RNAs are extremely important molecules inside the cell, which perform many different functions. For example, messenger RNAs, transfer RNAs and ribosomal RNAs are involved in protein synthesis, whereas noncoding RNAs have numerous regulatory roles. Ribonucleases (RNases) are the enzymes responsible for the processing and degradation of all types of RNAs, having multiple roles in every aspect of RNA metabolism. However, the involvement of RNases in disease is still not well understood. This review focuses on the involvement of the RNase II/RNB family of 3′–5′ exoribonucleases in human disease. This can be attributed to direct effects, whereby mutations in the eukaryotic enzymes of this family  are associated with a disease, or indirect effects, whereby mutations in the prokaryotic counterparts of RNase II/RNB family (RNase II and/or RNase R) affect the physiology and virulence of several human pathogens. In this review, we compare the structural and biochemical characteristics of the members of the RNase II/RNB family of enzymes. The outcomes of mutations impacting enzymatic function are revisited, in terms of both the direct and indirect effects on disease. Furthermore, we also describe the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) viral exoribonuclease and its importance to combat the COVID‐19 pandemic. As a result, RNases may be a good therapeutic target to reduce bacterial and viral pathogenicity. These are the two perspectives on RNase II/RNB family enzymes that are presented in this review. Ribonucleases (or simply RNases) are the enzymes responsible for the processing and degradation of all types of RNAs, having multiple roles in every aspect of RNA metabolism. Here, we review what is known about the involvement of the RNase II/RNB family of 3′–5′ exoribonucleases in human disease. As RNases may constitute an efficient approach to fight pathogenicity, this is also discussed in the context of bacterial and viral infections. CSDcold‐shock domainDis3defective in sister chromatid joiningDis3L1Dis3‐like exonuclease 1Dis3L2Dis3‐like exonuclease 2MLSMarfan‐like syndromensp10nonstructural protein 10nsp14nonstructural protein 14PINPilT N‐terminus domainPRLMNSPerlman syndromeRNaseribonucleaseRNBribonuclease B (renamed as RNase II) catalytic domainSARS‐CoV‐2severe acute respiratory syndrome coronavirus 2WTWilms' tumourThe amount of RNA present in the cell is modulated by cellular needs and environmental demands. The final RNA levels are determined by the balance between RNA synthesis and degradation. Ribonucleases (RNases) are the enzymes responsible for the degradation of all types of RNA, ensuring RNA processing and turnover , 2. RNasEscherichia coli), but only a few have an essential activity . Th. Th28]. The degradation can then proceed in two pathways. If decapping occurs, then decay proceeds in the 5′–3′ direction: the 5′–3′ exoribonuclease 1  is able to completely degrade the RNA starting from its 5′‐monophosphate end , 34. WhiThe above are the most widely studied general processes of RNA decay. However, the discovery of Dis3L2, another member of the RNase II/RNB family, revealed a new alternative eukaryotic RNA decay pathway independent of deadenylation and decapping, but rather mediated by uridylation. This major finding took place in fission yeast , and almThe RNase II/RNB family of enzymes display hydrolytic 3′–5′ exonucleolytic activity, and they are present in all domains of life. Changes in their catalytic functions or alterations in their cellular levels can relate to pathogenesis and disease. As such, we will revisit the main catalytic properties of these enzymes to contextualize their importance.In prokaryotes, the representative enzymes of this family are RNase II and RNase R. Both are key players in RNA degradation and quality control, being present in a wide range of bacteria, including pathogenic agents. In what concerns their catalytic properties , both prE. coli rnb gene that encodes the family prototype RNase B (renamed as RNase II). In fact, the RNB domain is considered the main characteristic of this family, giving it the name.Notably, all members of the RNase II/RNB family have a comparable protein domain organization Fig. . From th2+‐dependent manner. This second feature relies on four essential residues in the PIN domain active site . However, while Dis3 can cleave RNA molecules endoribonucleolytically, Dis3L1 harbours an inactive PIN domain due to the absence of two of the four active site residues (E97 and D146 in hDIS3), making it unable to cleave RNAs internally.While this arrangement is shared by prokaryotic RNase II and RNase R, the eukaryotic Dis3/Rrp44 and Dis3L1 evidence two additional N‐term domains, both related to the association of these proteins with the RNA exosome: the CR3 and the PilT N‐terminus (PIN) domains , 63, 64 Schizosaccharomyces pombe [In Dis3L2, the CR3 and PIN domains are missing, so this enzyme acts independently of the RNA exosome in an alternative RNA decay pathway  , 66, 67;S. pombe (SpDis3L2) and humans (hDIS3L2) demonstrated that these RNases just require two protruding nucleotides at the 3′‐end of the structured RNAs in order to unwind them, and even manage to degrade them if they have blunt ends [There is not yet evidence of the ability of Dis3L1 to cleave double‐stranded RNAs (dsRNAs), but it has already been reported that Dis3 can degrade structured RNAs, as long as they bear a single‐stranded 3′ overhang with at least 4 nt (corroborated both in budding yeast and in human Dis3 orthologues) , 66, 67.unt ends , 42.A recent study on the landscape of functional interactions of human processive RNases, including Dis3‐like enzymes, identified new functional interactions between them. Despite the limited interpretation of data, this study provided insights into the multiplicity of processes in which these enzymes are involved and how cellular processes can be interrelated .The involvement of hDIS3, and to a smaller extent hDIS3L1, in human disease is well established . InhDIS3L2) is annotated in chromosome 2, and it has fourteen possible splicing variants and five confirmed isoforms. The canonical isoform 1 possesses 21 exons, resulting in 885 amino acids and a molecular weight of 99.3 kDa. Interestingly, when consulting the Human Protein Atlas (HPA) platform, it is possible to ascertain that the protein ‘DIS3 mitotic control homolog‐like 2’ has detectable RNA expression in all analysed cell types, cell lines and tissues (including the brain), but protein expression levels vary among tissues [Considering the diversity of processes in which Dis3L2 is engaged, it is conceivable that it is intimately related to human health and disease. The human Dis3L2 gene , encoded by the gene NPPC that regulates bone growth, subsequently leading to excessive bone growth and skeletal malformations, which are characteristic of this syndrome.Moreover, it was also discovered that patients with Marfan‐like syndrome (MLS) display a chromosomal translocation that results in partial deletion of the iewed in )  and forms a functional complex with RhpA, a DEAD‐box RNA helicase, to better degrade structured substrates [Shigella flexneri, which is the causative agent of dysentery in humans and monkeys, also relies on RNase R for its virulence. Namely, RNase R was shown to influence the production of virulence‐associated antigens at the post‐transcriptional level . It also affected the capacity of bacteria to adhere and spread inside cells [Legionella pneumophila is a ubiquitous bacterium in freshwater environments and in several manmade water systems that causes a potentially lethal pneumonia: Legionnaires' disease. Therefore, this bacterium needs to survive at a wide range of temperatures, including the low temperatures of air conditioners [Legionella pneumophila codes for an RNase R homologue, which was also shown to be important for cell growth at lower temperatures [L. pneumophila  maturation [M. genitalium to discriminate between 2′‐O‐methylations can be further used as a powerful analytical tool for the research of RNA modifications in biological and clinical samples [In recent years, research into RNA metabolism and RNases in foodborne and gastrointestinal pathogens has increased considerably. Most foodborne pathogens can withstand refrigerated temperatures, and the role of RNase R under cold‐shock conditions led to studies targeting this RNase. In eratures , confirm in mice   in E. coli under cold‐shock conditions [trans‐translation [E. coli associates with a ribonucleoprotein complex that contains an RNA molecule named tmRNA and the RNA‐binding protein SmpB, reinforcing the role of RNase R in trans‐translation . The triability , 121. Thbacteria , 125.Streptococcus pneumoniae is involved in translation by affecting the amount of 70S ribosomes required for proper mRNA translation, and by regulating the expression level of important translation factors implicated in ribosome recycling [Besides its role in protein quality control, it was also shown that RNase R from ecycling  (Table 2rnr message), there is an increase in the amount of RNase R protein in the cell [S. aureus [gmr (gene‐modulating RNase II), on the basis that RNase II protein turnover is slower in the gmr deletion strain [gmr [Considering the crucial role of RNase R, it is expected that the levels of this protein must be tightly controlled. Indeed, RNase R levels increase in cold‐shock conditions, as already addressed above, but the enzyme can be regulated in other ways. In the stationary phase, although there is a decrease of its mRNA levels  were shown to impact several RNA viruses, such as the model coronavirus mouse hepatitis virus (MHV). Yet, the opposite also happens when NMD is inhibited upon viral infection , 132. ThCoronaviridae family and to play essential roles in the life cycle of coronaviruses (CoVs) [In the context of the current COVID‐19 pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), particular attention was given to its 3′–5′ hydrolytic exoribonucleolytic activity. This enzymatic activity was found to be highly conserved within the s (CoVs) . This acs (CoVs) , 137. ThCoVs contain by far one of the largest and most complex RNA virus genomes (approximately 30 kb). The nsp14 ExoN domain belongs to the DEDD 3′–5′ exonuclease superfamily, which includes the proofreading domains of many DNA polymerases , 138. ThDespite the existence of vaccines, there is concern about the threat posed by the emergence of new SARS‐CoV‐2 variants. The generation of these variants might be explained through the occurrence of genomic recombination events . Paradoxet al. [The pleiotropic nonstructural protein 10 (nsp10) was found to form a heterodimer with the ExoN domain of nsp14 to stabilize it and stimulate its activity, similar to what was verified in other CoVs. Therefore, nsp14 relies on nsp10 for its ExoN activity , 153. Saet al.  identifiet al. . Strikinet al. . This sh2+ and Mn2+, although maximal activation is observed with Mg2+. The ions Co2+ and Zn2+ promote residual activity, and catalysis is not supported by Ca2+, Ni2+ or Cu2+ [2+, were considered helpful in the evaluation of the dynamic of COVID‐19 infection [The ExoN activity of SARS‐CoV‐2 nsp14 is assisted by a two‐metal ion mechanism; different divalent ions modulate its activity, with specific selectivity towards Mg or Cu2+ , 155. Th or Cu2+ . Recentlnfection .In fact, nsp14 protein is a multitasking enzyme, not only involved in viral life cycle but also capable of interfering with host cells, namely with hindering the host innate immune system. Its ExoN activity cleaves double‐stranded RNA intermediates that are generated during viral replication, which would otherwise activate the type I interferon (IFN‐I) response , 160. ThSARS‐CoV‐2 has an exceptionally high level of transmissibility and adaption to new conditions. This has been translating into an exorbitant number of infections and deaths worldwide. The existence of nsp14 3′–5′ exoribonuclease activity largely contributes to this, since it is a key component of recombination and high‐fidelity replication, and contributes to immune system evasion, virulence and resistance to antivirals.The major features of the exoribonucleases from the RNase II/RNB family impact human disease both directly and indirectly. The eukaryotic Dis3 and Dis3L1 are known to interact with the exosome complex, but in different cellular locations. They can act on a wide range of substrates in eukaryotic cells, and their direct involvement in cancer development and progression is also well documented (mainly for hDIS3) , 16, 164The role of the bacterial counterparts of RNB family in disease was also reviewed. The interaction of pathogenic bacteria with host immune cells triggers a series of events that include rapid genetic reprogramming. In this context, RNases act as key modulators of RNA levels for this rapid adaptation and have been shown to be virulence‐associated factors. Prokaryotic RNases have been shown to modulate the virulence and performance of many human pathogens. The gene encoding RNase R was the first to be linked with bacterial virulence , 94. HowIn the context of the current COVID‐19 pandemic caused by the RNA virus SARS‐CoV‐2, we considered it was also important to discuss the importance of the role of a viral hydrolytic 3′–5′ exoribonuclease in viral infection. The viral protein nsp14 harbours 3′–5′ exoribonucleolytic activity, is responsible for high‐fidelity RNA synthesis, and also confers resistance to NAs, leads to immune evasion and contributes to virulence .Since bacterial and viral exoribonucleases seem to be necessary for the virulence of many pathogens, their study will be a stepping stone for the development of novel antibacterial and antiviral therapeutics that target them and impair their activity.All the evidence presented here, as two perspectives of the same story, reinforces the message that the understanding of RNase II/RNB family of RNases in RNA metabolism, both in microbes and in higher organisms, is required to identify and combat many mechanisms that lead to human disease.The authors declare no conflict of interest.SMC, MS, RGM and SCV wrote the original draft. SMC and MS designed the figures. SCV and CMA conceptualized and coordinated the work and edited the final version of the manuscript. All authors contributed to the discussion of the work and final revision, and approved the final manuscript.


