Climate plays a central role in coral-reef development, especially in marginal environments. The high-latitude reefs of southeast Florida are currently non-accreting, relict systems with low coral cover. This region also did not support the extensive Late Pleistocene reef development observed in many other locations around the world; however, there is evidence of significant reef building in southeast Florida during the Holocene. Using 146 radiometric ages from reefs extending ~ 120 km along Florida’s southeast coast, we test the hypothesis that the latitudinal extent of Holocene reef development in this region was modulated by climatic variability. We demonstrate that although sea-level changes impacted rates of reef accretion and allowed reefs to backstep inshore as new habitats were flooded, sea level was not the ultimate cause of reef demise. Instead, we conclude that climate was the primary driver of the expansion and contraction of Florida’s reefs during the Holocene. Reefs grew to 26.7° N in southeast Florida during the relatively warm, stable climate at the beginning of the Holocene Thermal Maximum (HTM) ~ 10,000 years ago, but subsequent cooling and increased frequency of winter cold fronts were associated with the equatorward contraction of reef building. By ~ 7800 years ago, actively accreting reefs only extended to 26.1° N. Reefs further contracted to 25.8° N after 5800 years ago, and by 3000 years ago reef development had terminated throughout southern Florida (24.5–26.7° N). Modern warming is unlikely to simply reverse this trend, however, because the climate of the Anthropocene will be fundamentally different from the HTM. By increasing the frequency and intensity of both warm and cold extreme-weather events, contemporary climate change will instead amplify conditions inimical to reef development in marginal reef environments such as southern Florida, making them more likely to continue to deteriorate than to resume accretion in the future. Although the rate and magnitude of coral loss over the last 50 years may be without precedent in recent millennia3, climate has been a primary control on the rate, duration, and spatial extent of reef development throughout geologic history10. Coral-reef development is generally most extensive and most rapid in tropical environments, where temperatures are warm and stable12. Although recent coral-reef degradation has been driven primarily by elevated ocean temperatures2, many declines in the past have been attributed to cooling trends16. In environments where thermal conditions are marginal, even minor cooling has the potential to suppress or even shut down reef-building by species that evolved in the tropics18. Whereas a number of recent studies have suggested that marginal environments—high-latitude habitats, mesophotic reefs, and locations with elevated turbidity or upwelling—could serve as refugia from warming for thermally sensitive coral taxa22, most of these ecosystems do not support reef accretion at present20. An important question, therefore, is whether and how the response of marginal reefs to climatic trends in the past can be used to project the future development of today’s degrading reef ecosystems7 in response to anthropogenic warming trends and the impact of those recent trends on both warm and cold thermal extremes.Anthropogenic climate change is now considered to be the primary cause of coral-reef degradation globally23. The Holocene epoch was nonetheless characterized by significant thermal variability over multidecadal to millennial timescales24. Average global temperatures peaked ~ 10–6 thousand years ago (ka)—an interval known as the Holocene Thermal Maximum (HTM)—before declining significantly over the Middle to Late Holocene25. Whereas tropical environments are buffered from the most extreme thermal variability, subtropical habitats are sensitive to broad-scale climatic oscillations28. For example, the relatively warm climate of the HTM drove temporary poleward expansions of corals in Florida29, the northern Gulf of Mexico30, and the high-latitude Pacific .Climatic variability during the Holocene was moderate compared with changes over longer geologic intervals33, and periodic winter cold events frequently push them below this threshold35. As a result, contemporary reef growth throughout southern Florida is negligible36; however, recent range expansions of reef-building corals in Florida and other high-latitude locations have led to the suggestion that subtropical environments may serve as refugia for these corals in a future, warmer world30. It is unclear whether such range expansions would translate into expansions of reef-building in subtropical habitats. Here, we test the hypothesis that the latitudinal limits of reef-framework construction on Florida’s subtropical reefs were controlled primarily by climate during the Holocene. We track the changing geography of reef accretion along the northern extent of the Florida reef tract over the last ~ 10,000 years and compare the climatic drivers of Holocene and Anthropocene reef development to project the future of reef-building in marginal environments such as southern Florida.Florida’s subtropical reefs currently exist near the thermal minimum for reef development33, which extends southwest along the Florida Keys to the Dry Tortugas37. Whereas the FKRT has recently supported abundant populations of reef-building corals, the SFCRT to the north is a ‘relict’ reef system38, characterized by low-relief, hardbottom habitats with low coral cover40 .The Florida Reef Tract extends more than 500 km along Florida’s Atlantic coast from Dry Tortugas National Park to northern Palm Beach County . Listed in order of distance from shore , these habitats are the Nearshore Ridge Complex , Inner Reef , Middle Reef , Outer Reef , and Deep Ridge . Whereas all five reef habitats are present south of Hillsboro Inlet, the IR is absent north of the inlet, the MR terminates in southern Palm Beach County, and the OR only extends to central Palm Beach County40  The DR, which is a low-relief, hardbottom habitat rather than a true reef terrace, extends the furthest north, terminating near the boundary between Palm Beach and Martin Counties40. The distance between these habitats also varies with latitude, with the IR, MR, and OR converging in south Miami, just north of Fowey Rocks. The best developed habitats on the SFCRT are the OR and IR42, which are the focus of this study  and the data used in this study are summarized in Table We collected and radiometrically dated 62 new samples, including 52 51, there is no evidence of significant Pleistocene reef growth along the SFCRT. Indeed, the northern end of the FKRT is the limit of both contemporary reef development in southern Florida and of Late Pleistocene reefs31 (~ 400–125 ka). From Fowey Rocks north, a combination of coquina ridges and mixed carbonate/siliciclastic sandstones serve as the bedrock for the Holocene SFCRT51. The absence of Pleistocene reef deposits within the bedrock of the SFCRT suggests that reef growth was largely restricted to the lowest latitudes of southern Florida for much of the late Quaternary.Unlike the FKRT, which largely grew atop antecedent, late-Pleistocene reef frameworkA. palmata expanded into shallow-water environments from south Miami to central Palm Beach County  and averaged 8.3 m ky−1 . The youngest ages for this final period of reef growth at each location were 7.2, 8.1, and 7.8 ka, respectively. There was a second peak of A. palmata ages on the OR in Miami at ~ 5.5 ka  the SFCRT backstepped inshore and contracted to the south Fig.  to const53, allowing the IR to initiate 1–2 km inshore of the OR in some locations42; however, the extent and duration of the inshore expansion of the SFCRT varied by latitude  to the depths of the initiation surfaces on the IR of Broward (− 9 to − 12 m MSL)42, suggests that the IR likely established around the same time in both locations  in southern Palm Beach County interpreted by Finkl et al.54 suggested that the shallowest antecedent substrate available inshore of the OR there is at a depth of approximately 25 m. That would have been too deep for A. palmata reefs to establish during the Middle Holocene 53. The only shallower inshore, consolidated substrate is the NRC, which is presently at a depth of around -4 m MSL40, but this feature would not have flooded until ~ 5 ka53.The IR is absent north of the Hillsboro Inlet in northern Broward County Fig. 40, likel56 suggested that the > 500-yr gap and difference in elevation between published ages from the OR47 and IR48 of the SFCRT indicated that rapid, 6- to 7-m sea-level rise at ~ 8 ka shut down the development of the OR by reef-drowning. We revaluate this hypothesis in the context of our expanded dataset of A. palmata ages from southern Florida and recent sea-level reconstructions for the region57. The data we present on sea level in south Florida during the Holocene . Although we present the predictions of this model for the south Florida subregion, the model incorporates spatial correlations