggested by Lirman58 projected over a decade to produce a decadal net fragmentation rate of 55%. Those rates are comparable to rates found in a similar study by Vardi et al.56. We note that storms would result in a higher net fragmentation rate, which could be either beneficial or detrimental to A. palmata populations depending on their frequency58. Because of the uncertainties in future storm climates11, we did not explicitly incorporate storm-related fragmentation into our model; however, the potential for increasing storm impacts is somewhat accounted for in our increasing mortality scenario described below.Low population sizes have significantly limited sexual reproduction of 74 with the 55% fragmentation rate58 to produce population-level survival rate of 58%, which we round to 60% for simplicity. We ran the model with three theoretical scenarios of how mortality could change over time: 1) a fixed, 60% population survival rate over time; 2) increased climate-change impacts over time for which mortality was increased by 5% each decade ; and 3) decreased climate-change impacts over time through increased resilience, acclimatization, and/or adaptation of A. palmata for which mortality was decreased by 5% each decade . We acknowledge that these scenarios present a simplified version of reality because of the uncertainties in the most likely carbon-emission scenarios and in the capacity of corals to acclimate/adapt to long-term climate impacts. Our aim was to encompass a large range of possible mortality rates (20‒56%) in our models, while accounting for fragmentation as an important part of the life history of A. palamta58. We converted projected surface area from the models to percent cover, by dividing the sum of colony areas by the total area of reef-crest habitat on BIRNM of 166,407 m2 based on the assumptions that (1) all A. palmata remained within the bounds of that habitat area, and (2) that there was no overlap among colonies.Change in population size in our model was parameterized by combining the 3% survival rate suggested by Garrison and WardA. palmata was increased by 5 to 30% at +5% intervals. We consider an increase of 30% to be a conservative ecological limit for our scenarios based on the observation that cover of A. palmata on the reef crest at BIRNM was >50% historically29 and maximum A. palmata cover at our sites is ~20% at present. For this analysis, we used the mean calcification rate of A. palmata of 29.07 kg m−2 y−1 measured in this study to provide a more conservative estimate of reef-accretion potential than the value of 36.24 kg m−2 y−1 we used in the baseline carbonate-budget model.We also re-ran the carbonate budgets to estimate reef-accretion potential using eight theoretical restoration scenarios wherein the cover of 48. Indeed, by comparing empirical, geological records of recent reef accretion to carbonate-budget assessments of reef-accretion potential, Toth et al.22 suggested that carbonate-budget models in south Florida may have underestimated net reef erosion by ~1 mm y−1. We used a similar approach in this study by calculating the difference between a carbonate-budget derived estimate of historic reef-accretion potential for the reef-crest environments at BIRNM based on the maximum observed A. palmata cover in the 1970s of 62%35 and our estimates of bioerosion in 2016 and the maximum Holocene reef accretion by A. palmata in St. Croix of 13.4 mm y−1 . Our estimate of historic carbonate production by A. palmata based on that analysis was 16.48 kg CaCO3 m−2 y−1, which is remarkably similar to Gladfelter’s35 estimate of A. palmata production at BIRNM at the time of 15.18 kg CaCO3 m−2 y−1. Finally, whereas these simple, conceptual models are intended to illustrate the potential of restoration to increase coral cover and reef-accretion potential at BIRNM in theory, we acknowledge that important uncertainties remain regarding the success of restoration outcomes in practice.Reef-accretion potential quantified in our study should be considered an optimistic, maximum estimate of long-term reef accretion77 is a process-based numerical model that resolves key hydrodynamic processes on coral-reef-lined coasts79. A two-dimensional (2D) XBeach surfbeat model of Buck Island Reef was constructed to simulate wave and water-level transformation across the reef (details provided in80). A curvilinear grid was set up with grid resolution varying 4‒40 m in cross-shore direction, and alongshore resolution ranging 9‒50 m. The 2014 lidar-derived topo-bathymetry of St. Croix, U.S. Virgin Islands provided the model bed levels. To account for the effect of coral-reef roughness on waves and currents, spatially varying hydrodynamic roughness was included in the model following the methodology by Storlazzi et al.81. Modeling of hydrodynamics and the resulting different water-level components and total water levels followed the methodology of Quataert et al.5.XBeach82. Wave boundary conditions were generated by a SWAN83 model with a regional domain around northern St. Croix, nested in a large-scale domain covering the U.S. and British Virgin Islands. The wave model was forced by ERA5 wave time series and wind fields84 and validated against four months (July–October 2010) of CDIP (2021) wave-buoy measurements at Christiansted Harbor and Fareham, St. Croix, VI. The modeled mean water levels, sea-swell (5–25 s periods) wave heights , and infragravity (>25 s periods) wave heights  by XBeach agree well with the observations  and wave and storm-surge conditions (10- and 50-year storms). Wave conditions were based on Storlazzi et al.81 and included a Hs = 4.01 m and Tp = 15.8 s for the 10-year event  and Hs = 4.78 m and Tp = 18.7 s for the 50-year event ; the incident wave direction was from 65° for both return periods. Storm surge levels of +0.37 m and +0.64 m were imposed for the 10- and 50-year event, respectively85, and added to the MHHW level of 0.40 m for a 1-h simulation period. This same set of forcing conditions were run using modeled bathymetry of the BIRNM fringing reef at 2100 based on our estimated rates of reef accretion (and erosion). For all 20 simulations, maximum total water levels along the shoreline were extracted from the model results to assess the impact of future climate scenarios on the nearshore hydrodynamics at Buck Island.The validated XBeach model was then forced with a range of sea-level-rise scenarios (+0.0, +0.2, +0.5, +1.2, and +2.0 m; cf.A. palmata restoration scenario for northern and southern reef sectors from the sea-level-rise scenarios We used linear interpolation to predict total water levels at the adjusted sea levels for each storm scenario in our model. Because our models do not include scenarios of lower-than-present water depths over the reef crest, in cases where the predicted increase in reef elevation with restoration was greater than sea-level rise, we applied the total water levels for the +0.0 sea-level-rise scenario.We also assessed the potential impact of coral restoration on total water levels by subtracting the increase in reef elevation at the reef crest predicted by our carbonate budgets under the +30% Supplementary InformationPeer Review File