Acropora palmata and Orbicella spp. have been the most common corals. The abundance of competitive, fast-growing acroporids was constant over multi-millennial timescales. A decline in A. cervicornis abundance, however, and three centennial-scale gaps in A. palmata occurrence, suggest that the modern decline in acroporids was not unprecedented. Stress-tolerant corals predominate at the beginning of Holocene successions. Following the improvement of environmental conditions after inundation of the reef pedestal, their abundance has decreased. The abundance of weedy corals has increased during the Holocene underlining the importance of fecundity for the coral community. Reef-accretion rate, as calculated based on 76 new U-series age dates, has decreased over the Holocene and the mean value of 3.36 m kyr−1 is at the lower end of global reef growth compilations and predicted future rates of rise in sea level.Tropical coral reefs, as prominent marine diversity hotspots, are in decline, and long-term studies help to improve understanding of the effects of global warming, sea-level rise, ocean acidification, deterioration of water quality, and disease. Here, we evaluated relative coral abundance and reef accretion rates over the past 9000 years in Belize barrier and atoll reefs, the largest reef system in the Atlantic Ocean.  In the Caribbean realm, live coral cover has diminished by 80% in three decades7, many reefs are no longer dominated by corals but fleshy algae8, reef-building acroporid and massive faviid corals are reduced in abundance and have been replaced by weedy, generalistic taxa11, and reef accretion potential is low (mean of 1.87 ± 2.16 mm yr−1)12. These dramatic changes threaten ecological functionality balances, may lead to the collapse of habitat complexity with a concomitant loss in biodiversity, a decrease in calcium carbonate production and reef accretion, decline in framework resilience, and eventually compromise environmental service provisions12. Millions of people are living along tropical coasts and on low-lying reef islands and are therefore dependent on coral reefs socio-economically. The alliance of the 39 small island states (AOSIS) alone, all of them situated adjacent to coral reefs and many of them located just a few meters above sea level, provide the homelands for some 80 million people, i.e., one percent of the world's population. Therefore, the issue of understanding and addressing coral reef decline is not purely scientific.The status of tropical coral reefs has been declining due to warming, increase in cyclone strength and frequency, ocean acidification, pollution, and diseaseThe study of drill core material extracted from modern coral reefs allows detailed and systematic reconstructions of environmental conditions during the Holocene based on sedimentological, paleoecological, and geochronological analyses. Based on these data, previous ecological and environmental changes can be reconstructed that may allow scientists and managers to decide whether or not current declines observed in corals and coral reefs are unprecedented and may serve as a baseline for future predictions. Therefore, we have revisited 22 drill cores extracted from the barrier and atoll reefs of Belize Fig. , which r13  at the Belize Barrier Reef ranged from 25 (winter) to 32 °C (summer) during 2000–2002 (Smithsonian field station). The offshore Belize area is microtidal. Tropical cyclones have passed the Belize reef system repeatedly during the Common Era and caused considerable damage in the reefs21.The climate of the area is tropical with average air temperatures from 25 °C in the winter to 29 °C in the summer, as measured on the central barrier reef at the Smithsonian field station on Carrie Bow Cay25. Those studies indicated that reef accretion rates average ca. 3 m kyr−1 and show a decreasing trend during the Holocene26. Several environmental factors have been influencing Holocene reef growth in Belize including sea-level rise, variation in siliciclastic input from the mainland, exposure to waves and currents, and precipitation, as well as differential subsidence25.Shallow coring on the marginal barrier and atoll reefs during previous studies recovered > 20 m of Holocene reef accretion, which started around 9 kyrs BP28 ice and earth model exhibiting the best overall fit  and decrease during the Holocene   and massive  coral reef accretion is in the same range and cannot be distinguished statistically based on a Kruskal–Wallis-test . Accretion rate correlates with the mean rate of sea-level rise  based on output from the optimal ANU model , but significant in case of the temperature anomaly of Osman et al.31   and corals from the Orbicella group (25.2 ± 1.7%). They are followed in decreasing abundance by A. cervicornis (9.5 ± 1.0%), as well as species of Millepora, Porites, Agaricia, Pseudodiploria, and Siderastrea, with abundances of < 8%  as compared to the younger 6–3 and 3–0 ka time bins   and Orbicella (n = 6) are most common . The abundance of the competitive group of corals exhibits no statistically significant trend through time . The abundance of stress-tolerant taxa is generally declining throughout the Holocene . In this group, Orbicella shows a significant decline in abundance from 29.5 ± 3.2% in the 6–3 ka to 13.8 ± 2.6% in the 3–0 ka time bin. Pseudodiploria abundance is decreasing at first from 5.8 ± 1.1% to 2.4 ± 0.8% and then increasing to 6.7 ± 1.7% within the three time bins. Conversely, the abundance of weedy taxa such as Agaricia and Porites is increasing over time   are close to the values calculated based on previous data27 and confirm the observation that reefs dominated by branched corals accrete at comparable rates as compared to reefs dominated by massive corals, even though the growth rates of these different morphologies differ by one order of magnitude39. The latter has been explained by common breakage of branched corals in shallow water and the steady growth of massive corals in somewhat deeper water with lower impacts of physical and biological disturbance36. Accretion rates of reef margins in Belize are in the same range as compared to other regions in the western Atlantic (Caribbean average 3.37 ± 1.56 m kyr-1), and somewhat lower when compared with data from the Indo-Pacific 40 and at the lower end of predictions of future sea-level rise by the IPCC (26–98 cm per century or 2.6–9.8 m kyr−1)29. This would confirm predictions of future reef accretion potential based on carbonate production estimates, which have shown that reefs in Belize, the wider Caribbean, and parts of the Indian Ocean are likely losing the capacity to track predicted rises in future sea level12.The new reef accretion rates from Belize barrier and atoll reefs  phase shift from A. cervicornis to Agaricia tenuifolia has been documented41. White band disease is common in Acropora, and the taxon has been shown to be rather vulnerable to anthropogenic stress42. Data from the present study suggest that comparable changes have also occurred, at least on longer timescales, during the Holocene. Weedy taxa in Belize reef margins show a statistically significant increase in abundance throughout the Holocene (from 8.5 ± 1.4 to 16.3 ± 2.8%), and stress-tolerant corals exhibit significant decline (from 35.0 ± 3.1 to 21.3 ± 3.3%). No significant change appears to have occurred in the relative abundance of all the competitive coral taxa , which underlines the deleterious effect of turbid and nutrient-rich waters created after the initial flooding of subaerially exposed reef banks with soil cover44. This effect has been potentially responsible for the fact that carbonate-producing organisms need a certain amount of time to become established on inundated platforms and start what is commonly called the carbonate factory46. Observations on St. Croix47 have challenged the inimical bank water hypothesis by showing that Holocene reefs had in fact drowned only well after platform inundation and the presumed presence of inimical waters for reasons not entirely clear. But the observation of this study that 86% (12 out of 14 cores) of the basal Holocene corals directly overlying Pleistocene reef pedestal are stress-tolerant, especially to sedimentation (Pseudodiploria)32, lends strong evidence for the validity of the inimical bank water model for Belize. For comparison, a Pseudodiploria sp. coral was also recovered at the very base of the Holocene succession overlying Pleistocene pedestal, recovered at the barrier reef margin at Carrie Bow Cay, Belize23. The observation that the relative abundance of weedy taxa has increased throughout the Holocene suggests that fecundity appears to become a more important trait in the high-energy reef margins of Belize as compared to maximized survival rate as in stress-tolerant corals such as Orbicella48. Likewise, a previous study has shown that the abundance of Orbicella sp. in cores from the fore reef at Carrie Bow Cay was relatively high in the older core parts and decreased towards younger (< 2 ka BP) parts where the branched coral A. cervicornis predominated22. Interestingly, in this study, the relative abundance of the stress-tolerant Pseudodiploria has almost tripled (2.4 ± 0.8% to 6.7 ± 1.7%) whereas that of Orbicella was divided in half (29.5 ± 3.2% to 13.8 ± 2.6%) from the 6–3 to the 3–0 ka time bins, possibly reflecting a deterioration in water quality as exerted by increased storm-induced and anthropogenic run-off49.At the base of Holocene coral reef successions, lags in reef accretion have been observed that apparently occurred after initial inundation of the reef pedestal. These lag times, which may occur between basal mangrove peat and overlying basal Holocene corals, lasted 2.04–2.19 ka in Belize26 as well as south Florida50. Coral sclerochronology51 and vegetation data52 from offshore Belize suggest also that warm and wet conditions during early Holocene times were followed by cooler and drier conditions in the mid-late Holocene. There are opposing data and models of the trends in Holocene temperature, described as the Holocene temperature conundrum53, such that it is not entirely clear how reef accretion in Belize fits in these patterns. The data of Marcott and others suggest a mid-late temperature fall following the Holocene Climate Optimum30, which would fit the vegetation and sclerochronology data from Belize. However, the correlation of our accretion-rate data and the Marcott-anomaly is insignificant , suggesting a deterioration in the environmental conditions for reef development over time, as also indicated by the increase in weedy coral taxa during the Holocene. It cannot be ruled out completely though, that additional age dating would potentially fill the existing acroporid gaps as it did in the case of A. palmata in the Florida