in sea level and, therefore, is reflective of regional trends from sea-level proxy data collected throughout the tropical western Atlantic53.Before high-resolution data from the Broward IR were available, some researchers57 60. Together, these reconstructions suggest that although high rates of sea-level rise at ~ 8.2 ka have been implicated as a driver of reef demise in a number of locations around the world56, the evidence for an abrupt sea-level jump at this time that would have been large enough to cause reef drowning is not robust in many locations59, including southern Florida49.Several recent studies of Holocene sea level from the western Atlantic support the conclusion that sea level rose gradually over the transition from the Early to Middle Holocene57 Fig. A, with m42  and growth of the OR was keeping pace with sea level at the time of its shutdown , because of the relatively lower rates of sea-level rise after ~ 8 ka (3.9–2.1 m ky−1 from 8.1–5.8 ka)53; however, rates of A. palmata accretion on the IR continued to keep pace with sea level throughout the Middle Holocene .Similarly, although rising sea level promoted the initiation of the IR in some locations by flooding antecedent surfaces inshore of the OR, sea level cannot explain the demise of the IR. Accretion during the Middle Holocene on the IR was significantly slower than during the Early Holocene on the OR in Broward  suggests a common, yet unidentified, driver62.Another hypothesis for the shutdown of Early Holocene reefs in the western Atlantic was that resuspension of terrigenous sediments from sea-level flooding of inshore areas caused reef drowning due to light limitation and eutrophications62 Fig. . AlthougA. palmata reef growth off Miami was continuous from ~ 10–3 ka, in Broward, reef development ceased by ~ 5.7 ka. At the northern limits of the SFCRT in Palm Beach, reefs only grew until ~ 7.8 ka . Although the development of the SFCRT was controlled by the interaction between antecedent geomorphology, sea-level rise, and climate, we argue that climate was the primary driver of its contraction and eventual demise.Whereas ka Figs. , 3. This11. In southern Florida, temperatures typically remain above this minimum, but winter cold-fronts periodically push reefs below that threshold31, limiting modern reef development in the region29. For example in January of 1977 and 2010, Florida’s reefs experienced two of the most extreme cold events on record, with prolonged low-temperature excursions causing significant coral mortality throughout much of the region35. Because A. palmata is particularly cold-sensitive, even more moderate cold fronts would have been sufficient to limit its historic distribution to habitats south of the SFCRT33 and suppress significant reef accretion over millennial timescales29. Currently, extreme cold fronts impact southern Florida with a period of ~ 20 years18; however, it is likely that climate modulated the periodicity and geography of cold-front impacts during the Holocene.The global distribution of coral reefs is generally limited to locations where minimum seawater temperatures exceed ~ 18 °C 26  is associated with increased meridional circulation and a southerly shift of the polar jet stream27. Conversely, a more northerly position of the ITCZ is associated with increased zonal circulation and a northerly shift of the polar jet stream27. High-resolution reconstructions of local, millennial-scale temperature variability from the marine environments of southeast Florida are not available at present and we were unable to directly reconstruct climate variability in this study due to limited sampling of the western Atlantic corals that have been shown to produce high-fidelity paleoclimate data: Orbicella faveolata and Siderastrea siderea28. For these reasons, and because our A. palmata ages only record the timing of reef shutdown rather than the full history of reef development in many locations, we were not able to statistically evaluate the relationship between climate and reef-building. Instead, for the following discussion we rely on broad correlations between reef development and Holocene variability in global-scale temperature24  and the ITCZ had continued to move south25 , a negative North Atlantic Oscillation65 (4.5–2.0 ka), and extreme southern excursions and high variability in the position of the ITCZ25 . The two Late-Holocene ages we obtained from the MR in Miami (1.4 and 0.5 ka50) were from unattached Mancinia areolata colonies that are commonly found in sea grass habitats, suggesting that the MR may represent a back-reef habitat that formed when the OR was accreting, rather than a well-developed fore-reef like the IR and OR.Although reef communities established on the less-developed MR habitats44, primarily Siderastrea siderea, Porites astreoides, Millepora alcicornis, Stephanocoenia intersepta, and Montastraea cavernosa in southeast Florida68; however, most of these species have relatively low capacity for carbonate production and they have not contributed significantly to the construction of Florida’s reefs44. Furthermore, in southeast Florida, recent cold-stress events were associated with increases in the abundance of non-calcifying organisms including gorgonians and fleshy macroalgae32. It is likely that the same suite of taxa would have colonized the relict reefs of the SFCRT during the Holocene following cold-related mortality of A. palmata, but unlike A. palmata, these non-reef-building taxa would have made little if any subsequent contribution to the reef framework.Most modern reefs throughout southern Florida are dominated by generalist or weedy corals69. In the marginal, high-latitude environments of southeast Florida, sea-level rise facilitated backstepping of reefs into inshore habitats during the Middle Holocene, but latitudinal expansion and contraction of Florida’s reefs appears to have been most strongly modulated by climatic variability29. The relatively warm, stable climate of the HTM allowed reefs in Florida and other high-latitude locations30 to expand their ranges poleward during the Early Holocene. As the climate cooled and the frequency of winter cold fronts increased, however, the SFCRT contracted equatorward. The shutdown of reef-building at the southernmost limits of the SFCRT at ~ 3 ka coincides with the termination of reef-building throughout the FKRT, which also appears to have occurred as a result of cooling9. A recent reconstruction of reef development from subtropical reefs in southeastern China16 likewise implicated cooling following the HTM as a likely cause of declines in development, which, in combination with the records from southern Florida, suggests that subtropical reefs may generally be more sensitive to climatic variability than those in more tropical locations. In addition, marginal reefs in the eastern tropical Pacific that experienced strong, seasonal, cold-water upwelling were more vulnerable to millennial-scale climatic perturbations during the Holocene than reefs with more stable annual temperatures . Whereas moderate levels of thermal variability can increase the resilience of reefs to modern climatic extremes71, these studies of Holocene reef development suggest that in marginal habitats, where variability in the physical environment is especially high, periodic disturbances may be more likely to push reefs past critical environmental thresholds for continued survival and reef-building71.Most reefs in the tropical western Atlantic grew more-or-less continuously during the Holocene, and reef development in many parts of the world was more strongly influenced by sea level than by climate29, and historic records suggest that many reefs on the FKRT had high coral cover as recently as the early 1970s31, carbonate production and reef accretion have remained negligible in most locations throughout southern Florida36. Furthermore, recent thermal- stress events and the devastating outbreak of stony coral tissue loss disease have significantly reduced the abundance of reef-building corals throughout the SFCRT72. As a result, the reef framework constructed over millennia is now rapidly eroding74. Active management and the restoration of reef-building corals has the potential to re-establish some key ecological processes and mitigate the problem of erosion on Florida’s reefs44, but a resumption of reef-building on the SFCRT is unlikely, at least on decadal to centennial scales. Whereas coral populations have the potential to respond to favorable conditions in the short-term, the geological process of reef-building is more vulnerable to environmental variability and more difficult to restore once it is lost9.Although populations of thermally sensitive acroporid corals have periodically expanded their ranges north to the SFCRT in recent decades in response to contemporary warming30, analogizing future climate to the HTM may be a false comparison. Anthropogenic climate change is not simply driving global warming: it is amplifying changes in the frequency, intensity, and geography of extreme-weather events76. For