RNA editing has been described as promoting genetic heterogeneity, leading to the development of multiple disorders, including cancer. The cytosine deaminase APOBEC3B is implicated in tumor evolution through DNA mutation, but whether it also functions as an RNA editing enzyme has not been studied.Here, we engineer a novel doxycycline-inducible mouse model of human APOBEC3B-overexpression to understand the impact of this enzyme in tissue homeostasis and address a potential role in C-to-U RNA editing. Elevated and sustained levels of APOBEC3B lead to rapid alteration of cellular fitness, major organ dysfunction, and ultimately lethality in mice. Importantly, RNA-sequencing of mouse tissues expressing high levels of APOBEC3B identifies frequent UCC-to-UUC RNA editing events that are not evident in the corresponding genomic DNA.This work identifies, for the first time, a new deaminase-dependent function for APOBEC3B in RNA editing and presents a preclinical tool to help understand the emerging role of APOBEC3B as a driver of carcinogenesis.The online version contains supplementary material available at 10.1186/s13059-023-03115-4. ApoB) mRNA which results in a stop codon (UAA) and the generation of the truncated protein ApoB48  were fed with 625 ppm dox impregnated food pellets. For control, littermates with the genotypes (+ / +)(+ /rtTA); (+ /A3B)(+ / +) were fed with doxycycline. For the mutant mice, only this genotype was used (+ /A3BE255A)(+ /rtTA).For inducing the transgene in vivo, different genotypes of the APOBEC3B:rtTA alleles [  and following the manufacturer’s instructions. Primers were designed to create targeted specific changes  in the plasmid containing the wild-type A3B. This plasmid was used for electroporating the ES cells to generate mutant Tissues were fixed in formalin overnight and embedded in paraffin. Antigen retrieval was performed using 0.09% (v/v) unmasking solution  for 30 min in a steamer. Inactivation of endogenous peroxidases was done using 3% Hydrogen Peroxide  for 10 min. Secondary antibody staining and biotin-streptavidin incubation were performed using species-specific VECTASTAIN Elite ABC kits . DAB Peroxidase Substrate kit  was utilized for antibody detection. Primary antibodies used were anti-pH3 Ser10 , cleaved caspase 3 , γH2AX , APOBEC3B  , tGFP  following the manufacturer’s protocol.DNA deaminase activity was measured in whole lysates of different tissues using previously described protocols . A FluorFor protein extraction and immunoblotting, mouse tissues were lysed in RIPA lysis buffer . Samples were then boiled for 10 min and cleared by centrifugation. Protein expression was assessed by immunoblotting using 40–90 μg of the lysates and probed using the following specific antibodies: anti-APOBEC3B  ; anti-tGSnap-frozen tissues were ground with a mortar and pestle on dry ice. For total RNA and genomic DNA extraction, AllPrep DNA/RNA Mini  was used and cDNA synthesis was done using the QuantiTect Reverse Transcription Kit  according to the manufacturer’s instructions.Quantification using real-time PCR was initiated using 10 ng of cDNA with SYBR Green PCR Master Mix (2 ×)  in a LightCycler II®480 (Roche) Relative mRNA levels were calculated according to the ΔCt or ΔΔCt relative quantification method and were normalized to the examined house-keeping genes  levels.A3B-dependent RNA editing events in mouse pancreas and liver tissues were identified from matched RNA-seq and WES data of individual A3B mice and a pool of control littermates.WES libraries were prepared from 0.5 μg of genomic DNA using Agilent Low Input Exom-Seq Mouse kit for Illumina platforms and sequenced with Illumina HiSeq 2000 v4 technology (100-nucleotide paired-end reads). WES data were aligned to the mouse reference genome assembly (mm10) using SpeedSeq . PCR duphttps://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Quality control, including per-base quality, duplication levels, and over-representative sequences, passed all the checkpoints. RNA-seq reads were aligned to mouse reference genome assembly mm10 using STAR/2.7.1a with basic two pass mode for realigning splice junctions enabled. Picard tools (version 2.18.16) were then used to mark duplicate reads and split CIGAR reads with Ns at the splice junctions. Mutect2 from GATK (3.6) was used to call RNA variants in tissues from A3B-overexpressing animals relative to the pool of normal RNA-seq data from normal tissues of control littermates. RNA variants that passed Mutect2 internal filter with at least 6 reads supporting the variant, a minimum of 20 total reads at the altered site and a variant allele frequency greater than 0.05 were used for downstream analysis. RNA editing levels at Apobec1 target sites were extracted from the raw tables of the aligned reads before applying any filters for further analysis.Bulk RNA-seq libraries were prepared from 1.2 μg of total RNA with TruSeq Stranded kit for Illumina platforms and sequenced with Illumina HiSeq 2000 v4 technology (100-nucleotide paired-end reads). Before and after trimming of the RNA-seq data, the RNAseq quality was evaluated using FastQC (https://github.com/temizna/plotRNAedits).Variants detected at the RNA level were considered an RNA editing event when the same variant was not present on the DNA level in the matched exome data from each sample and used for subsequent signature analysis. Relative contribution refers to the contribution that each single base substitution has to the overall base substitution spectrum in A3B mice. All sequence logos were generated and visualized using ggseqlogo package in R. Full context RNA editing events were displayed using PlotRNAedits  were calculated using the DEseq2 package in R. Gene set enrichment analysis (GSEA) of differentially expressed genes was performed using ‘ClusterProfiler’ package of R and javaGSEA Desktop Application v2.2.2.Pathways were considered significantly enriched at an FDR equal to or smaller than 0.25.TPMs levels of various genes were extracted by generating a read-count matrix with the Bioconductor packages GenomicAlignments and GenomicFeatures in R.https://github.com/temizna/plotRNAedits).All sequences logos were generated and visualized using “ggseqlogo” package in R. Full context RNA editing evens were displayed using PlotRNAedits . Additional file 1. Supplementary figures.Additional file 2:Table S1. RNA editing events per chromosome.Additional file 3:Table S2. RNA editing validation.Additional file 4:Table S3. Mice.Additional file 5:Table S4. Sequences of primers used for the validation.Additional file 6. Data source.Additional file 7. Peer review history.