Reef Tract57.Other than the discussed changes in the abundance of stress-tolerant and weedy corals, competitive taxa, including acroporids and milleporids, do not seem to exhibit considerable change during the Holocene. The abundance of competitive taxa in Belize reef margins was constantly high at approximately 50%. It could be questioned why the abundance has not decreased due to the decrease in accommodation space with lower rates of sea-level rise starting in the mid-late Holocene. Presumably, acroporids and milleporids responded to this by lateral growth, i.e., progradationA. palmata in particular during the second half of the 20th Century, exacerbated by the combined effect of the high sea-surface temperatures and the 1998 landfall of Category 5 Hurricane Mitch58. A comparison of the trends observed in this study to these recent changes in coral community structure in reef margins of Belize is hampered though, because quantitative monitoring programs either did not collect data on coral composition around the shallow reef margin59, or monitored entities such as “branching coral” or “massive coral” that included several undesignated taxa60.Qualitative observations and anecdotal reports from along the Belize barrier and atoll reef margins clearly suggest a decline in acroporids in general and A. palmata and Orbicella spp. were the most common reef-building corals in Holocene reef margins of Belize barrier and atoll reefs. The abundance of competitive corals including acroporids appears to be relatively constant during the past 9000 years. Stress-tolerant taxa such as Pseudodiploria spp. predominate during Holocene reef initiation, and then subsequently decline. Meanwhile, the abundance of weedy taxa including Agaricia spp. and Porites spp. increases over time. A decline in the abundance of A. cervicornis and the existence of repeated gaps in A. palmata suggest that the recent demise in acroporids had precedents in the pre-Anthropocene Holocene. Also, reef-accretion rate is comparably low and has been decreasing during the Holocene.In summary, this study has shown that The twenty-two cores revisited during this project were collected during four expeditions  using a portable rotary drill with wireline system  using standard guidebooks62. Coral abundance was quantified by counting the numbers of drilled coral fragments > 2 cm diameter in core. In total, 1110 coral fragments were analyzed and identified. We excluded fragments with a diameter < 2 cm from our analysis due to the challenge of correctly attributing them to a taxon. Fragments that evidently belonged to the same coral as seen in fitting fracture surfaces were naturally counted as one specimen. This pitfall is usually unproblematic in massive corals. In branched corals, in our case largely A. cervicornis, each fragment > 2 cm encountered in core was counted separately. We used nine categories including A. palmata, A. cervicornis, Orbicella spp., Pseudodiploria spp., Porites spp., Siderastrea spp., Agaricia spp., Millepora spp., and a final catch-all category “miscellaneous coral”. Subsequently, categories were summarized using the live-history-trait concept of Cramer and others including fast-growth competitive , low-growth and stress-tolerant , and weedy, high-fecundity  corals32. The traits were characterized as maximizing growth (competitive), survival (stress-tolerant), and fecundity (weedy). The distribution and availability of age data in the 9 kyr-time period investigated, allowed the definition of three robust time bins during the Holocene  in order to meaningfully estimate temporal change.Cores were analyzed sedimentologically, taphonomically, and rates of reef accretion calculated14C-dates of corals (n = 127) and 14C-dates of basal mangrove peat (n = 14). Uncertainties are reported as 2-sigma (95% probability). Of the coral data, 69 are from acroporids, 33 from Orbicella, 12 from Pseudodiploria and 10 from Porites, Millepora, and Siderastrea , because the data were not normally distributed. Regression analysis was done using the OriginPro software package. Correlation analysis was used to compare accretion rates with environmental factors such as rate of sea-level rise and sea-surface temperature anomalies. We applied linear regression to test the temporal trends in reef accretion and in the observed changes in relative abundances of coral taxa composition. Slopes and their uncertainties (m kyr−1 and %·kyr−1) were calculated based on the principle of least squares to identify significant trends. We used the Poisson distribution as a model for coral count distribution. The uncertainty of the Poisson distribution is given with σ = √µ where µ denotes the mean value of the distribution65, which is identified with the number of corals per time bin. The relative abundance of the coral groups was computed and the uncertainties were propagated by Gaussian error propagation accounting for the uncertainty of the number of corals in the given group and of the sum of all corals found in the respective time bin. Uncertainties are reported as 1 standard error (SE). The time uncertainties used, represent the maximum dating errors in the three time bins .Other statistical methods include correlation analysis as well as Chi-Square and Kruskal–Wallis-tests performed using the PAST softwareSupplementary Information.


Acropora nana, Pocillopora acuta, and Porites lutea. Both Durusdinium and Cladocopium were present in all coral species at both reef sites across all seasons, but general trends in their detection (based on qPCR cycle) varied between sites and among species. Photochemical efficiency  was relatively similar between reef sites but differed consistently among species; no clear evidence of seasonal trends in v/FmF was found. Quantifying natural Symbiodiniaceae dynamics can help facilitate a more comprehensive interpretation of thermal tolerance response as well as plasticity potential of the coral holobiont.Ocean warming and marine heatwaves induced by climate change are impacting coral reefs globally, leading to coral bleaching and mortality. Yet, coral resistance and resilience to warming are not uniform across reef sites and corals can show inter- and intraspecific variability. To understand changes in coral health and to elucidate mechanisms of coral thermal tolerance, baseline data on the dynamics of coral holobiont performance under non-stressed conditions are needed. We monitored the seasonal dynamics of algal symbionts (family Symbiodiniaceae) hosted by corals from a chronically warmed and thermally variable reef compared to a thermally stable reef in southern Taiwan over 15 months. We assessed the genera and photochemical efficiency of Symbiodiniaceae in three coral species:  Further, species within a genus of Symbiodiniaceae can also show different responses to high temperature, which can vary based on coral host-algal symbiont pairing  . Symbiod pairing .in situ physiological/molecular data under unstressed conditions . Such data are needed to put monitoring studies  and short-term laboratory experiments in context. This is especially pertinent when assessing the potential role of coral traits and reef site characteristics  on the acquisition of thermal resistance and resilience.In general, a challenge to pinpointing potential mechanisms of thermal tolerance in corals is the lack of long-term, Acropora nana, Pocillopora acuta, and Porites lutea, at both sites. We predicted that in the absence of large thermal anomalies, Symbiodiniaceae genus/genera associations would remain stable across seasons but would show site- and species-specific differences due to the distinct thermal regimes of the reef sites and species life-histories, respectively. We predicted that photochemical efficiency would vary across seasons, but that seasonal trends among species would be similar.We assessed the seasonal dynamics of algal symbionts hosted by corals from a chronically warmed and thermally variable reef compared to a thermally stable reef in southern Taiwan over 15 months. We examined the dynamics of Symbiodiniaceae genus associations and photochemical efficiency across three common coral species with different life-history traits: This study was initially reported in e.g., e.g., tourism and nutrient pollution  and Wanlitung reef , are located in southern Taiwan . Outlet ollution ). Live collution , and ∼17ollution .5), nitrite (NO2−), nitrate (NO3−), ammonia (NH3), and phosphate (PO43−). Water samples were taken monthly from the same location and depth (∼3 m) at each site; the two study sites were sampled on the same day and within 1 h of each other.A temperature logger  was deployed at ∼3 m depth at each site and recorded seawater temperature every 10 min from June 2018 to August 2019. Some temperature data collected in this study have been published as supplementary information for a complementary laboratory experiment using the same sites and coral species see . Water cA. nana, P. acuta, and P. lutea, situated at ∼2–4 m depth, were tagged at each reef in May 2018. The same colonies were sampled five times : summer 2018 (July 30 and August 1), fall 2018 , winter 2019 (February 22 and 21), spring 2019 (April 27 and 23), and summer 2019 (August 29 and 27) . In the rare instances where a colony had died or could not be relocated, a new colony was tagged and sampled  or a hammer and chisel (P. lutea). Colonies of each species were situated at least 5 m away from each other  and P. acuta . DNA extraction was accomplished using a salting-out method modified from A subset of six colonies per species from each site was selected for Symbiodiniaceae analysis see  and S4 aCladocopium sp. and Durusdinium sp. in coral samples was determined using the primer pairs for Cladocopium and Durusdinium-specific ITS1 in qPCR assays , and deionized sterile water to a total volume of 10 µL. Based on the protocol described in i.e., dominant vs. background genus) was not conducted in this study. Instead, we provide a coarse assessment of the presence/absence of Symbiodiniaceae upon which we hope future research can investigate in more detail.The presence or absence of R assays . The detR assays . Each qPFv/Fm), a measure of photochemical efficiency (Fv/Fm were undertaken at our lab facilities (rather than in the field) to allow for effective dark adaptation, and because accurate in situ measurements would have been challenging during summer sampling due to the high wave action from typhoons and tropical storms commonly affecting the waters surrounding Taiwan. Another fragment from each colony was placed in ethanol for Symbiodiniaceae genera assessment. The last fragment from each colony was placed in liquid nitrogen for lipid assessment, but due to an error in sample storage  we have omitted these data due to concerns of possible sample