example, the increasing frequency and severity of high-temperature extremes will continue to cause widespread coral bleaching and outbreaks of infectious coral disease around the world76, and even putative cooler-water coral refugia such as high-latitude reefs, mesophotic reefs, and upwelling zones are not immune to these impacts20. Because the coral assemblages in these marginal environments are already depauperate and are isolated from the diminishing source populations elsewhere in the Caribbean, they may be even less resilient to climate change and other anthropogenic impacts than most ‘mainstream’ reefs20. Additionally, as a result of the disproportionate contemporary warming of the high-latitude northern Hemisphere , climate change may be amplifying the trend of suppressed zonal circulation78, favoring meridional flow patterns that are reminiscent of the longer-term shift that occurred during the Middle to Late Holocene78  and from a wastewater-pipe excavation through the IR at − 9.8 m MSL .The earliest data from the SFCRT reef subsurface were provided by Lighty et al.42 detailed the internal composition of the IR. A trench caused by the grounding of the submarine USS Memphis80 afforded yet another opportunity to observe and date intact reef framework , which marks the northernmost extent of the OR in Palm Beach County. Five samples were collected just south of that location from a 2-m-deep trench in the reef surface that had previously been dredged for a sewage outfall .Between August and November 2013, ABM and AEO collected a total of 12 reef-surface samples from four randomly selected sites on the OR offshore of Boynton Beach in Palm Beach County . At each site, exposures of internal reef framework were haphazardly sampled within two randomly selected 10 × 10 m quadrats using a hammer and chisel. Eight representative samples from the site were selected for radiometric dating in this study. On 7 July 2019, ABM and AEO collected eight additional 81. There were nine stations on the IR and eight on the OR. At each site, three permanent transects were established by installing markers at 0, 10, and 20 m along each transect. In addition, three sediment stations were established adjacent to each transect at each site. To install permanent markers at each of the monitoring stations, a total of 12 holes were drilled into the reef surface with a hydraulic-powered rotary drill using a 2.5-cm-diameter coring bit , one A. palmata from the OR in the same area, and two Ma. areolata samples from the MR near Government Cut .During the Fall of 2013, 17 permanent monitoring sites were established on the IR and OR off Miami Beach, Florida  as part of the compliance monitoring for the Port Miami (Government Cut) Deepening ProjectA. palmata we collected in this study were in their original growth positions, they were all collected from well-cemented reef framework, suggesting that they are likely in situ and not storm-ridge deposits. Furthermore, our observations of the exposed IR and OR in Miami , located 8 km SW of the site. For this study, 11 of the best-preserved samples of A. palmata and five samples of other coral species, representing a depth range of − 10.7 to − 17.1 m MSL, were selected for radiometric dating.On 8 May 2017, WFP, LTT, and MLR collected a total of 29 subfossil coral fragments by hand from the southern channel-wall of Government Cut, where the channel cuts through the in situ reef framework of Miami’s OR , deionized water for 15 min to remove detrital material from the skeletal matrix and were acid-etched to remove surficial contaminants prior to radiocarbon analysis. The samples from Boynton Beach were also soaked in a diluted 8% sodium hypochlorite solution to remove organics and pre-screened for evidence of diagenetic alteration using a combination of X-ray diffraction and petrographic analysis of thin sections. Those samples contained no detectable calcite and only minimal secondary aragonite.83. Although this method results in higher analytical uncertainties  than for samples dated using the standard AMS procedure, it has been shown to produce ages with high accuracy84. Dating methods for the ages derived in previous studies85 can be found in those publications . Conventional radiocarbon ages were corrected for the fractionation of δ13C based on measured δ13C, or δ13C = 0 ± 4‰ if δ13C was not measured. All radiocarbon ages were calibrated to years before present (where ‘present’ is 1950 C.E.) using the Marine13 calibration curve86 in Calib 7.0.2 software . In order to account for the temporally variable, local radiocarbon reservoir-age offset (ΔR) in southern Florida67, each radiocarbon age was assigned a predicted ΔR  from an empirical model of Holocene ΔR variability developed by Toth et al.87. Two U-series ages from Stathakopoulos and Riegl42 were excluded from our analysis because elevated 232Th indicated the possibility of contamination by detrital thorium, and an additional sample from that dataset was excluded because it was likely transported from its original depositional environment82. All radiometric age data, sample depths (and uncertainties), and sample metadata are provided in Toth et al.50 (10.5066/P9Z21NMU).All of the surface samples from West Palm Beach, eight of the reef-surface samples collected around Government Cut, and two of the Boynton Beach samples were dated by accelerator mass spectrometry (AMS) at the Woods Hole Oceanographic Institution’s National Ocean Sciences AMS Center (NOSAMS). The remaining six corals from Boynton Beach were dated by AMS at the Center for Applied Isotope Studies at the University of Georgia. The samples from south Miami were processed at the USGS Radiocarbon Laboratory in Reston, Virginia and were AMS-dated at the Lawrence Livermore National Laboratory. The remaining surface samples from around Government Cut were AMS dated at the Keck Carbon Cycle AMS Laboratory at UC Irvine using the rapid dating methodology described by Bush et al.A. palmata framework collected from Government Cut. For the previously published records85, we used published core logs58 to identify which intervals in the cores were dominated by A. palmata, massive corals, or mixed assemblages. Although we did not find a significant difference in accretion by massive, A. palmata, or mixed facies  using the IsoplotR package v.3.388. KDE creates a smoothed probability density curve based on the weighted distances of data within a sliding probability distribution function (kernel) window. The shape of the kernel (width of the window) is defined by a bandwidth parameter within the KDE. The IsoplotR package uses an adaptive bandwidth modifier89 that increases the bandwidth where data are sparse and decreases the bandwidth where the data are dense88. In our KDE analysis, we used a starting bandwidth of 300 years, based on the mean total (positive plus negative) 2σ uncertainty in the radiometric ages of 287 years in the dataset. The peaks in the KDE represent clustering in the distribution of A. palmata ages near the reef surface, which we assume to represent the last period of reef development in any given location. Termination of reef accretion most likely would have occurred sometime between this peak and the youngest A. palmata age at that location. We used this range of ages as a conservative estimate of the timing of reef shutdown rather than simply relying on the youngest A. palmata ages at each location because minimum ages are subject to sampling biases.The time at which reef development terminated at each site was determined based on the ages of A. palmata ages from the OR of Miami that were significantly younger (by > 2.3 ky on average) than the rest of the ages in the dataset . These ages represent a separate, later interval when A. palmata was present on the Miami OR that would have biased the comparison among subregions. Without additional sampling it is not possible to determine if these ages represent the resumption of reef development on the Miami OR or a short-lived, isolated population. The timing of IR-termination in Broward and Miami was compared using a Mann–Whitney U test because the data were not normally distributed even after natural-log and square-root transformation. Final termination ages  were compared along a latitudinal gradient using a linear regression. The residuals of the model were normally distributed  with untransformed data. Accretion rates of A. palmata reefs were compared between the Early and Middle Holocene in the Broward subregion using an independent t-test. The data were normal  and homogeneous  after natural-log transformations. We also compared the rates of reef accretion during the Late Holocene by massive corals on the IR to the single estimate of reef accretion on the MR, 0.8 m ky−1 (Supplementary Table 90.OR-termination ages were compared among subregions using a Kruskal–Wallis test and Nemenyi post-hoc test because the data were not normally distributed even after natural-log or square-root transformation. For this analysis, we did not include three Supplementary Information.