Telomeres are terminal structures that define the ends of linear chromosomes. They harbour specialized ribonucleoprotein complexes which play a major role in genome integrity by preventing unscheduled DNA damage repair events. Genes located adjacent to telomere repeat sequences are repressed by a phenomenon called telomere position effect (TPE) via epigenetic silencing. RNA surveillance pathways post-transcriptionally regulate any leaky transcripts arising from the telomeres. Recently, multiple non-coding RNA species originate from telomere ends, namely, TERRA (telomeric repeat‐containing RNA), ARRET, sub-telomeric XUTs and sub-telomeric CUTs have been identified. In this study, we report a role for the transcription termination complex (Rtt103-Rai1-Rat1) in regulating the abundance of the sub-telomeric transcripts in a transcription-dependent manner. We show that the Rtt103 mutants have elevated levels of TERRA and other sub-telomeric transcripts that are usually silenced. Our study suggests that Rtt103 potentially recruits the exonuclease, Rat1 in a RNA polymerase II dependent manner to degrade these transcripts and regulate their levels in the cell. Saccharomyces cerevisiae consist of a short array of tandem repeats (C1–3A/TG1–3) that averages approximately 300 ± 75 bp in size [Telomere-Associated Sequences). TAS are of two classes, namely, X and Y’. The Y′ elements are either long (6.7 kb) or short (5.2 kb) and are present in 0–4 copies per chromosome end [Telomeres, the terminal structure of eukaryotic linear chromosomes, are an array of specialized nucleoprotein complexes. They play a crucial role in maintaining genome integrity by protecting the ends from being recognized as DNA-double strand breaks (DSBs) . Like mo in size . The endpeats C1–A/TG1–3 ther long .7 kb or her long .7 kb or 1–3 repeats (Type II) or TERRA (Telomere Repeat containing RNA) transcripts to lengthen the critically shortened telomeres [Telomeres are subjected to continuous attrition with each cell division because of the end replication problem . This iselomeres .Another salient feature of the telomeres is the establishment of heterochromatin region. The telomeric tract is non-nucleosomal and has a distinct set of telomere specific proteins ,14. The Recently, it has become clear that despite the epigenetic silencing of sub-telomeric regions, several RNA species arise from the telomeric and sub-telomeric region. These include TERRA (telomeric repeat‐containing RNA), sub-TERRA, ARRET, ARIA, sub-telomeric XUTs, sub-telomeric CUTs and several other RNA species which originate from telomere ends ,28. SomeS. cerevisiae, TERRA is an RNA Pol II product which is regulated both epigenetically [In etically  and postetically ,30. In Xetically . The tur1-S2-P3-T4-S5-P6-S7) that are dynamically phosphorylated and dephosphorylated [The transcription cycle of RNA Pol II is a highly coordinated event which is regulated via its C-Terminal Domain (CTD) . In yeasorylated . At the orylated . As it torylated ,41. Ser5orylated . Towardsorylated . Rtt103porylated . Apart forylated .A genome-wide distribution analysis of RNA Pol II revealed its enrichment in various ‘epigenetically silenced’ regions of the genome like the telomeres, rDNA ‘E-Pro’ region, centromeres, HML and HMR loci. Interestingly, it was shown that the transcriptional regulation at the rDNA is via the alternative transcription termination pathway Nrd1-Nab3-Sen1 ,49. The In our study, we report a novel role of transcription termination complex Rtt103, Rai1 and Rat1 in sub-telomeric gene silencing and in the regulation of the long non-coding RNAs arising from the sub-telomeric and telomeric tracts. Further, we demonstrate that this complex is physically enriched at the telomeres. The recruitment of the Rat1 exonuclease to the terminus is dependent on Rtt103 and requires active transcription suggesting a potential mechanism for the enrichment of this complex at the telomeres.S. cerevisiae to identify suppressors for yku70Δ temperature-sensitive phenotype yielded high copy expression of RTT103 as a partial suppressor for temperature sensitivity [RTT103 does not have a role in repairing non-chromosomal substrates via non-homologous end joining (data not shown), a key function of Yku70, we extended the study to test whether Rtt103 has any role in telomere metabolism and gene silencing, another important function of Yku70. Transcription of genes located adjacent to telomeres is usually repressed by the telomere position effect (TPE) [rtt103Δ in a strain that harbours URA3 at the telomeric region of chromosome VIIL. The expression of URA3 in this locus is silenced by TPE [URA3 gene encodes for an enzyme orotidine-5’-phosphate decarboxylase, which converts the 5-FOA into a toxic substance 5’ fluorouridine monophosphate. Hence, loss in silencing restricts the growth of the cell in 5-FOA, and conversely, robust growth on plates containing 5-FOA indicates silencing. Wild-type cells exhibit TPE. As silencing at the telomeres is stochastic, wild-type cells grow on both SC-URA and SC + 5-FOA plates. But in an rtt103Δ, we found reduced silencing, as indicated by the reduction in growth on 5-FOA plate, compared to wild type  or multicopy (2 micron) RTT103 (Previous investigation in sitivity . Althougct (TPE) . To assed by TPE . The meaild type . yku70Δ,) RTT103 .Figure ADE2 marker at the telomeric region of chromosome VIIL. ade2Δ cells display a red colony colour phenotype on rich yeast growth medium, YPD. The red colour is due to the accumulation of intermediate metabolites of the adenine biosynthesis pathway. When ADE2 is inserted proximal to the telomere sequences, wild type cells exhibit TPE and do not continuously express ADE2. So