degradation. We do, however, think it is important to mention our error here as a cautionary tale; see Coral fragments were immediately transported to the research facilities of the National Museum of Marine Biology and Aquarium  for sampling and photochemistry measurements. Corals were held in coolers filled with seawater from the source reef during transportation; transit duration was approximately 10 min (Wanlitung reef) or 20 min (Outlet reef). One fragment from each colony was dark adapted for 30 min and three replicate measurements of maximum quantum yield . Linear mixed-effects model assumptions were visually checked by plotting model residuals and through calculation of generalized variance inflation factors; GVIF(1/(2*df) was used to calculate generalized variation inflation factors as this approach is more suitable for categorical variables. We report the presence/absence of Cladocopium and Durusdinium detected in coral colonies at each site across seasons for each species. We used either a Student’s t-test or a Mann–Whitney U test (depending on data distribution) to assess difference between reef sites for each water chemistry parameter; model assumptions of normality and equal variance were tested using Shapiro–Wilk and Levene’s tests, respectively. All analyses were conducted in R  to assess (1) the effect of reef site and season, and their interaction, on water temperature , and (2) the effect of reef site, season, and coral species, and their interactions, on ted in R  using thted in R , lmerTested in R , emmeansted in R , car  and spring   , with the exception of fall 2018 when no difference was found between sites  and summer 2019    at Outlet and Wanlitung reefs over the study period of June 2018-August 2019 was 27.9 ± 1.8 °C and 27.8 ± 1.7 °C, respectively  & 1B. Me< 0.001) . Outlet < 0.001) . Outlet en sites . Lower t< 0.001) . Water cor NO3−) . In briei.e., based on qPCR amplification cycle) showed site- and species-specific patterns that remained relatively consistent across seasons. Colonies of A. nana at Outlet reef  showed the presence of both Cladocopium and Durusdinium starting at ∼15 qPCR cycles, whereas A. nana from Wanlitung reef  showed the presence of Cladocopium at ∼15 qPCR cycles and Durusdinium at ≥ 30 qPCR cycles  than Cladocopium (≥ 25 qPCR cycles) in colonies from Outlet reef, but the opposite pattern was observed for colonies from Wanlitung reef   in colonies from Outlet reef than Wanlitung reef (≥ 30 qPCR cycles)  . In P. l cycles) . Coral sFv/Fm between reef sites occurred periodically in some seasons, with colonies from Outlet reef  having higher Fv/Fm than colonies from Wanlitung reef  , for P. acuta in fall 2018 and spring 2019 , and for P. lutea in summer 2018 only .Intraspecific differences in le reef) –5C. ThisFv/Fm showed similar trends at both reef sites. Colonies of P. acuta and A. nana did not differ in Fv/Fm in any season, and both species periodically had higher Fv/Fm than P. lutea   but only P. acuta was higher than P. lutea in summer 2018 . At Wanlitung reef, both P. acuta and A. nana had higher Fv/Fm than P. lutea in summer 2018, winter 2019, spring 2019, and summer 2019 , but only P. acuta was higher than P. lutea in summer 2018 . There was no difference in Fv/Fm among species at Wanlitung reef in fall 2018.Interspecific differences in  ± 0.03) –5C. At OFv/Fm but some variation over time was observed in each species at each site , whereas at Wanlitung reef Fv/Fm was higher in spring 2019 than all other seasons . In P. acuta, Fv/Fm was higher in spring 2019 than summer 2019 at Outlet reef , in contrast to Wanlitung reef where Fv/Fm was only higher in winter 2019 compared to fall 2018 . In P. lutea, Fv/Fm at Outlet reef was lower in summer 2019 than all other seasons except for spring 2019, and was higher in fall 2018 compared to spring 2019 , whereas at Wanlitung reef Fv/Fm was higher in fall 2018 and spring 2019 than in summer 2019 .Overall, there were no clear seasonal trends in ach site –5C. In AA. nana, P. acuta, and P. lutea colonies at a warmed and thermally variable reef (Outlet reef) and a thermally stable reef (Wanlitung reef) showed site- and species-specific patterns. In general, Durusdinium was detected earlier in the qPCR cycle than Cladocopium in colonies from the warmed and thermally variable reef than at the thermally stable reef. Symbiodiniaceae associations remained relatively consistent across seasons. Reef site patterns in Symbiodiniaceae associations were not clearly mirrored by similar site patterns in photochemical efficiency. Intraspecific Fv/Fm differed between sites in only one or two seasons for each coral species, with corals from the warmed and thermally variable reef periodically having higher Fv/Fm. Interspecific differences showed similar patterns at both sites, with A. nana and P. acuta typically having higher Fv/Fm than P. lutea. Although there was some subtle variation in Fv/Fm over time for each species at each site, no consistent seasonal trends in Fv/Fm were observed.Our comparison of algal symbiont dynamics in e.g., genetic adaptations  of Symbiodiniaceae genera in coral tissues can change after stress events  with more thermally tolerant genera typically replacing thermally sensitive ones  throughout our study, with no heatwaves or summer mass bleaching observed. Symbiodiniaceae genus fidelity may also be attributed to acclimatization/adaptation to reef site characteristics , and seasonally variable at Wanlitung reef but not at Outlet reef. In our study, typical seasonal differences in temperature and light may not have been enough to elicit significant changes in Fv/Fm for our coral species. Future investigation into other photochemical metrics may be useful to better assess photosystem dynamics . This iciennesi , algal sciennesi , and/or ciennesi .i.e., genera or species-level quantification) to better assess the influence of thermal regime on Symbiodiniaceae associations among coral hosts. Baseline seasonal data under non-stressed conditions are pertinent to improve our understanding of energy provision sources relevant to coral thermal tolerance. Identifying typical ranges of normal variability in coral and Symbiodiniaceae physiology, coupled with an appreciation of the role that species traits and reef characteristics play, will allow for a better understanding of coral holobiont resistance and resilience in a warming ocean.We did not detect clear evidence of seasonal trends in dominant Symbiodiniaceae genera or photochemical efficiency in our study species, rather differences were more apparent among sites with contrasting thermal regimes and among coral species—highlighting the importance of species-specific studies. Reef site patterns that we observed in Symbiodiniaceae genera detection, using a coarse presence/absence qPCR approach, merit more comprehensive investigation (10.7717/peerj.15421/supp-1Supplemental Information 1Click here for additional data file.


Fusarium graminearum. Live-cell imaging showed that all the components of the RNA exosome complex are localized in the nucleus. FgEXOSC1 and FgEXOSCA were successfully knocked out; they are both involved in the vegetative growth, sexual reproduction, and pathogenicity of F. graminearum. Moreover, deletion of FgEXOSC1 resulted in abnormal toxisomes, decreased deoxynivalenol (DON) production, and downregulation of the expression levels of DON biosynthesis genes. The RNA-binding domain and N-terminal region of FgExosc1 are required for its normal localization and functions. Transcriptome sequencing (RNA-seq) showed that the disruption of FgEXOSC1 resulted in differential expression of 3,439 genes. Genes involved in processing of noncoding RNA (ncRNA), rRNA and ncRNA metabolism, ribosome biogenesis, and ribonucleoprotein complex biogenesis were significantly upregulated. Furthermore, subcellular localization, green fluorescent protein (GFP) pulldown, and coimmunoprecipitation (co-IP) assays demonstrated that FgExosc1 associates with the other components of the RNA exosome to form the RNA exosome complex in F. graminearum. Deletion of FgEXOSC1 and FgEXOSCA reduced the relative expression of some of the other subunits of the RNA exosome. Deletion of FgEXOSC1 affected the localization of FgExosc4, FgExosc6, and FgExosc7. In summary, our study reveals that the RNA exosome is involved in vegetative growth, sexual reproduction, DON production, and pathogenicity of F. graminearum.The RNA exosome complex is a conserved, multisubunit RNase complex that contributes to the processing and degradation of RNAs in mammalian cells. However, the roles of the RNA exosome in phytopathogenic fungi and how it relates to fungal development and pathogenicity remain unclear. Herein, we identified 12 components of the RNA exosome in the wheat fungal pathogen IMPORTANCE The RNA exosome complex is the most versatile RNA degradation machinery in eukaryotes. However, little is known about how this complex regulates the development and pathogenicity of plant-pathogenic fungi. In this study, we systematically identified 12 components of the RNA exosome complex in Fusarium head blight fungus Fusarium graminearum and first unveiled their subcellular localizations and established their biological functions in relation to the fungal development and pathogenesis. All the RNA exosome components are localized in the nucleus. FgExosc1 and FgExoscA are both required for the vegetative growth, sexual reproduction, DON production and pathogenicity in F. graminearum. FgExosc1 is involved in ncRNA processing, rRNA and ncRNA metabolism process, ribosome biogenesis and ribonucleoprotein complex biogenesis. FgExosc1 associates with the other components of RNA exosome complex and form the exosome complex in F. graminearum. Our study provides new insights into the role of the RNA exosome in regulating RNA metabolism, which is associated with fungal development and pathogenicity. Rrp6 and Exo11Rrp44+Rrp6  proteins, namely, AtRrp6L1, AtRrp6L2 and AtRrp6L3 , while silencing of dMTR3, dRRP6, dRRP41, and dRRP42 by RNA interference (RNAi) resulted in death of the adult insects   of transcripts having developmental and virulence-associated functions  –32. In Ugenicity –35. In Munctions . MoCwf15unctions . In Botrirulence . As an iFusarium head blight (FHB) which is caused by Fusarium graminearum can result in serious economic losses, and mycotoxin contamination of wheat grains seriously threatens the health of humans and animals  revealed that FgExosc1 is involved in ncRNA processing, rRNA and ncRNA metabolism, ribosome biogenesis and ribonucleoprotein complex biogenesis. Furthermore, GFP pulldown and coimmunoprecipitation (co-IP) experiments showed that FgExosc1 associates with the other components of the RNA exosome complex in F. graminearum.In this study, we identified 12 