The convolutional neural network (CNN) has been proven to have better performance in hyperspectral image (HSI) classification than traditional methods. Traditional CNN on hyperspectral image classification is used to pay more attention to spectral features and ignore spatial information. In this paper, a new HSI model called local and hybrid dilated convolution fusion network (LDFN) was proposed, which fuses the local information of details and rich spatial features by expanding the perception field. The details of our local and hybrid dilated convolution fusion network methods are as follows. First, many operations are selected, such as standard convolution, average pooling, dropout and batch normalization. Then, fusion operations of local and hybrid dilated convolution are included to extract rich spatial-spectral information. Last, different convolution layers are gathered into residual fusion networks and finally input into the softmax layer to classify. Three widely hyperspectral datasets  have been used in the experiments, which show that LDFN outperforms state-of-art classifiers. The technology of hyperspectral remote sensing makes full use of high-altitude detection equipment with visible light, infrared light and microwave and other technical methods through information processing and transmission, which can carry out the remote non-contact classification and recognition of ground objects. Hyperspectral image (HSI) has hundreds of adjacent narrow bands  that havIn the early age of hyperspectral image classification, traditional machine learning methods were widely used, for example, support vector machines (SVM) ,10, k-neIn recent years, with the development of technology, deep learning methods can provide more dynamic automation features. The basic idea of deep learning is that the training model resolves which features are more significant than others in the case of fewer human constraints. Therefore, deep learning methods have been widely used in HSI classification, for instance, M. He et al.  proposedThese models use different methods on the basis of deep learning. However, with the depth of network layers becoming deeper, they face difficulties with training and accuracy decline.Considering the problems above, the paper proposed a multi-scale feature fusion network based on local and hybrid dilated convolution (LDFN), which uses a fusion strategy that not only picks up the local information of details but also collects the rich spatial features by expanding the perception field. A residual fusion network was designed to integrate the local convolution and hybrid dilated convolution (HDC), which have a deeper structure of networks and fast connection with other layers. Therefore, our methods have great robustness and excellent ability to learn spatial-spectral information classification.In summary, the main contributions of this paper are three-fold.(1)The proposed hybrid dilated convolution stacked with different sizes of dilation rates is used to extract the spatial information.(2)Local and hybrid dilated convolution methods are integrated which can simply replace the traditional standard convolution. Local convolution connects local pixels closely, which can make our convolution layer more flexible and expressive. Then hybrid dilated convolution is able to raise the field of vision without raising the amount of computation, which can fully collect the spatial-spectral features of hyperspectral images.(3)The proposed new model also uses the specific residual network  to fuse The rest of this paper is arranged as follows. Traditional CNN consists of several normal operations, such as convolution operations, activation operations, batch normalization operations and pooling operations. The details of the convolution operations are as follows.(1)Convolutional Layersk means kernel. The formula of the convolutional layer . The other consists of deep learning methods, including 3D-CNN[19], 3D-CAE[20], D-CNN[22] and SSRN[21]. The different methods have the same input size of patch blocks as our LDFN model.The first one is carried out on the dataset of the Indian Pines. All methods choose 10% samples to train the model and 90% samples to test. The second one is based on the dataset of Salinas. A 1% sample was chosen to train the model and 99% to test all the methods. The third experiment is carried out on the dataset of Pavia University for which 2% of the samples are selected to train all methods’ models and 98% to test all the methods. In this section, different structures of local and HDC fusion models are compared to prove the effectiveness of the LDFN model. Since the local spatial-spectral contents are extracted by local convolution, the size of the HDC starts at 2 instead of 1. In general, the extensive experiments with three HSI datasets prove that our LDFN model is not only steady and convenient for training, but also effective and advanced in technology.In this paper, a novel deep learning method called the local and hybrid dilated convolution fusion network was proposed for HSI classification. The proposed local and hybrid dilation fusion network fuses local convolution and hybrid dilation convolution, local convolution connects local pixels closely, which can make our convolution layer more flexible and expressive. Hybrid dilation convolution stacked with different dilation rates of 2, 3 and 5 can raise the field of vision and consider the spatial correlation of hyperspectral images in adjacent areas without increasing the amount of computation. It also uses the specific residual fusion network to integrate the previous HDC and standard convolution into the main channels, which can not only solve the problem of the insufficient receptive field but also can extract multi-scale feature information. Experimental results demonstrate that the LDFN model can achieve a satisfactory classification accuracy for hyperspectral images under the lightweight standard.The proposed LDFN model still has great room for improvement. At present, the LDFN model still has redundant parameters and needs to spend some time training to extract spectral-spatial features. In future research, more attention will be paid to multi-scale information fusion and reducing model parameters, which may help optimize the LDFN model and can better integrate spectral features and spatial features for HSI classification.