they appear red like an ade2Δ mutant, stochastically switching to white for a few generations, leading to the appearance colonies with red and white sectors . However, this strain was extremely sick and was not used for further studies.Further, we used a colour-based assay to study the loss of silencing by integrating the  sectors . We notiURA3 or ADE2 gene immediately adjacent to the telomeric TG1–3 tract by removing the adjacent X and Y′ elements [rtt103Δ is not specific to modified truncated telomeres, we also assessed the silencing defect of URA3 marker inserted at the core X of chromosome XI-L of unmodified natural telomeres [rtt103Δ strain has lowered growth in 5-FOA while growing robustly on SC-URA plates, indicating reduced silencing at the native telomeres as well. In order to further validate the silencing defect in rtt103Δ, we also assessed the relative levels of two native sub-telomeric genes namely YIR042-C and YFR057-W via quantitative reverse transcriptase PCR (qRT-PCR) [YIR042-C is located at chromosome IX and 3.9Kb away from the telomere end, whereas YFR057-W is located at chromosome VI at 645bp away from the end and have been shown to be silenced in a Sir-protein-dependent manner [rtt103Δ, there was a 12-fold increase in the relative amounts of YFR057-W in comparison with WT. We could only detect a modest increase in YIR042-C transcripts that was not statistically significant  . YIR042-t manner . qRT-PCRnificant . Complemrai1Δ and rat1–1 at the two native sub-telomeric genes, namely, YIR042-C and YFR057-W, via quantitative reverse-transcriptase PCR (qRT-PCR). Rat1 is an essential gene and the rat1–1 allele is temperature sensitive. Therefore, the silencing was assessed at 39°C in rat1–1. All three mutants revealed a varying range of silencing defects for YFR057-W in comparison with wild type  to non-permissive temperature (39°C), whereas RNA Pol I and Pol III transcription status remains unaltered. We analysed the stability of transcripts arising from YFR057-W and YIR042-C along with ACT1 at regular intervals after inhibiting transcription. Total RNA was isolated from samples harvested at 0 minutes (just before the temperature shift) and every half an hour after the temperature shift to 39°C. Relative abundance of sub-telomeric transcripts and ACT1 transcripts was obtained by normalization to 7S RNA and plotted as a function of time. 7S RNA is a non-coding RNA arising from the SCR1 locus, is transcribed by RNA polymerase III, and is unlikely to be affected by defective RNA polymerase II function at higher temperatures. In comparison with wild type (rpb1–1), rtt103Δ rpb1–1 revealed greater stability of sub-telomeric transcripts, whereas stability of ACT1 transcript remained the same in both . These data suggest that in an rtt103Δ mutant, it is the stability of the transcript, not the rate of transcription, which is altered and it is also specific to sub-telomeric transcripts.From the existing literature, it is evident that sub-telomeric transcripts are regulated epigenetically , transcrrtt103Δ strain (Kathirvel and Mishra unpublished), we found several of the putative Y’ helicases encoded within Y’-element of the sub-telomere region were up-regulated by more than twofold. In S. cerevisiae there are 11 Y′-Long elements and eight Y′-Short elements [rtt103Δ strains via RT-PCR using single primer set to measure highly conserved Y’ helicases from seven distinct loci. We find that Y’ helicases are upregulated in rtt103Δ and rai1Δ .Recent discoveries of regulatory ncRNAs controlling cis and trans gene silencing in budding yeast have led to speculation that these RNAs might be directly involved in heterochromatin regulation at the telomeric regions . In the elements . In telortt103Δ, rai1Δ and rat1–1 as described by Iglesias et al., 2011 via quantitative real-time PCR protocol [rai1Δ had a larger level of accumulation compared to both rtt103Δ and rat1-1. While it is known that Rat1 plays a key role in keeping TERRA levels low, we find that both its partners, Rtt103 and Rai1, also contribute to regulating TERRA levels. These results suggest that, as proposed for transcription termination, the Rat1-Rai1-Rtt103 could work together in the regulation of TERRA.In addition to ORFs for putative helicases, the sub-telomeric region also contains promoter like elements for TERRA ,63. The protocol   which has reduced interaction with RNA Pol II as determined by anisotropy and NMR measurements [RTT103 (R108N) in comparison with the strain with wild type RTT103 . Similar to their role in transcription termination, both Rtt103 partners, Rai1 and Rat1, are also involved in regulating the levels of these transcripts. Of note, we show that Rtt103 could recruit Rat1, the exonuclease that that has been implicated in the degradation of these transcripts. We show that association of Rtt103 with the RNA polymerase II is required for regulating the levels of transcripts and that this association is likely mediated via interaction of the C terminal domain of RNA polymerase with Rtt103.In this work, we demonstrate that Rtt103, a transcription termination factor, is required for efficient silencing of telomeric and sub-telomeric transcripts. The telomere length of Several recent high-throughput transcriptional analyses have now established that transcription of telomeres and sub-telomeres is a conserved phenomenon among different phyla ,63,70,71rtt103Δ, silencing at the sub-telomeres is compromised at both modified and natural telomeres  to the sub-telomeric transcripts leads to degradation. As Rat1 can only act on uncapped 5’ ends, we think that once the RNA is capped, it has to be decapped and then Rat1 can degrade. This is possibly post-transcriptional. Alternately