candidate components of the RNA exosome complex in F. graminearum, we obtained the protein sequences of the components of RNA exosome in Saccharomyces cerevisiae and humans and identified 11 potential candidate proteins via BLASTp analysis, which we designated FgExosc1 (FGSG_13120), FgExosc2 (FGSG_09961), FgExosc3 (FGSG_08645), FgExosc4 (FGSG_01091), FgExosc5 (FGSG_09363), FgExosc6 (FGSG_04277), FgExosc7 (FGSG_10879), FgExosc8 (FGSG_08578). FgExosc9 (FGSG_05509), FgExosc10 (FGSG_06049), and FgExosc11 (FGSG_01184) (Table S1). In addition, we found a protein annotated as exosome-associated family (FGSG_08866) in the database FungiDB (https://fungidb.org), which we designated FgExoscA. Analyses of FgExoscA homologs revealed that it has no homologs in humans or S. cerevisiae, but it is present in a number of other filamentous fungi (Table S2), including Fusarium oxysporum, Magnaporthe oryzae, Sclerotinia sclerotiorum, and in some basidiomycete fungi (such as Ustilago maydis) as well as Aspergillus niger and Verticillium dahliae. These results suggest that ExoscA, a specific component of the RNA exosome, is widely distributed in fungi.To identify the components of RNA exosome in F. graminearum, we analyzed the conserved domains and motifs of the 12 RNA exosome components. As shown in Fig. S1 in the supplemental material, FgExosc1, FgExosc2, and FgExosc3 have the typical conserved RNA binding domain (RBD) S1; FgExosc4, FgExosc5, FgExosc6, FgExosc7, FgExosc8 and FgExosc9 have the typical RNase_PH domain; FgExosc10 contained the PMC2NT domain at the N terminus and HRDC  domain at the C terminus. FgExosc11 contains a PilT N terminus (PIN) and a Vac domain. FgExoscA contains only one SAS10-UTP domain.To further determine the architecture of RNA exosome components in F. graminearum in comparison with those in Saccharomyces cerevisiae, Magnaporthe oryzae, Aspergillus niger, Arabidopsis thaliana, and Homo sapiens, where we constructed a phylogenetic tree of the RNA exosome components in these species (Table S2). As shown in Further, we analyzed the phylogenetic relationship of RNA exosome components in F. graminearum, we tagged each of the protein components with a green fluorescent protein (GFP), under the control of their native promoters. As shown in F. graminearum.To investigate the subcellular localization of RNA exosome in F. graminearum, FgEXOSC1 and FgEXOSCA were successfully knocked out by homologous recombination . However, we could not obtain mutants of the other 10 genes after several attempts. Phenotypic analysis of the ΔFgexosc1 mutant shows that its growth rate was severely decreased compared to that of wild-type strain PH-1  agar plates and in glass tubes containing CM agar. In addition, the ΔFgexosc1 mutant displayed aberrant mycelia with swelling and vacuolization in liquid CM compared to wild-type strain PH-1  into the protoplasts of the ΔFgexosc1 and ΔFgexoscA mutants, respectively. We succeeded in generating the complemented ΔFgexosc1-C and ΔFgexoscA-C strains, respectively. Consistently, ΔFgexosc1-C and ΔFgexoscA-C strains were both found to have the phenotypic defects observed in all the mutants restored to the levels observed in the wild type.To investigate the biological functions of RNA exosome in ain PH-1  and B. Ominearum  and D. Main PH-1 . These rF. graminearum are known to be the main inocula infecting flowering wheat heads  medium. As shown in Fgexosc1 mutant is clearly reduced in comparison to that produced by wild-type strain PH-1, while the ΔFgexoscA mutant had no significant difference. In fact, at 6 days postinoculation in CMC medium, the ΔFgexosc1 mutant failed to produce any conidia. However, very few conidia were recorded when the incubation time of the mutant culture was extended to 9 days. The number of septa per conidium in the ΔFgexosc1 and ΔFgexoscA mutants was similar to that observed in the wild-type strain conidia  of the ΔFgexosc1 and ΔFgexoscA mutants are 1.42 and 4.74, respectively, while the wild-type strain PH-1, ΔFgexosc1-C, and ΔFgexoscA-C strains have indices of 13.44, 13.00, and 13.33, respectively  medium. After 7 days of static culture at 28°C, DON production was determined. As shown in Fgexosc1 mutant was significantly low (0.89 mg/g) compared to the amount produced by the wild type (34.74 mg/g). In addition, the amount of DON produced by the ΔFgexoscA strain was nearly half of that produced by the wild type  in the wild-type strain PH-1 and the ΔFgexosc1 mutant cultured in TBI medium. We found that the expression levels of these genes in the ΔFgexosc1 mutant were significantly downregulated compared to those in the wild type , while the fluorescence intensities of FgTri1-GFP- and FgTri4-GFP-labeled toxisomes were not significantly different from those in wild-type strain PH-1 . Together, our data indicated that FgExosc1 and FgExoscA are both required for normal DON production and pathogenicity of F. graminearum.Deoxynivalenol (DON) is one of the virulence factors of nfection . To veriild type . To furtild type . Consistain PH-1 . In addiF. graminearum . To analyze the roles of this domain, we generated the GFP fusion constructs GFP-FgEXOSC1RBDΔ (ΔRBD), GFP-FgEXOSC1NΔ (ΔN), and GFP-FgEXOSC1CΔ (ΔC) with deletions of the RBD (aa 83 to 173), N-terminal domain (aa 1 to 82), and C-terminal domain (aa 174 to 208), respectively (Fgexosc1 mutant to obtain the GFP-Fgexosc1RBDΔ (ΔRBD), GFP-Fgexosc1NΔ (ΔN), and GFP-Fgexosc1CΔ (ΔC) mutants, respectively. Analyses of the phenotypes of these mutants revealed that the full-length RBD is required for the full functions of FgExosc1 in vegetative growth, conidiation, and pathogenicity, while the N-terminal region is only partially required  in ectively . Next, tNΔ appeared diffuse in the cytoplasm and did not accumulate in the nucleolus (RBDΔ displays a ring- or crescent-shaped appearance around the nucleolus but does not concentrate in the nucleolus (CΔ (ΔC) was not significantly affected when its C-terminal region was deleted  assay was performed to visualize the interactions in living cells. As shown in Fig. S5, FgExosc1 directly interacts with each of FgExosc2, FgExosc6, FgExosc7, and FgExoscA in the nucleus of F. graminearum. However, FgExosc1 does not directly interact with other RNA exosome components. Furthermore, BiFC was also used to check for possible interaction of FgExoscA with FgExosc2, FgExosc3, FgExosc4, FgExosc5, FgExosc6, FgExosc7, FgExosc8, FgExosc9, FgExosc10, and FgExosc11. The results showed that FgExoscA directly interacts with FgExosc1, FgExosc2, FgExosc3, and FgExosc8 in vivo  , 5, 56. AtCSL4 (EXOSC1) does not cause significant phenotypic changes, but homozygous mutations of the remaining components are deadly, while heterozygous mutations lead to different phenotypic defects (ScRRP6 (EXOSC10) in yeast leads to slow growth of the mutants, while the other components  are all essential for the survival of the organism  could not be detected in the cytoplasm. In yeast, Dis3/Rrp44 (Exosc11) is localized in both the nucleus and cytoplasm, while Rrp6 (Exosc10) is present only in the nucleus; in humans, Rrp6 is similarly present in both the nucleus and cytoplasm  and snoRNAs, bound to approximately 80 ribosome proteins , including OSP24, OSP25, and OSP44 . All the strains were cultured on CM, starch yeast medium (SYM), and minimal medium (MM) at 28°C for 3 days (r 3 days . For culF. graminearum were performed by following standard protocols (FgEXOSC1 (FGSG_13120) and FgEXOSCA (FGSG_08866) genes were deleted using split-marker approach  and native promoter of FgEXOSC1 and GFP genes using the primer pair in Table S4. The amplicons were ligated and then cloned into a pKNT vector and the product was sequenced for verification. The same method was used to construct the mCherry-FgEXOSC1 vector. The FgEXOSCA-GFP vector was generated by amplifying the ORF and native promoter of FgEXOSCA using the primer pair in Table S4. The gene sequence was tagged with GFP at its C terminus and then cloned into a pKNTG2 vector and finally sequenced for verification. The same method was used to generate the GFP fusion constructs for other RNA exosome complex subunits.Protoplast preparation and fungal transformation of rotocols . FgEXOSCapproach . The priTo assay asexual reproduction, PH-1 and the various mutants were inoculated into liquid carboxymethyl cellulose (CMC) medium to induce conidiation . The numInfection assays on flowering wheat heads and wheat coleoptiles were conducted as previously described , 71. MycFgexosc1 mutant were inoculated in liquid TBI for 3 days. Total RNA was isolated from mycelia using an Eastep total RNA extraction kit, and first-strand cDNA was synthesized using Moloney murine leukemia virus (M-MLV) reverse transcriptase as previously reported (−ΔΔCT (cycle threshold) method (F. graminearum housekeeping genes for actin (FGSG_07335) and β-tubulin (FGSG_06611) were used as the endogenous reference genes. A similar method was used to analyze the relative expression levels of FgEXOSC2, FgEXOSC3, FgEXOSC4, FgEXOSC5, FgEXOSC6, FgEXOSC7, FgEXOSC8, FgEXOSC9, FgEXOSC10, and FgEXOSC11 in ΔFgexosc1 and ΔFgexoscA mutants. All experiments and qRT-PCR assays were repeated three times.For quantitative real-time PCR (qRT-PCR) of DON biosynthesis genes, PH-1 and the Δreported . For rel) method . The F. Fgexosc1 mutant were cultured in CM for 2 and 4 days, respectively. Mycelia from the resulting cultures were then harvested and stored at −80°C for future use. RNA was extracted and detected using the RNA Nano 6000 assay kit of the Bioanalyzer 2100 system . The RNA library was sequenced on an Illumina NovaSeq platform and 150-bp paired-end reads were generated. Reference genome and gene model annotation files were downloaded directly from genome website. The mapped reads of each sample were assembled by StringTie (v1.3.3b) (Mycelia from PH-1 and the Δv1.3.3b)  in a refBriefly, GFP-FgExosc1 and PH-1-GFP strains were cultured in liquid CM for 2 days with shaking (180 rpm) at 28°C. Mycelia were then harvested and stored at −80°C. An affinity purification assay was then carried out as previously reported . For immFgEXOSC1 and FgEXOSCA were cloned into pCX62-cYFP vector . FgEXOSC1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, and -A were cloned into the pKNT-nYFP vector . The resulting constructs were verified by sequencing and then cotransformed into the protoplasts of PH-1 in pairs . YFP signals were observed using a Nikon A1R laser scanning confocal microscope. GFP and YFP excitations were detected at 488-nm light .All the primers used for BiFC assays are listed in Table S4. PRJNA894853.The RNA-seq data were deposited in the NCBI BioProject database under accession number