Sequence variation in a widespread, recurrent, structured RNA 3D motif, the Sarcin/Ricin (S/R), was studied to address three related questions: First, how do the stabilities of structured RNA 3D motifs, composed of non-Watson–Crick (non-WC) basepairs, compare to WC-paired helices of similar length and sequence? Second, what are the effects on the stabilities of such motifs of isosteric and non-isosteric base substitutions in the non-WC pairs? And third, is there selection for particular base combinations in non-WC basepairs, depending on the temperature regime to which an organism adapts? A survey of large and small subunit rRNAs from organisms adapted to different temperatures revealed the presence of systematic sequence variations at many non-WC paired sites of S/R motifs. UV melting analysis and enzymatic digestion assays of oligonucleotides containing the motif suggest that more stable motifs tend to be more rigid. We further found that the base substitutions at non-Watson–Crick pairing sites can significantly affect the thermodynamic stabilities of S/R motifs and these effects are highly context specific indicating the importance of base-stacking and base-phosphate interactions on motif stability. This study highlights the significance of non-canonical base pairs and their contributions to modulating the stability and flexibility of RNA molecules. In fact, the standard subunits . The remsubunits .Previous work on RNA adaptation to different environments has focused on the ratio of GC to AU Watson–Crick base pairs  as GC baEscherichia coli (mesophile) and Thermus thermophilus (thermophile) by aligning the structurally conserved base-paired positions in the 5S, 16S and 23S rRNAs  base pairs and later studies were included non-canonical base pairs . ParticuIn this work, we use a different approach. We chose a recurrent, well-structured and relatively large internal loop (5 × 4 nucleotides not counting the closing bases or 6 × 5 with closing bases) for detailed study. We have chosen the sarcin-ricin (S/R) motif as the focus of study of phylogenetic and ecological sequence variation and thermodynamic measurements for these reasons:(i) The motif is recurrent and widespread in structured RNA molecules ; (ii) ItWe began by cataloguing the sequence variations observed in atomic-resolution 3D structures, and then we examined sequence variations of the motif at corresponding positions in aligned sequences. We collected these data to choose sequence variants for thermodynamic characterization. We also studied sequences containing single mutations that are predicted to disrupt the motif, guided by base pair isostericity matrices .Thermodynamic analysis of S/R motif variations found in organisms occupying different temperature niches and present in 3D structures indicates that a single change in a non-WC pair can significantly affect the overall stability of the motif. To verify whether sequence variants that have different thermodynamic stabilities still form the same motif, we probed their solution structures using single-strand specific nucleases T1 and A, double strand specific nuclease V1, as well as, DEPC. We found that isosteric base pair substitutions preserve the overall conformation of the motif, while single base substitutions that disrupt key non-WC base pairs destabilize the entire S/R motif.RNA oligonucleotides used for thermodynamic studies were obtained from IDT DNA (www.idtdna.com) and purified using preparative denaturing polyacrylamide gel electrophoresis (PAGE) or preparative 20 cm × 20 cm thin-layer chromatography (TLC) . RNA was eluted from gel slices or TLC matrix using a buffer containing 0.3 M NaCl, 10 mM Tris pH 8.0, 0.5 mM EDTA, then ethanol precipitated with 2.5 volume of cold 100% ethanol, rinsed twice with cold 80% ethanol, dried, and dissolved in 30 μl of double deionized (dd H2O) water. Purified RNAs produced single bands on a 16.5 cm × 20 cm analytical denaturing 20% polyacrylamide gel. For UV-melting measurements, the RNA stock solutions were extensively dialyzed first against 10 mM EDTA, 100 mM NaCl, and 10 mM sodium cacodylate and then against dd H2O and dissolved in dd H2O for storage. For each melting profile, a small volume of the oligomer stock was dissolved in 0.1 ml of buffer containing 10 mM sodium cacodylate (pH 6.9), 0.5 mM EDTA and either 10 mM MgCl2 and 100 mM NaCl or just 1.0 M NaCl.The RNA hairpin molecules for structural probing were prepared by runoff transcription of PCR amplified DNA templates. Synthetic DNA molecules coding for the anti-sense sequence of the desired RNA were purchased from IDT DNA (www.idtdna.com) and amplified by PCR using primers containing the T7 RNA polymerase promoter. Template strands for T7 transcription were designed using the online program written by Jesse Stombaugh (link: http://rna.bgsu.edu/oldwebsite/rnatodna.html). PCR products were purified using the QiaQuick PCR purification kit . RNA molecules were prepared by in vitro transcription using T7 RNA polymerase  and purified on denaturing PAGE as describe above.in vitro transcription, were labeled with gamma 32P at the 5′ terminus. Molecules were first dephosphorylated using Calf Intestinal alkaline phosphatase—10 000 U/ml  using the manufacture's procedure. The dephosphorylated RNAs were 5′-end labeled with [γ-32P] ATP (PerkinElmer Co.) using T4 polynucleotide kinase (New England Biolabs Inc.) according to procedure adopted from Ambion Applied Biosystems INC (http://www.ambion.com/techlib/misc/RNA5_labeling.html). Labeled RNAs were purified on 20% denaturing PAGE before use.RNA hairpins, prepared by 2 or just 1 M NaCl. Single-stranded extinction coefficients were calculated from the extinction coefficients for dinucleotide monophosphate and nucleosides using the literature parameters , 0.5 mM EDTA and either 0.1 M NaCl/10 mM MgClrameters . Strand rameters . AbsorbaTM were observed. However, transitions in the presence of Mg2+ were not reversible since Mg2+ catalyzes the hydrolysis of RNA backbone  for duplex formation of RNA S/R motif constructs were calculated from the melting profiles by curve fitting using the MeltWin (version 3.0) software. This program fits the shape of each curve to the two-state model with sloping base lines given by Equation (Thermodynamic parameters (ΔEquation  using a Equation .(1)\docuAT is the total absorbance, ASS is the absorbance of RNA single strands, ADS is the absorbance of the RNA double strand complex and f is the fraction of total RNA in the duplex form. Assuming the transitions from the duplex to single strands conform to the two-state model, then, the fraction of RNA duplexes can be related to equilibrium dissociation constant KD according to Equation \docu° values :(3)\docuR2) of ≥0.99 were observed for the fits to the melting curves using Equation \docuH° values from the two methods generally agree within 10% error  and snap-cooled on ice. To this we added 1 μl of 10 × Tris–HCl buffer  and the resulting mixtures were incubated at 30°C for 10 min . Finally, 1 μl of 50 mM MgCl2 was added and the mixture incubation continued for 30 min (assembling step). RNA samples were digested using 1 μl of T1 (1 U/μl), or A (0.011 ng/μl) or V1 (0.0005 U/μl) for 1 min at 30°C. The digestion reactions were stopped by adding 10 μl of colorless gel-loading solution . The products of digestions were analyzed on 8 M urea–15% sequencing PAGE.Prior to digestions, samples of radiolabeled hairpins 1 μl  containing 1 μl yeast tRNA (10 mg/ml) were