or additionally, Rai1 could also independently exhibit this activity on nascent transcripts co-transcriptionally once recruited to the transcribing polymerase as it possesses a decapping and exonuclease activity on unmethylated 5’ caps [How might Rtt103 regulate sub-telomeric transcript levels? We envision multiple possibilities . At the  5’ caps . As loss 5’ caps . In sum,rtt103Δ [rtt103Δ and it is known that Y’ helicase is incorporated into the viral like particles produced in the Ty1 transposition cycle [Interestingly, Rtt103 was initially isolated in a screen for mutants that elevated Ty1 transcription and it was demonstrated that there was a moderate increase in Ty1 transcripts in rtt103Δ . This raS. cerevisiae, sub-TERRA XUT and sub-TERRA CUT are complementary to each other and can form dsRNA. As RNAi does not exist in S. cerevisiae, could RNA degradation pathways be more critical to regulate the levels of such non-coding telomere transcripts?There are a number of key questions that remain to be addressed. Induction or overexpression of TERRA from a single telomere induces early-onset senescence . Does sttlh which is regulated under nitrogen starvation, cri-TER a temperature-dependent non-coding RNA, genes responsible for biofilm formation [The sub-telomeric regions reveal higher levels of recombination leading to faster evolution of gene families residing in this locus . This alormation . It remaSaccharomyces cerevisiae strains used in this study are derivatives of W303 or BY4741 and are listed in Table 1. Strains were grown under standard conditions in YPD (Yeast Peptone Dextrose) or synthetic complete (SC) medium at 28°C. For temperature-sensitive (ts) mutants, further specifications are mentioned in the methods section. Standard procedures were followed for yeast manipulations. Either micromanipulation or PCR-based homology-dependent transformation was employed to generate knockouts [The nockouts .Strain 600 − 1 and ten-fold serial dilutions were made and 5 µl spotted onto appropriate agar plates. Plates were then incubated for 2–3 days at 28°C and photographed. The FOA concentration used is 1 mg/ml. For the ADE2 colour-based silencing assay, cells were directly plated on YPD agar plates, incubated at 30°C for 2 to 3 days and photographed.For spotting assays, yeast cells were grown overnight at 28°C in appropriate selection media. Cells were harvested at ODrat1–1 ts mutant, the cells were sub-cultured and grown up to OD600 0.5 ~ 0.8 (28°C) and shifted to non-permissive temperature (39°C) for 3 hours. For data normalization of rat1–1, similarly treated wild-type cells were used. For DNaseI digestion, 3 μg of RNA was subjected to digestion with 10 units of DNaseI (New England Biolabs) enzyme at 37°C for 3.5 hours to achieve an RNA which is completely devoid of telomeric DNA. Before c-DNA synthesis, a normal PCR (40 Cycles) was performed with primers specific for telomere and ACT1 to assess the DNA contamination. RNA alone was also employed as a template for qRT-PCR to confirm the purity of the samples. The reverse transcription was performed using Verso cDNA Synthesis Kit (Thermo Scientific) as per the manufacturer’s protocol at 55°C for 60 min followed by inactivation at 95°C for 2 min.Total RNA was isolated by extraction with hot acidic-phenol (pH-5) as described by Collart and Oliviero . For RNAACT1 oligonucleotide in a final volume of 20 μl. For qPCR, the cDNA was diluted with equal volume of nuclease-free H2O. A volume of 1 μl cDNA was quantified in a final volume of 10 μl reaction by real-time PCR with the Power SYBR Green PCR Master mix (Applied Biosystems) using an Quanstudio3 Real-Time PCR System. The final concentration for each primer set differs from 0.2 to 0.6 μM as described by Iglesias et al., 2011 [a /12 R-YP2a /13 L/15 R) (4Y’-9 L/10 R/12 R-YP2a /15 R) (Y’3-12 L-YP1a /12 R-YP2 a /15 R). Whereas for X-only containing telomeres, individual primers have been designed − 4 L, 7 L, 10 R, 13 R, 15 L, 10R14R.TERRA reverse transcription was done using 10 μM CA oligonucleotide and 2 μM l., 2011 . The rea600 0.8–1 and crosslinked for 10 mins with formaldehyde  and quenched with glycine (360 mM) for 15 mins. Cells were pelleted and washed twice with 1× TBS, resuspended in lysis buffer  and lysed using 0.5-mm glass beads in a vortex mixer for 20 min at 4°C. The chromatin lysate was recovered and sheared 10 sec on/off  for 5 cycles. An input sample representing 10% of the ChIP extract was employed as input for normalizing the qPCR. 80 μl bed volume of IgG Sepharose beads were washed with lysis buffer and IP was performed overnight at 4°C. Beads were washed with lysis buffer P500 , + LiCl detergent buffer  + 2× TBS. The bead-bound DNA was eluted in 100 μl (1% SDS/1× TBS) + 150 μl (1% SDS/1× TBS) for 10 min at 65°C. For reversing the crosslink, input and IPs were treated overnight at 65°C with proteinase K and RNase A. DNA from bound (IP) and unbound fraction (input) was purified by Phenol-Chloroform-Isoamyl alcohol (PCI- 25:24:1) extraction followed by ethanol precipitation. qPCR was performed as mentioned above and % input calculation was employed to assess the relative enrichment.ChIP was performed as described . Briefly600 were subject to protein extraction by TCA method. To the cell pellet 200 µl of 20% TCA was added and lysed by vortex at high speed for 5 min. The lysate was then collected onto a fresh tube and the glass beads were washed twice with 150 µl of 5% TCA. The washed elutes were added to the previous lysate and spun at 13000 rpm for 10 min. To the pellet 200 µl of Laemmli buffer was added and pH was adjusted using 2 M Tris.Cells corresponding to 2 units of ODThe dissolved protein pellet was then denatured by boiling