In the last decades, technological advances for RNA manipulation enabled and expanded its application in vaccine development. This approach comprises synthetic single-stranded mRNA molecules that direct the translation of the antigen responsible for activating the desired immune response. The success of RNA vaccines depends on the delivery vehicle. Among the systems, yeasts emerge as a new approach, already employed to deliver protein antigens, with efficacy demonstrated through preclinical and clinical trials. β-glucans and mannans in their walls are responsible for the adjuvant property of this system. Yeast β-glucan capsules, microparticles, and nanoparticles can modulate immune responses and have a high capacity to carry nucleic acids, with bioavailability upon oral immunization and targeting to receptors present in antigen-presenting cells (APCs). In addition, yeasts are suitable vehicles for the protection and specific delivery of therapeutic vaccines based on RNAi. Compared to protein antigens, the use of yeast for DNA or RNA vaccine delivery is less established and has fewer studies, most of them in the preclinical phase. Here, we present an overview of the attributes of yeast or its derivatives for the delivery of RNA-based vaccines, discussing the current challenges and prospects of this promising strategy. Vaccination is the most effective prophylaxis measure for infectious diseases . AccordiWithin this scenario, the mRNA-based vaccine production strategy proved to be a safe, fast, and effective alternative. Primarily developed decades ago, RNA vaccines took a long time to be widely employed due to inherent problems such as low stability, excessive immunostimulation, and low efficacy in some trials ,4. HowevSaccharomyces cerevisiae, Schizosaccharomyces pombe, and Pichia pastoris have been proposed as promising delivery systems for acid nucleic-based vaccines [In parallel, the interest in more efficient delivery systems has also received attention. Several methods have already been adopted, such as lipid nanoparticles used in mRNA vaccines developed for SARS-CoV-2 ,7. Howevvaccines ,10. Amonvaccines ,11. Yeasvaccines ,14,15,16vaccines . Althougvaccines ,18,19. UThe mRNA vaccines comprise synthetic single-stranded molecules that direct the translation of the antigen responsible for activating the immune response. Like endogenous mRNA, the in vitro assembly includes one or more antigen-encoding ORFs, untranslated regions, a 3′ poly-A tail, and a 5′ methylated cap that prevents unintended immune responses and degradation  Figure .Current classifications include three main types of RNA vaccines: conventional (mRNA), without modifications and self-amplifying capacity; base-modified non-amplifying mRNA (bmRNA) vaccines; and auto-amplifying mRNA (saRNA) vaccines . RNA vacUnlike most traditional vaccines, mRNA vaccines are temperature sensitive, which can reduce their efficacy. RNA is inherently unstable, and external factors like temperature, pH, and enzymatic activity can cause it to degrade quickly ,26. FurtThe success of mRNA vaccines also depends on delivery techniques. The vehicle must protect the RNA from possible digestion by ribonucleases and allow for effective uptake by the target cell, dissociation of genetic material from the delivery vehicle, and escape from endosomes ,31. In aSome microorganisms, such as yeasts, have demonstrated promise as biofactories for therapeutic proteins and carriers of biological molecules ,10. ThesSaccharomyces cerevisiae and Pichia pastoris have been proposed as delivery systems for protein antigens or nucleic acids in the development of prophylactic and therapeutic vaccines [Besides being recognized biotechnological platforms for the obtaining of immunobiological products, yeasts such as vaccines . This apvaccines ,9,54. Thvaccines . Althougvaccines ,18,19,34vaccines . Some covaccines . On the vaccines ,57.S. cerevisiae and P. pastoris, the exposure of β-1,3-glucan can be favored by heating the yeast at 60 °C for at least 1 h, expanding their adjuvant property [An advantage of the use of yeasts as vaccine vehicles is their immunostimulatory property due to the presence of β-1,3-glucans and mannoproteins in their wall, that, when detected by Pattern Recognition Receptors (PRRs), promote the release of cytokines, chemokines, and eicosanoids that modulate the inflammatory response in addition to leading to the development of specific adaptive immune responses against the pathogen ,59,60. Aproperty  , the capture and phagocytosis of yeasts are mediated by the presence of the Dectin-1 receptor that recognizes the β-1,3-glucans. Among the different receptors responsible for yeast recognition and phagocytosis, Dectin-1 is the receptor on APCs that recognize β-glucans, the major component of the yeast cell wall ,70 FiguB. ActivaPGK1, derived from the phosphoglycerate kinase (PGK) pathway. As well as PGK1, other constitutive promoters such as GAP  mediate transcription before the yeast phagocytosis [MLS1), nmt1 (thiamine-repressible) and isocitrate lyase (ICL1), whose activation and transcription mediation happen just after phagocytosis [AOX1), commonly used to regulate the expression of heterologous proteins in P. pastoris [GAL promoter that is inducible by galactose and can have its expression reduced or increased, depending on the carbon source that the yeast is using, glucose or ethanol [The ability to use yeast as a vaccine vector has been explored in different studies for the therapy of different types of cancer, as immunomodulators, and for the treatment or prophylaxis of infectious diseases ,17,72. Iocytosis . On the ocytosis ,73. Furtpastoris ,75, as w ethanol .The simultaneous carrying of DNA and RNA vaccines by yeast is a promising strategy. In this case, the vector used for yeast transformation must present a promoter for each vaccine antigen . In addiAnother component of the expression cassette, upstream to the target gene, is an IRES  sequence derived from EMCV . This region prevents antigen translation in yeast cells and increases translational levels in host cells, mainly in the macrophages ,73. The S. cerevisiae may be a useful tool for altering the profile of these TAMs within the context of the tumor microenvironment [One of the main applications of mRNA delivery by yeast has been immunomodulation in therapeutic approaches . Polarizironment . In addiThe yeast-based biodelivery goes beyond using the whole cell as a vaccine vehicle. Yeast cell particles (YCP) obtained from the mannoprotein layer removal have also been used for this purpose ,79,80 F. These pThe capsules can be recognized as a pathogen-associated molecular pattern (PAMP), induce and modulate immune responses, and can carry proteins, DNA, RNA, and small molecules. Antigen delivery based on these particles allows oral administration and promotes antigenic targeting to APCs due to the presence of β-1,3-D-glucan receptors in these immune cells ,84,86,87GPs are 2–4 µm hollow and porous structures that allow the encapsulation of a high load of antigens through central polyplexes and synthetic structures that help maintain the conformation and integrity of the particles for targeted delivery of antigens to APCs . GPs as The delivery of siRNA by GP NPs demonstrates high intracellular delivery capacity, even when compared to commercial carriers such as Lipofectamine, and has low toxicity .These NPGPs lack reproduction activity and are smaller than whole yeast cells . The morWhen assessing the efficacy of a vaccine strategy, the route of administration is an essential factor in immunogenicity as it determines effective antigen presentation, potency, and effector response . Thus, iP. pastoris administered intravenously in mice (about 48 h after its inoculation) since the absence of pathogenicity and ability to colonize leads to its elimination from the organism. Furthermore, it did not show the capacity to induce the secretion of cytokines and immunoglobulins, such as TNF-α, IL-6, IgM, and IgG, or a cellular response in the serum of the inoculated animals. Despite this, this lack of response to the vector minimizes the chances of adverse events occurring because of the innate immunity reaction against the yeast. The results showed no pathologies or alterations in the body, highlighting its safety [Oral, subcutaneous, intramuscular, and administration are common routes for yeast-based vaccines . The ints safety .Saccharomyces cerevisiae cell wall with sulfate to treat Newcastle disease (ND) in chickens [Regarding the intramuscular route, the currently studied vaccines use a portion of the structures found in the cell wall of yeasts known as B-glucans ,98. Theschickens ,101.S. cerevisiae to express human carcinoembryonic antigen (CEA) as a therapeutic strategy against cancer. Patients received the vaccine subcutaneously every two weeks for three months [Another typical route is subcutaneous administration, explored in the preclinical and clinical phases. Bilusic et al. (2014) used the yeast e months . The rese months .While vaccines are commonly administered through intramuscular or subcutaneous routes in numerous studies, these traditional methods have limitations. They present challenges in achieving a consistent and specific cellular and humoral response, providing inadequate protection in mucosal areas, and causing discomfort during the application, all contributing to low adherence rates . On the Despite this, some hurdles must be overcome in oral vaccines since natural local barriers, such as those found in the gastrointestinal tract, make accessing and distributing vaccine components problematic, resulting in limited antigen availability. Physical structures such as the mucus and glycocalyx layer, as well as low pH and the presence of proteases and nucleases on the mucosal surface that cause the breakdown of nucleic acids and antigenic proteins included in the formulation are among the reasons ,105.Alexander (2021) describes how yeast-based delivery systems for oral gene delivery can be explored both as vaccine strategies and for immunotherapy. Furthermore, the antigen can be delivered to the target tissue via the intestinal mononuclear phagocytic system, which then transports it to the systemic circulation . In thisIn addition to mRNA-based vaccines, different experimental models have demonstrated that it is possible to silence or modulate the expression of target genes in a specific way in eukaryotic cells through dsRNA (double-stranded RNA) or shRNA (short hairpin RNA) molecules ,112. TheIn recent years, the understanding of RNAi has enabled its use as a gene therapy tool against infections caused by different viruses such as coronaviruses associated with severe acute respiratory syndrome, influenza, hepatitis B virus, and Dengue virus ,116,117.However, the success of this strategy involves both the effectiveness of silencing and the non-occurrence of toxic effects. Thus, some