heated to 94°C in 5 μl of dd H2O, frozen, and dried again on the SpeedVac to remove organics. Finally, cleaved RNA products were resuspended in 6 μl of denaturing loading dye solution  bromophenol blue) and analyzed on 8 M urea–15% sequencing PAGE.RNA chemical modification by a diethylpyrocarbonate (DEPC) was performed under native conditions. Briefly, labeled RNA hairpins  were annealed in SC buffer (pH 6.9) in presence of 10 μg yeast tRNA. Next, 15 μl of DEPC (SigmaAldrich) was added to each RNA sample. Samples were incubated for 30 min at 20°C with mixing every 10 min. Treated RNA strands were precipitated with 2.5 volume of cold ethanol and dried RNA pellets were resuspended in 20 μl aniline-acetate buffer . The cleavage reaction was performed by incubating the mixture for 15 min at 60°C in the dark. Samples were frozen on dry ice and then lyophilized in the SpeedVac. Pellets were resuspended in 50 μl of dd HLike many other ‘loops’ the nucleotides of S/R motif are represented as single-stranded in 2D structure. The standard S/R motif is an asymmetrical 5 × 4 internal loop Figure , left, ahttp://rna.bgsu.edu/rna3dhub/nrlist) using geometric discrepancy 0.8 Å/nucleotide as the similarity cutoff  TheWhile substitution of isosteric base pairs does not distort the 3D structure of the motif, it may affect the thermodynamic stability or the rigidity/flexibility of the structure. This aspect of isosteric substitutions in conserved motifs has not been addressed adequately in previous work.T. thermophilus, and T. maritima) the upper tHS base pair is always AG, whereas in mesophilic groups AA also occurs  relative to the prototype duplex. For example, the '#2_U2C' duplex U at position 2 changed to C. Each nucleotide substitution is colored in red Figure .E. coli.Motif location in rRNA, indicated by the helix number and organisms name, e.g. 23S-H13 Optimal growth temperature class .www.mfold.bioinfo.rpi.edu, Zuker 2003) to avoid the formation of self-complementary secondary structures, internal hairpin structures or misaligned duplexes. We used duplexes for melting experiments rather than hairpin constructs to avoid specific interactions of hairpins with divalent metal ions  of the #10_U2G/C12A duplex and obtained agreement with calculated parameters within 10% for the two methods, as shown in UV-melting experiments of S/R motif RNA duplexes were performed by monitoring of the absorbance change at 260 nm. Some RNA melting curves were also recorded at 280 nm as a check and gave similar results. Melting controls of single strands did not exhibit distinct transitions. Table 2+ ions, in particular, a melting temperature increase in the presence of 50 mM MgCl2 has been demonstrated for eukaryotic 5S loop E . The data suggest that majority of RNA duplexes generally have higher stability in Mg2+ containing buffer. The differences in thermodynamic parameters obtained between two buffers are summarized in Table The differences in thermodynamic parameters of RNA duplexes from 10 mM Mg2+ concentration for the S/R motif formation we measured TM of the #10_U2G/C12A RNA duplex as a function of [Mg2+] from 1 to 50 mM. Figure 2 the TM curve reaches its plateau and further increase in [Mg2+] does not significantly affect the TM. Thus, we used 10 mM MgCl2 in all experiments involving magnesium. Typical thermal denaturation curves of five representative RNA duplexes containing S/R motif sequence taken at 10 mM MgCl2 are shown in Figure 2+ were only melted once due to Mg2+ driven RNA cleavage.To find the optimal MgThe tHS base pair family frequently occurs in functional RNA motifs including S/R motif, Kink-turn, loop E, multi-way junctions as well as hairpin loops (e.g. GNRA hairpin loop). The most well known member of this family is the tHS A–G pair, also known as sheared AG base pair which often contributes to RNA stability by extensive cross-strand purine stacking.Sarcin/Ricin motifs contain two conserved tHS base pairs with opposite orientations. We refer to the tHS pair between nts 6 and 9 as the ‘upper tHS’ pair and the pair between 2 and 12 as the ‘lower tSH’ pair.Four variants of the upper tHS pairing position (nt 6 and 9) were studied:G°37 of –15.9 kcal/mol in the presence of Mg2+ ions. This tHS base combination is the most common in S/R motifs according to WebFR3D results  > tSH A2–C12  > tSH G2–A12  > tSH C2–C12  > tSH A2–A12  >> tSH U2–A12 .tSH U2–C12 (ΔAll lower tSH pairs are isosteric (see above). And only tSH U2–A12 is ambiguous. The A at position C12 could form a Watson–Crick base pair with U2, which is expected to disrupt the motif. However, UA is also an allowed base combination for tSH, and so it is possible that the tSH pairing geometry is conserved. This sequence variant tests the resilience of the motif to ambiguous base substitutions. In fact, this sequence variant occurs in the central junction of yeast 26S rRNA as shown in Figure G°37 of –11.5 kcal/mol and suggests that this substitution significantly reduces the stability of the S/R motif.On the other hand, the control molecule #4_ U2G comprises tSH G2–C12. The tSH GC combination has never been observed in any 3D structure and thus is not expected to form this combination. UV-melting data shows that this RNA duplex has a Δ2+:Comparison of the lower tSH isosteric sequence combinations of the S/R motif RNA duplexes melted in the presence of 1 M NaCl shows a slightly different order of stability as compared to the experiment with 10 mM MgG°37 = –13.1 kcal/mol) > tSH U2–C12  > tSH G2–A12  > tSH C2–C12  > tSH U2–A12  > tSH A2–A12 .tSH A2–C12 . However, comparison between molecules #10_U2G/C12A and #12_U2G/G9A/C12A with upper tHS A6–A9 and lower tSH G2–A12 shows a much larger difference in ΔΔG° of –6.8 kcal/mol (see Table TM are in green colored cells)Interestingly, all isosteric combinations at the lower tSH depend on the presence of Mget al.  interactions ,37. 3D sG°37 value of –16.6 kcal/mol, while ΔG°37 for the S/R motifs with bulges A4 and C4 were found to be –10.6 kcal/mol and –11.5 kcal/mol respectively. The free energy difference of the bulged G4 compared to A4 and C4 is ∼–5.5 kcal suggesting that the prototype duplex much more stable than A4 and C4 (duplexes G4A and G4C). However, mutations in bulged nucleotides result in significant differences in calculated entropy values. The calculated entropy values for these duplexes are shown in Table S° of –260.5 e.u. while substitution to bulged A4 or C4 resulted in significantly higher ΔS° values of –114.2 e.u. and –158.3 e.u. Even larger entropy change was observed in presence of 1 M NaCl for A4 and C4  at equivalent position from S/R motif of 16S rRNA of H17  that have differences only in the bulged nt G4, A4 and C4  were studied in which only tHH the base combination differed: tHH A3-A11, tHH G3-A11 and tHH A3-C11 .To directly probe the RNA duplexes used in UV-melting studies is not feasible, so we designed longer hairpin shaped molecule which included a control motif for probing experiments. The sequences of twelve of the sixteen RNA constructs designed for structure probing are shown Figure 32P phosphate. All RNAs were probed in their native conformations in the presence of 10 mM MgCl2. The buffer conditions and annealing procedures were identical to the UV-melting experiments described above to ensure that the RNA molecules adopt the same conformation. The cleavage data averaged over several experiments are summarized in Figure RNA structure probing was carried out with the helix