at 95°C for 5 min. SDS-PAGE was performed followed by a semi-dry method of transfer (Power Blotter system/Invitrogen) to PVDF membrane for western blotting. The blots were then probed with primary TAP-tag antibody  and HRP conjugated secondary anti-rabbit . For probing Rtt103 × 13myc, rabbit Myc (Abcam Ab9106 1:1000) primary was used. The signal was detected by ECL reagent (BioRad) and imaged in ChemiDoc Imaging system by BioRad.30 was used.For RNA slot blots, 10 μg of denatured RNA was loaded onto Hybond N+ membranes (Amersham) using slot blot manifold Hoefer PR648 (Amersham). For southern blot, 3 μg of over-night digested XhoI genomic DNA was loaded. After transfer, blots were auto cross-linked via Stratagene UV Stratalinker 1800. The probes and whole hybridization procedure were carried out according to the manufacturers instruction for DIG-High prime DNA labelling and detection starter kit I . For Tel probe, terminally digoxigenin labelled d(GT)Click here for additional data file.


Saccharomyces cerevisiae and Candida albicans. We show that within glyoxal family of compounds, phenylglyoxal (PGO) is the best guanine probe for structural probing in S. cerevisiae and C. albicans. Further, we show that PGO treatment does not affect the processing of different RNA species in the cell and is not toxic for the cells under the conditions we have established for RNA structural probing. We also explore the effectiveness of uracil modification by Cyclohexyl-3-(2-Morpholinoethyl) Carbodiimide metho-p-Toluenesulfonate (CMCT) in vivo and demonstrate that uracils can be modified by CMCT in S. cerevisiae in vivo. Our results provide the conditions for in vivo probing the reactivity of guanine and uracil nucleotides in RNA structures in yeast and offer a valuable tool for studying RNA structure and function in two widely used yeast model systems.RNA structure can be essential for its cellular function. Therefore, methods to investigate the structure of RNA in vivo are of great importance for understanding the role of cellular RNAs. RNA structure probing is an indirect method to asess the three-dimensional structure of RNA by analyzing the reactivity of different nucleotides to chemical modifications. Dimethyl sulfate (DMS) is a well-established compound that reports on base pairing context of adenine (A) and cytidine (C) in-vitro and in-vivo, but is not reactive to guanine (G) or uracil (U). Recently, new compounds were used to modify Gs and Us in plant, bacteria, and human cells. To complement the scope of RNA structural probing by chemical modifications in the model organism yeast, we analyze the effectiveness of guanine modification by the glyoxal family in  As RNAs can be both single- and double-stranded and are highly flexible, they can adopt diverse secondary and tertiary structures in physiological conditions. An example of simple RNA three-dimensional structures includes base-paired doubled-stranded areas such as hairpin stems. More complex RNA motifs comprise three-dimensional structures such as ribose zippers, kink turns, and pseudoknots . The verRNA structure-function relationships inside the cell are affected by the transcription rate, the local solution environment, and the presence of small molecules or RNA-protein interactions. The physical state of RNA in vivo can provide insight into the function of the RNA . StructuSelective 2’- hydroxyl acylation analyzed by primer extension (SHAPE) uses an electrophilic carbonyl derivative that can be attacked by the strong nucleophile 2’-hydroxyl group on the ribose sugar [RNA chemical probing is the fastest way to indirectly investigate RNA structures by using chemical reagents to modify nucleotides at specific positions and analyze the modification efficiency of each site. RNA structure probing techniques can target ribose sugars or nitrogen bases. se sugar . Modificse sugar . While Sse sugar . DMS canse sugar , 10. DMSse sugar . Howevera of the imino group of the amidine moiety of adenines and cytosine (N1 of adenine or N3 of cytosine) is much lower than that of guanine, making adenine and cytosine less reactive nucleotides with glyoxal family compounds than guanine [Oryza sativa and gram-negative bacteria Bacillus subtilis and Escherichia coli in vivo [In the glyoxal family, glyoxal (GO), methylglyoxal (MGO), and phenylglyoxal (PGO) are potential candidates for probing guanines. These chemical probes are carbonyl derivates that are electrophilic towards the nucleophilic amidine group in adenine, cytosine, and guanine. As uracil lacks this functional group, it does not react with glyoxal family compounds. The pK guanine . Glyoxal in vivo . The useUracils are probed by carbodiimides family reagents, including 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and Cyclohexyl-3-(2-Morpholinoethyl) Carbodiimide metho-p-Toluenesulfonate (CMCT). EDC reaction in vitro is slower than CMCT due to hyperconjugation, weakening the electrophilicity of the compound . HoweverSaccharomyces cerevisiae and Candida albicans are two well-studied fungi that also serve as model organisms for studies of conserved biological processes. S. cerevisiae is a simple eukaryotic organism whose RNA biology shares many features with higher eukaryotes, making it a suitable model organism to study different aspects of gene expression [C. albicans is an opportunistic fungal pathogen and the most prevalent cause of fungal infections [Saccharomyces cerevisiae and in the human fungal pathogen Candida albicans. We compare three different glyoxal derivatives and show that PGO yields the highest modification rate of guanines in yeast without affecting cell viability or RNA processing. We also present the conditions for in vivo modification