adaptations are performed, including chemical modifications and changes in size, charge, and hydrophilicity that can interfere with the cellular uptake and biodistribution of RNAi molecules . The admIn this sense, the efficiency of this approach also depends on the use of efficient delivery systems ,121. LipS. cerevisiae, as observed by analyzing the dendritic cells from the intestine of immunized animals [In contrast, the yeasts in their various presentations  are feasible vehicles for the protection and specific delivery of therapeutic vaccines based on shRNA or dsRNA ,123,124. animals . AlthougAfter oral administration, the yeasts are phagocytosed by macrophages and dendritic cells in the gut-associated lymphatic tissues (GALT). These phagocytic cells, which are APCs, can migrate and infiltrate other lymphatic tissues and induce specific responses even in sites distant from the point of administration . This meAs already mentioned, the size of the yeast and the binding to specific receptors such as Dectin-1 and CD206 favor the process of phagocytosis by antigen-presenting cells . These iS. cerevisiae as a vehicle for a vector designed for a DNA vaccine encoding the myostatin gene and for delivery of an IL-21 shRNA vaccine as therapeutic targets. Studies like this open the way for improving vaccines with more than one type of vaccine antigen that can complement or amplify the desired immune response [In therapies based on RNAi transport by yeast, the main targets explored for silencing and immunomodulation are cytokines that play an effector role in the differentiation or maturation of APCs , cellular receptors , repressor molecules , or even specific antigens of infectious agents ,82,129. response .S. cerevisiae have a vast set of expression vectors that can be adapted for this type of application. The main components of these cassettes are the promoter, the selection mark, and the integration sites if desired. Both episomal and integrative vectors have been used, and integration has been seen to ensure more stable delivery. However, how this stability and the number of RNAi copies per cell can influence the effect or duration of gene silencing is still unclear [In addition to selecting therapeutic targets, it is necessary to consider how to construct the expression cassettes to be inserted into the yeast. Species like  unclear ,125. Con unclear  or activ unclear . When usS. cerevisiae, do not express Dicer and Argonaute. Thus, the bioengineered RNAi molecules can be accumulated without degrading [On the other hand, when using adopted native yeast promoters, it is expected the transcription and accumulation of RNAi in the yeast cell, such as egrading ,124. It egrading . It is aegrading . Inductiegrading ,123.S. cerevisiae). In addition, most of the thermal stability and duration of vaccine antigen expression studies have been tested for yeasts carrying protein antigens. Such adjustments are essential for yeast-delivered RNA vaccines to reach the pharmaceutical market. The development of RNA vaccines has gained a greater dimension due to the addition of optimizations in its structure and the discovery of new carriers capable of protecting the molecule, allowing their specific uptake to the target cells. Yeast cells or their derivatives (β-glucan particles) have these features appropriated for a vaccine delivery system besides their use as immunomodulators in therapeutic strategies. Despite the mentioned advantages, some points of study and investigation remain open for wide employment of yeast-delivered RNA vaccines. Appropriate methods for quantifying the carried RNA are still not well established, as well as the most effective protocols for incorporating RNA vaccines in yeast (whole yeast vaccines) and β-glucan capsules. To date, most of the published studies involve proofs of concept evaluated in a preclinical model. The analysis of the results of preclinical studies regarding the quality and safety of RNA vaccines carried by yeast indicates that in the medium term, these vaccines will advance to clinical phase studies. However, it is still necessary to obtain results from clinical studies evaluating the therapeutic action of this approach, to establish methods for quantifying yeast-delivered nucleic acids, standardize the concentration parameters of doses, and even to explore more yeast species that can be used (beyond


RNA modifications have emerged as a fundamental mechanism of post-transcriptional gene regulation, playing vital roles in cellular physiology and the development of various diseases. While the investigation of RNA modifications has seen significant advancements, the exploration of their implication in allergic diseases has been comparatively overlooked. Allergic reactions, including hay fever, asthma, eczema and food allergies, result from hypersensitive immune responses, affecting a considerable population worldwide. Despite the high prevalence, the molecular mechanisms underlying these responses remain partially understood. The potential role of RNA modifications in modulating the hypersensitive immune responses has yet to be fully elucidated. This mini-review seeks to shed light on potential connections between RNA modifications and allergy, highlighting recent findings and potential future research directions. By expanding our understanding of the complex interplay between RNA modifications and allergic responses, we hope to unlock new avenues for allergy diagnosis, prognosis, and therapeutic intervention. Canonical residues within RNA molecule can be modified in various ways including the addition of chemical group to either a base or a sugar, or both, isomerization, deamination etc. The number of possible RNA modifications described to date exceeds 170 . ModifieEnzymes and proteins that interact with RNA modifications can be roughly divided into three categories: (a) writers catalyze the inclusion of modifications, (b) readers recognize and bind them, and (c) erasers remove them. While the functions of the writers and erasers are obvious, the readers have various modes of action, such as direct implementation of certain catalytic activity like splicing  on the mH) cells, which then initiate T cell and B cell-based immune memory to the allergen. These allergic conditions are characterized by skewed systemic immune responses towards type 2 immunity, in which TH2 cells, high levels of IgE antibodies, but also activated type 2 innate lymphoid cells (ILCs) and eosinophils play important roles. This kind of immune responses are often characteristic to atopic dermatitis, allergic asthma, allergic rhinitis and food allergies  that is deposited by NSUN (NOL1/NOP2/SUN RNA methyltransferase) family proteins  Figure . m5C is Out of non-methylation RNA modifications, pseudouridines (Ψ) are strongly enriched in stable non-coding RNA species such as rRNA and tRNA where their main role is stabilization of secondary structure. Ψs are also present in other types of RNA, although with considerably decreased abundancy. Nevertheless, Ψ levels on mRNA can change significantly as well as expression and localization of pseudouridine synthases (PUS) , 56, 73 Inosine (I), a deamination product of adenosine, is another abundant RNA modification. It is catalyzed by adenosine deaminases acting on RNA (ADARs) –81 Figu. InosineConsidering the significance of RNA modifications for various regulatory cellular processes, recent research has extensively explored their implications in health disorders. Thus, RNA modifications and related proteins are strongly involved in cancer physiology as being markers for the disease, onco-suppressors or oncogenes, dependent on the type of cancer , neAlthough the molecular mechanisms underlying the allergic diseases are extensively studied, the role of RNA modifications remains unexplored and no direct link between RNA modifications and allergic diseases has been established. Therefore, here we only focus on those cell types, about which there is available research results correlating their function with RNA modifications. These include DCs, macrophages, T and B cells, and mast cells. While the implication of several other cell types, such as basophils, neutrophils, eosinophils and ILCs in allergy is widely recognized , 90, thein vitro model to differentiate them from peripheral blood CD14+ monocytes. Therefore, we discuss the effects of RNA modifications in these cell types here together (+/CD103+ DCs that are able to activate CD8+ T cells in the context of major histocompatibility complex (MHC) class I  mRNAs  mRNAs . Depleti2) mRNAs . One of 2) mRNAs  that is 2) mRNAs . Another2) mRNAs . As for 2) mRNAs  and NSUN2) mRNAs .H2 cells, promotes immunoglobulin class-switch recombination in B cells to activate the production of IgE antibodies. Maturation of B lymphocytes is a complex multi-stage process and depletion of ADAR1, performed on different stages, dramatically impairs consecutive development and strongly decreases the capacity of B cells to get activated by T cells (During IgE-mediated allergic reaction, activation of type 2 immune response conducted by T T cells –116. Def T cells . Class-s T cells .Mast cells and, to some extent, basophils are the major regulators of IgE-mediated allergic reaction. Aggregation of allergen-derived antigen-specific FcεRI receptors on the surface of MCs and basophils induces degranulation and consecutive inflammation, followed by the manifestation of symptoms, characteristic for allergic reaction . ComprehRNA modifications play a pivotal role in cellular function and regulation, impacting numerous biological processes essential for health and disease. Allergic diseases result from aberrant immune responses, involving various cell types and a complex interplay of cytokines, chemokines, and other immune mediators, which all lead to changes in adaptive and innate immune memory. We propose that RNA modifications play a critical role in these processes through their known contributions to the development and function of macrophages, DCs, T cells, B cells, and mast cells as well as by other yet undescribed mechanisms. As an example, we have generated a model, how m6A influences the development of allergic inflammation . Still, Furthermore, changes in RNA modification patterns could reflect disease status, progression, or response to treatment. Moreover, enzymes responsible for RNA modifications could be exploited as markers for disease –124 and In conclusion, we propose that RNA modifications may play crucial roles in allergic disorders, impacting disease pathogenesis, diagnosis, and treatment. Continued exploration in this field can uncover new diagnostic markers and therapeutic targets, potentially revolutionizing allergy management and offering hope for millions of individuals suffering from these conditions.