specific RNase V1 and two single-strand specific nucleases T1 and A. RNAs were 5′-end labeled with tSH non-WC base pair with A34  and T. thermophilus (PDB: 1J5E) using R3DAlign . Each species and its subspecies were represented with a single sequence selected based on the highest A, C, G and U content. This was done in order to reduce the bias due to over representation of some organisms in the dataset. This dataset contains 561 sequences; within these sequences 82 belong to psychrophilic, 328 mesophilic, 108 thermophilic and 44 hyperthermophilic organisms (Supplementary table). We used Uniprot's taxonomy to obtain the phylogenetic information for each organism.The representative dataset was created by extracting all sequences corresponding to each species and all of its subspecies with known optimal growth temperatures from the Green Genes 16S alignment updated on October 6, 2010 . The Green Genes sequence with the largest number of aligned columns was used to correlate positions in the Green Genes alignment with the PDB sequence.Since some nucleotides in crystal structures are not resolved, the Green Genes alignment and the sequence from the 3D structure may not match. Therefore, we need to determine the correlation between the columns of the sequence alignments and the structure positions. To this end, we extracted all rows from Green Genes, which came from Using the correlations between sequence alignments and 3D structures we now examine the variations in S/R-like motifs H17, H27 and 3WJ found in the 16S rRNA. We extracted the columns from the alignment which corresponded to each motif. We then grouped each sequence by the phylum of its source organism and optimal growth temperature class and then counted unique sequences. The resulting tables can be found in Examining the S/R motif in the H27 of the 16S shows a strong correlation between AG and AA in the upper tHS base pair and growth temperature. As temperature increases the fraction of tHS AG also increases (Figure The bottom panel in Figure Overall, we do see some variations which may be due to temperature adaptation. However, our current methods are not precise enough to detect subtle changes. Further statistical analysis on a larger dataset is needed to validate these initial, limited findings.In the present work, we systematically study sequence variation at structurally conserved positions of S/R motif in rRNA from different organisms. Herein, our effort is aimed at gaining insight into the thermodynamics of isosteric and non-isosteric substitutions within the S/R motif. Besides the stability investigation, a structural probing analysis is performed to observe which mutations would disturb the motif.This study demonstrates that isosteric substitutions do not disturb the S/R motif as it was expected from survey of 3D structures and survey of 16S rRNA from organisms adapted to different temperatures.The most frequent variation was observed at the ‘lower’ tSH base pair and any neutral substitutions at this place do not significantly impact on the overall stability of the motif. On the other hand, non-isosteric substitutions destabilize the motif but the conformation is appears to be modest as reflected from structural probing experiments.Substitutions at the ‘upper’ tHS pair are the most crucial for the stability and conformation of the motif. Non-isosteric mutations destroy the geometry of S/R motif. Isosteric mutations tHS A6–A9 or C6–U9 destabilize the motif but the geometry is preserved. Moreover, tHS A6–A9 makes the motif more flexible as reflected by RNAse T1 digestion pattern.Any mutations at bulged nucleotides participating in triplet interaction significantly impact on the stability of the motif as shown by UV-melting experiments.tHS AG is the most frequent base pair in S/R motifs, whereas tHS AA is prevalent in cold environments. In addition, the bulged G, which is conserved in most organisms, mutates to U, A or C mostly in psychrophiles and mesophiles. This suggests that organisms may adapt to different temperature environments not only by adjusting the number of GC and AU Watson–Crick pairs in the helical regions as demonstrated previously (Our phylogenetic survey suggests a correlation between non-canonical base pairs and optimal growth temperatures. In high temperature environments eviously , but alsUsing S/R motif as an example, we have demonstrated that non-canonical interactions in structured RNA motifs are very important to overall structure and stability. By changing base pair at certain non-canonical positions we can control stability, structure, and perhaps function of large RNAs.gkab703_Supplemental_FilesClick here for additional data file.


DAZ and PRM1/PRM2 ratio) in semen and testicular tissue. Thus, they might be used as predictor biomarkersto evaluate success rate of testicular sperm extraction (TESE) procedure, but confirmation of this hypothesis requiresmore extensive research. By comparing the number of RNAs attributed to each fertility disorder in men, it is possibleto trace the causes of disease or return fertility to some infertile patients by regulating the mentioned molecules. Further researches can provide a better understanding of the use of RNA expression profiles in the diagnosis and treatmentof male infertility.Semen analysis is usually the first step in the assessment of male fertility. Although analyzes provide valuable information about male fertility, success of cytoplasmic sperm injection using this method is not predictable. In the recentyears, studies have shown that sperm quality assessment helps clinicians predict male fertility status based on theexpression of biomarkers. To write this article, a comprehensive study was conducted on several RNA transcriptsby searching related words on medical information databases by 2018. According to the literature, spermatogenesisbased disorders in male infertility have a significant relationship with the expression level of some RNA molecules(like  Sperm RNA contains several coding and non-codingtranscripts that represent a picture of past events, suchas spermatogenesis and sperm maturation. This, theyprovide new insights for male fertility and infertilityresearch. On the other hand, a new scope in infertilitystudy is participation of sperm RNA in the epigenetictransgenerational inheritance of the altered phenotypictraits in the progeny associated with paternal exposure. One of This review study was conducted on over 95 articlespublished in the Google Scholar, PubMed, Scopus,IranMedex, MEDLIB, IranDoc and Scientific InformationDatabase (SID) for the comprehensive information on thebiomarkers introduced for male infertility. All articleswere reviewed by the keywords of transcript, sperm,semen, testicular tissue and infertility, until September2018 and among them, 74 related papers were included. RNA evaluation in sperms is recommended becauseit may show a historical record of spermatogenesis.Additionally, it can be considered as genetic backgroundas well as fingerprint of the individual. Therefore, someRNAs may be brought up as potential diagnostic toolsfor evaluating male infertility and they may also play animportant role in the development of fetuses and zygotes. Dynamic cellular diversity has been reported in the RNAprofiles of fertile and infertile men, and therefore scientistsrefer to it as biomarker of infertility. Round spermatidscontain numerous varieties of transcripts that are stored inthe spermatid cytoplasm before expression of