of uracils in yeast using CMCT.pression . C. albifections . While tfections , 9, 16, Saccharomyces cerevisiae and BWP17 strain of Candida albicans were grown in YPD media at 30°C to an optical density of 0.5–0.6 before applying the desired chemical probes.BY4741 strain S. cerevisiae and C. albicans were incubated with glyoxal family compounds . S. cerevisiae was treated with CMCT. In each case, the pH of the media was around 7.5. For each compound, three different concentrations were tested. A no-compound solvent-treated sample was used as the negative control. Two incubation times (5 and 15 min) were tested for all conditions, and two biological replicates were analyzed. GO concentrations were 30 mM, 60 mM, and 120 mM. MGO and PGO concentrations were 5 mM, 10 mM, and 20 mM. CMCT concentrations were 25 mM, 50 mM, and 100 mM. All incubations were performed at 30°C. After incubation, samples were cooled down immediately on ice, and cells were harvested by centrifugation. Cells were washed three times with ice-cold water before RNA extraction.GO and MGO were purchased as water-based solutions. PGO and CMCT were solubilized in DMSO and water, respectively. Total RNA was extracted from cell pellets harvested from 10 mL of culture grown to mid-log phase using hot acid phenol. The extracted RNA was treated with DNase I for 15 minutes at 37°C and further purified using a Quick-RNA miniprep kit (Zymo Research) according to the manufacturer’s protocol.32P-labeled primers. Annealing was performed by incubation at 65°C for 5 minutes followed by a gradual cool down at room temperature over 10 minutes and final incubation on ice. The reverse transcription was performed using SuperScript III (ThermoFisher) at 50°C for 5 minutes per the manufacturer’s manual using a primer annealing to the 3’-end of the 5.8S rRNA (AAATGACGCTCAAACAGGCATG). 1 μL of 4M NaOH was added before heating the RNA at 95°C for 5 minutes to remove the RNA templates. The cDNA products were mixed with the formamide loading dye, heated at 95°C for 5 minutes, and separated on a prewarmed 6% urea/acrylamide sequencing gel in 0.5X TBE buffer. The gel was dried and exposed to a phosphoscreen. Data were quantified using Image Lab Software (Biorad) and analyzed in GraphPad Prism 8.0.Approximately 1 μg of purified RNA in 10 μL was mixed with 2 μL of 0.6 μM 32P-labeled DNA oligos against U3-3’ (AAAGTGGTTAACTTGTCAG), tRNALeu (GCATCTTACGATACCTG) and scR1 (ATCCCGGCCGCCTCCATCAC).Total RNA from two biological replicates treated with PGO or CMCT was isolated using the hot phenol method. snoRNAs were separated on 8% acrylamide/urea gels, transferred to Hybond nylon membrane , and probed using S. cerevisiae was incubated with PGO as described above. The density of the culture was adjusted to a final concentration of 107 cells/mL, followed by four successive cascade dilutions in a 1:10 ratio. Dilutions were spotted onto YPD plates and grown at 25°C, 30°C, and 37°C for 48 hours.S. cerevisiae cells. Therefore, we tested these concentrations as well as 0.5X and 2X of each . PGO concentrations were chosen based on the effect on yeast mitochondrial ATP synthase [1-ATPase in S. cerevisiae. We, therefore, tested 5, 10 and 20 mM concentrations of PGO.To define the best glyoxal derivative for probing guanines in yeast, we tested different concentrations of glyoxal (GO), methylglyoxal (MGO), and phenylglyoxal (PGO) at two incubation times. The tested concentrations of GO and MGO were chosen based on their effect on yeast cell growth, as a proxy for their cell penetration . Based osynthase , 19, wheS. cerevisiae) [S. cerevisiae by these compounds . GO weakly modifies guanines in the 5.8S rRNA of S. cerevisiae in vivo, as evident from the weak modification of nucleotides at positions G78 and G85 . At the 5 min time, GO modifications do not show a noticeable concentration-dependent relationship, as band intensity across all GO concentrations appears similar . At the 15 min time, the band intensity for G78 and G85 increases as the GO concentration increases . The reactivity of MGO towards guanines in the 5.8S rRNA of S. cerevisiae in vivo is weaker than that of GO . G78 and G85 modifications are comparable across all MGO concentrations, as band intensity remains the same at each time point and at each MGO concentration . In contrast, PGO demonstrates high reactivity towards guanine nucleotides in the 5.8S rRNA of S. cerevisiae in vivo . Further, at G78 and G85, modifications by PGO appear to be concentration-dependent, as band intensity at both positions increases as the PGO concentration rises .The 5.8S rRNA is a part of the large ribosomal subunit. Several positions on 5.8S rRNA are subject to glyoxal modification in rice 5.8S rRNA, including G82, G89 and G99  . To probS. cerevisiae 5.8S rRNA. To understand the reason for this, we analyzed the position of 5.8S rRNA G95 in the structure of yeast ribosomes [S. cerevisiae 5.8S rRNA by the C-terminal tail of RPL37 provides an explanation for the lack of reactivity of this nucleotide with PGO . The high reactivity of PGO comes in part from its increased hydrophobicity conferred by the phenyl moeity in the molecule, which allows it to penetrate through the phospholipid membrane bilayer [Unlike in rice, PGO does not modify the G95 in ibosomes . In thisibosomes , 21. Thu bilayer . The hyd bilayer , thus reFig 2A). Total RNA extracted from yeast cells treated with various concentrations of PGO at two time points was used for the Northern blot analysis. We first analyzed the processing of U3 snoRNA, required for proper ribosome biogenesis. U3 is transcribed as a precursor with 5’- and 3’-extensions which need to be removed in order to form the mature U3 snoRNA [Fig 2B). As another