In recent years, research on long non-coding RNAs (lncRNAs) has gained considerable attention due to the increasing number of newly identified transcripts. Several characteristics make their functional evaluation challenging, which called for the urgent need to combine molecular biology with other disciplines, including bioinformatics. Indeed, the recent development of computational pipelines and resources has greatly facilitated both the discovery and the mechanisms of action of lncRNAs. In this review, we present a curated collection of the most recent computational resources, which have been categorized into distinct groups: databases and annotation, identification and classification, interaction prediction, and structure prediction. As the repertoire of lncRNAs and their analysis tools continues to expand over the years, standardizing the computational pipelines and improving the existing annotation of lncRNAs will be crucial to facilitate functional genomics studies.  Furthermore, other in silico tools able to predict the interactions between lncRNAs and other biomolecules can be widely used to provide insights into the study of lncRNA-mediated biological networks. Finally, integrating prior knowledge with lncRNA expression data and identifying differentially expressed lncRNAs that participate in specific cellular pathways are useful approaches for studying these molecules effectively.From these considerations, it follows that lncRNA studies can benefit the combination of molecular with genomics and transcriptomics approaches. Indeed, bioinformatics can provide a potent set of tools for lncRNA identification within the genomes of different species thus offering a new window to infer their function, in both health and disease states. Several tools have been developed to analyze massive data generated by Next Generation Sequencing (NGS) technologies for the In this review, we summarize the collection of computational resources available for facilitating lncRNA research, classifying them into several categories to support annotation, classification, RNA/protein interactions, and structure analyses. Some of these tools have multiple features or functions and will be classified according to their primary use.22.1in silico resources. Among the RNAcentral hosted databases, LncBook and LNCipedia represent invaluable tools for lncRNA annotation Public databases are pivotal sources of scientific content. Annotation tools and resources offer the possibility to classify, standardize nomenclature by cross-referencing other databases and retrieve functional information about any given manually or computationally curated biological entities. In the last decade, many databases have been implemented with multiple ncRNA notions, such as sequences, interactions, and gene ontologies. In this direction, a huge international effort recently led to build RNAcentral The functional importance of lncRNA-mediated networks and interactions has recently led systems biology to develop tools for data storing and integration. In this direction, the database RAIN (RNA-protein Association and Interaction Networks) taxa, including RNA-RNA, RNA-protein, RNA-DNA, RNA-compound, and RNA-histone modification interactions.Other freely available databases include NPInter Furthermore, RNA-Chrom in silico data taken from other sources, such as NPInter Some of the available resources let users also explore RNA-RNA interactions, thus helping in the relations between lncRNAs and other types of short and long RNAs. An example is RISE (RNA Interactome from Sequencing Experiments) Growing evidence suggests that subcellular localization of lncRNAs could offer insights into their functionality. On this direction, in 2018 the database lncSLdb 2.2De novo identification of lncRNAs poses a challenging and non-trivial task which often requires a combination of both experimental and computational methodologies. One popular approach involves utilizing transcriptome assembly techniques that rely on high-throughput RNA sequencing (RNA-seq) data. These methodologies rely on de novo assembly algorithms, such as Trinity bona fide non-coding RNAs, the assembled transcripts can be further filtered based on other specific criteria, such as transcript length, exon-intron structure, and protein-coding potential The tools available for lncRNA annotation and coding-potential assessment, which are currently operational at the time of manuscript writing, are enlisted in Most of the available tools for the assessment of the coding potential of lncRNAs utilize primary sequence and/or structural information. For instance, CPAT Other tools utilize RNAseq data for lncRNA identification. Among them lncEvo EVlncRNA-pred RNAcode Collectively, these tools can identify lncRNAs by incorporating diverse features, such as primary sequences of RNAs and other information, including multiple sequence alignments. Frequently, a machine learning methodology underpins each of these tools to facilitate the training and construction of a model, which can accurately infer the coding potential. Many of these methods have been extensively compared LncRNA genes can also be identified by other functional characteristics, such as the presence of neighboring transcription factor binding sites or their proximity to specific chromatin domains.Several web servers and tools have been developed to help in deciphering the functions of lncRNAs. These tools, which include Co-LncRNA Overall, various bioinformatics and biostatistics methodologies can be employed in conjunction with experimental approaches, even if an exploration of these methodologies is beyond the scope of this review. However, experimental approaches aimed at identifying lncRNAs at both genome and transcriptome levels could be needed for a definitive identification of lncRNAs and their and annotation on public repositories.2.3loci, and regulating protein function, in both the nuclear and cytoplasmic compartments LncRNAs regulate gene expression through interactions with other molecules. For instance, they can engage with proteins in several ways, including direct binding, recruiting proteins to specific genomic Henceforth, we list some available bioinformatic tools for predicting lncRNA-protein interactions , which aRPISeq BGFE RBPsuite DM-RPIs (Deep Mining ncRNA-Protein Interactions) IPMiner (Interaction Pattern Miner) HLPI-Ensemble LPGNMF (Predicting Long Non-Coding RNA and Protein Interaction Using Graph Regularized Nonnegative Matrix Factorization) catRAPID PrismNet  LncADeep One key aspect of lncRNAs relies on their ability to functionally bind not only to proteins but also to nucleic acid sequences, including other RNA molecules. This binding capacity is reflected in their functioning as post-transcriptional regulators, being able to influence the expression of distinct genes directly or indirectly. Notoriously, lncRNAs can function as sponges for miRNAs In line with this, the output may serve as a starting point for conducting further experimental validations, such as performing cross-linking immunoprecipitation (CLIP) or reciprocal RNA pull-down approaches All these tools enable the prediction of putative interactions between lncRNAs and proteins or nucleic acids. Since the accuracy of these tools can vary depending on several aspects, including the datasets used for training, it is advisable to proceed with caution when inferring interactions with other molecular entities.2.4The formation of RNA secondary structures has been shown to drive the scaffolding activities of lncRNAs Although necessary, experimental procedures aimed at assessing the RNAs secondary structure can be time-consuming and expensive, leading to an increasing demand for automated tools to facilitate structure prediction. Considering this need, a plethora of algorithms and online resources have emerged in recent years, and can be used for studying RNA, and more specifically, lncRNAs .Table 3TOne of the most popular resources is ViennaRNA Web Services One major problem in predicting the secondary structure of lncRNAs is the existence of pseudoknots, which are complex secondary structures that arise when a single-stranded loop in the RNA base-pairs with another RNA region which is not adjacent in the primary sequence. The result is a structure in which two or more stem-loop structures are interlinked and create a “knot-like” appearance. PknotsRG The existence of tools that allow the comparison between RNA secondary structures is advantageous for researchers aiming to understand and interpret the relationships between different RNAs as well as differences and similarities among secondary structures inside the same RNA molecule. Web servers for the comparison of RNA sequences and secondary structures include Web-Beagle The identification of sequences or structure motifs in the RNA serves as a fundamental step in the discovery of potential RNA interactors. From this perspective, Adinolfi and collaborators In summary, several tools and web services exist for the prediction and comparison of lncRNA secondary structures. These tools use various approaches, including MFE methods, pseudoknot prediction, and deep learning for their predictions, providing advantages for functional studies on lncRNAs. Care should be taken when using computational tools to infer lncRNA functions on secondary structure predictions. Recent papers have highlighted some pitfalls in statistical methodologies behind those predictions, including the use of comparative sequence analysis 3Over the past few years, increasing attention was given to link lncRNA expression and nucleotide variations in genetic and complex diseases. Although next-generation RNA sequencing approaches have revealed numerous alterations in their expression, the implication of lncRNAs in disease is still in its infancy and demands further annotation and targeted methodologies. The improvement of deep-sequencing technologies made possible to acquire lncRNA sequences and study their mutations in cancer-related processes. These alterations include single nucleotide variations, indels, and copy number amplifications affecting non-coding regions of the genome LncRNAs have also been associated with diseases other than cancer, as a consequence of mutations occurring in their sequence and regulatory regions. Many studies have linked lncRNAs to a broad spectrum of diseases, including cardiometabolic traits loci (eQTL).The discovery of disease-associated lncRNAs, which could act through either direct or indirect pathogenic mechanisms, has been enhanced by the increasing use of research methodologies aimed at identifying genomic variants associated with diseases or traits, such as GWAS studies. In this context, a recent work led to the development of lncRNASNP 4The availability of computational tools capable of predicting the structural attributes, functional characteristics, and intermolecular interactions of RNA represents a valuable source for a more comprehensive understanding of the “dark side” of the genome. In the case of lncRNAs, the application of specialized bioinformatic pipelines, exclusively designed for analyzing this class of transcripts, is helping scientists to bridge the gap between the existence of still poorly characterized non-coding sequences and their possible impact on gene regulation.A plethora of bioinformatics tools has been developed to analyze the expression and regulation of lncRNAs. Broadly employed for the analysis of RNA sequencing data, prominent tools include DESeq2 in silico tools, that are thought for their direct and immediate application, is accompanied by the need of new structural, molecular and biochemical bench-based approaches. These can be either RNA or protein-centric methods for the analysis of RNA-protein interactions Scientists are also aware that predictive tools would require extra validations by means of experimental approaches. In fact, the advance of One of the areas that will undoubtedly require time and resources in the coming years is the standardization of the nomenclature for lncRNAs. The lack of standardized nomenclature for lncRNAs can lead to confusion and hinder data sharing and collaboration. Establishing clear and consistent naming conventions is crucial to facilitate communication and ensure that findings can be effectively integrated into the broader scientific community. Another crucial aspect is related to understanding the tissue-specific and condition-specific roles of lncRNAs, which is vital for unraveling their functions in health and disease. However, obtaining relevant data can be challenging, especially for rare cell types or under specific conditions. Furthermore, considering that lncRNAs often exhibit functional redundancy, where multiple lncRNAs may regulate the same genes or pathways, deciphering the individual contributions of these lncRNAs to cellular processes and disease states can be complex and requires sophisticated experimental design and analysis.Interdisciplinary collaboration from molecular biology, bioinformatics and related disciplines is expected to increase to make scientists more trustworthy and to address various issues related to the study of lncRNAs. This will strength open communication and more critical discussion on the importance of wet-lab approaches, such as CLIP-seq and PAR-CLIP-seq for the analysis of lncRNAs interactions. Training on large datasets and experimental validation will also beneficial for implementation of more accurate computational predictors and for the use of NGS methodologies to comprehensively profiling lncRNAs.Conceptualization: A.P., M.B. Data curation, formal analysis, investigation and methodology: A.P., M.B., G.P., M.H.C. Writing – review & editing: A.P., M.B., G.P., M.H.C. Project administration: A.P., M.B. Funding acquisition. M.B. All authors have commented on and approved the final version of the manuscript.The authors declare no competing interest.