control, we analyzed the processing pathway of the leucine tRNA (tRNALeu) which is transcribed as a precursor and undergoes processing before entering the translation pool [Leu do not change in the presence of PGO relative to the loading control (scR1) . These data indicate that the tRNALeu processing pathway remains unchanged in the presence of PGO. Together, our data indicate that treatment of yeast cells with PGO under our established conditions for RNA chemical probing are unlikely to affect the processing of major RNA species in the cell.A critical concern for in vivo RNA structural probing is whether the RNA metabolic pathways in the cell change upon treatment with the chemical probing reagents. We therefore assessed how different RNA processing pathways are affected by PGO treatment by looking at the processing of small nucleolar RNA U3 (U3 snoRNA) and a transfer RNA (leucine tRNA) using Northern blot analysis . At concentrations below 20 mM, PGO does not cause toxicity for S. cerevisiae as judged by the similar size of individual colonies of treated cells compared to no PGO-treated cells . However, at the concentration of 20 mM and after 15 min incubation period, S. cerevisiae cells no longer withstand the toxicity of PGO, resulting in smaller colonies and slow growth . Although this can be due to the toxic effect of PGO on the mitochondrial ATP synthase [S. cerevisiae cells with 20 mM PGO appears to be the optimal condition to achieve efficient nucleotide modification without affecting the cell viability.As there are other pathways which can affect different RNA molecules in the cell and are not studied here, we analyzed the overall fitness of yeast cells upon PGO treatment. A serial dilution spot test was conducted on PGO-treated synthase , 19, we S. cerevisiae are applicable to other fungi. To this end, we tested the modification of 5.8S rRNA of Candida albicans, a human fungal pathogen. Post-transcriptional regulation of gene expression is important for the pathogenicity of C. albicans [C. albicans as a public health threat, it is important to develop tools to study gene expression in this organism. Therefore, establishing the effective condition for guanine probing in C. albicans is of great importance for future pharmaceutical and biochemical applications. Having already established that PGO is the best guanine probe in S. cerevisiae, we used PGO to probe the 5.8S rRNA in C. albicans. PGO shows effective modification reactivity towards G77 and G84 in C. albicans  . However, PGO modification of these two nucleotides in C. albicans demonstrates weak band intensities overall, compared to that in S. cerevisiae, representing lower frequency of guanine modifications by PGO in C. albicans than that in S. cerevisiae . This different can arise from differences in the local environment of the G77 in C. albicans 5.8S rRNA relative to its S. cerevisiae counterpart. An alternative explanation could be that overall PGO entry to C. albicans cells is less efficeint compared to S. cerevisiae. Nonetheless, these data suggest that PGO can be used for probing guanines in C. albicans.Next, we sought to determine whether the conditions for modifying guanines in albicans . Given tS. cerevisiae. While EDC effectively modifies several positions on rice 5.8S rRNA [S. cerevisiae in vivo at positions U81 and U82 . The modification intensity is the highest at 100 mM CMCT, irrespective of the incubation time. CMCT is too large to effectively penetrate cell wall due to the presence of a quaternary ammonium ion that constitutes a positive charge in CMCT [Fig 5). CMCT can react with Gs, albeit to a lesser extent than Us [Fig 5A and data not shown).Uracil probing chemical reagents and their reaction conditions have yet to be established in yeast in vivo. CMCT, a carbodiimide derivative, has been used to probe Us in vitro . However.8S rRNA , none of in CMCT . This ca than Us . HoweverS. cerevisiae using Northern blot . The processing of the 3’ end of U3 snoRNA is not affected in the presence of varying concentrations of CMCT after 5-minute treatment . However, extended treatment with CMCT results in a decrease in the level of U3 precursor . This effect is more pronounced for the pre-tRNA product, the level of which goes down dramatically upon treatment with various concentrations of CMCT after 15 minutes . While a defect in the processing of RNA precursors usually results in the accumulation of the processing intermediates, a decrease in the levels of these intermediates represents either a global destabilization of the RNA species or a defect in the RNA transcription. Based on these data long incubation periods with CMCT (>5 min) should be avoided.We next assessed the effect of CMCT treatment on the RNA processing pathways in In summary, this work establishes the optimal chemical conditions in which PGO and CMCT can effectively probe guanine and uracil nucleotides, respectively, in yeast cells. We found that PGO is a potent probe within the glyoxal family derivatives to probe guanine in yeast in vivo. PGO incubation with yeast does not affect its RNA processing pathways, and at the PGO concentrations less than 20 mM and at less than 15-minute incubation period, yeast cells can withstand the toxic effect of PGO. CMCT can be used to probe uracil in yeast in vivo at 100 mM for 5 min, however, proper controls must be done to ensure that it is not interfering with the levels of the RNA of interest.S1 FigPosition of 5.8S G95 and RPL37 Gln79 and Ser82 are marked (PDB id: 6TNU). Blue and reg colors represent nitrogen and oxygen, respectively. The phosphate backbone is in orange.(TIF)Click here for additional data file.S1 Raw images(PDF)Click here for additional data file.