the relatedproteins. In the middle of spermatogenesis, chromatinremodeling results in genome transcriptional inactivation. Therefore, most RNA transcripts were transcribedbefore the inactivation process. Using techniques suchas real-time PCR, presence of the transcripts in humanspermatozoa was confirmed . Data evPLCζ),postacrosomal sheath WW domain-binding protein (PAWP)phospholipase C ζ ( and KITproto-oncogene receptor tyrosine kinase (KIT-Tr proteins),which initiate the cascade of oocyte activation signal. PLCζ gene inhumans, located at 12p12.3, is a family of phospholipase C enzyme. PLCζ protein is aspecial sperm protein with catalytic and domains catalytic X, Y and the Y-X bindingregion. At present, researchers often identify PLCζ as the most likely candidate forSAOAFs  in the posterior acrosomal region of thesperm head. During normal fertilization, when the sperm enters the egg, the egg isactivated. This is associated with an increase in the concentration of calcium in thecytoplasm. Studies showed that increased intracellular calcium concentrations of oocytesare due to spermatozoa SAOAFs, including the rSAOAFs .PLCζ was related to theoxidation of DNA sperm in human. Heytens et al.  treatment method can be used toincrease chance of improving fertilization rates in theseindividuals. Javadian-Elyaderani  and then with protamine (nd PRM2) .Protamind PRM2) . InIrany showed  thatPRMd et al.  study, itile men .PRM1/PRM2 mRNA ratio in ejaculatedspermatozoa could differentiate infertile from fertile groups. In terms ofTNP2, Savadi-Shiraz et al.  and sperm-head defects (P<0.05).Results of study performed by Liu et al. showed that normal development of sperm requiredmicroRNA-122 to control frequency of TNP2 mRNA and its subsequenttranslation  are members of thisfamily and expressed in the testicles of mature males. However, their function is unknownand needs to be investigated  compared to control group.Y-Box proteins are DNA and RNA-coupled proteins that play role in controlling geneexpression. According to the animal studies, expression of the pression . Moghbelpression  evaluated et al.  explainer et al.  showed aDuplication and unsuccessful differentiation of germcells are the main causes of infertility and they areaccomplished by regulating transcription of particulargenes. Non-coding RNAs, such as microRNAs and longnon-coding RNA (lncRNAs) are the main regulators ofthe expression of genes. The data obtained from deep-sequencing recently shows that lncRNAs are far morenumerous than protein-coding RNAs, thus proving thatthe human genome is more active in terms of transcriptioncompared to the previous view. Human testis tissue andimmature sperm have 7% miRNAs and 17% piRNAs.These small RNAs regulate gene expression at thetranscriptional, post-translational and chromatin levels.So far, it has been shown that one-third of human genesare regulated by miRNAs. In terms of numbers, morethan 200 miRNAs have thus far been found in humansperm, which indicates the important role of these RNAsin morphogenesis and sperm maturation . Somenomicro-RNAs (miRNAs) have been introduced as thekey regulators of gene expression at translation leveland control of post-translation changes. Several studiesshowed that these miRNAs interfere with spermatogenesisin controlling pathways that affect human reproduction,such as the survival of primordial germ cells andspermatogenesis. miRNAs are existed in various stagesof spermatogenesis and they have great expression inspermatid and spermatocyte pachytene cells. They exist in the body fluid in combination with lipoproteins or theyare enclosed in packages of double-layer membranescalled exosomes. Focus on the role of microRNAs in malereproductive disorders can further explain the molecularmechanisms of male infertility and it can create a newpathway as an effective biomarker for treating infertilityin men and for contraceptive pills . So far,For instance, by examining two miRNAs (miR-100and let-7b) regulating the alpha estrogen receptor gene inoligospermia men, Abhari et al.  found thCurrent investigations disclosed that let-7b has an inhibitoryeffect on cell proliferation  56). Th. Th56). Bouhallier et al.  demonstrHIWI2 rs508485 (T>C)with non-obstructive azoospermia and HIWI2 rs508485 (T>C) withnon-obstructive azoospermia  was previously confirmed in theIranian and Chinese populations. Figure 3 summarized the most prominent study carried outon piRNAs and male infertility: moloney leukemia virus 10-like 1  technology, one of which is RNAseq, shed light onthe molecular mechanism involved in infertility disorders, especially structural andfunctional defects in sexual cells. It is also believed that lncRNAs affect the proteinsinvolved in sperm motility and its entry into the egg as well as the pathways andmechanisms involved in fertility. Therefore, by studying and recognizing theserelationships as well as the manipulating and molecular interventions, new approaches anddiagnostic-therapeutic kits can be designed. In the study performed by Zhang et al. ,expressn et al. , demonstProper and complete spermatogenesis requiressimultaneous expression of a very large number ofcoding and non-coding genes. So that stopping ordisrupting expression of each one can lead to disruptionof the spermatogenesis process. Identifying such genesand evaluating their performance provides valuableinformation about the role of these genes in adult sperm,process of spermatogenesis, their function in embryofertilization and causes of idiopathic infertility. Geneticand epigenetic factors are elements contributed to thistype of infertility.AKAP, PRM2 andDAZ, it was showed that presence of DAZ andPRM2 genes can be used as a noninvasive molecular marker in seminal fluidof non-obstructive azoospermia patients to predict the presence or absence of sperm ormature spermatids (Therefore, this aspect needs to be considered for the investigation of infertile men. Themain emphasis for molecular evaluation and use of RNA sperm is the wide range contributionof male factor in infertility and testicular biopsy, which is a problem in the study ofinfertile men . Those wermatids . Recent ermatids .In the scope of infertility investigations, there are severalavailable tests evaluating sperm quality and function.But, there is still a demand for better and more reliableprocedures, considering that male factor infertility isinvolved in at least 45-50% of idiopathic cases. Use ofsperm transcripts in molecular analysis of spermatogenesisand infertility treatment is also important, especiallyin patients with non-obstructive azoospermia. Studiesshowed that in these patients expression of genes in thepathway for sperm production is changed.in vitrospermatogenesis can be suggested in the future.Today, the most common method for evaluating spermatogenesis in these individuals istesticular biopsy which is an invasive practice and recommended as an infertility study toolin the final stage. However, sperm may not be found due to regional spermatogenesis in thetesticular tissue taken from the biopsy and need for multiple biopsies of the patient tofind and extract the sperm. This procedure can cause tissue atrophy or infection. Thus,using sperm RNA content and preterm sexual cells can evaluate spermatogenesis molecularevents. Clinical application of sperm RNA is very valuable because if sperm RNA can providesimilar information compare to testis tissue, semen samples are a better and more acceptablechoice for the patient than a biopsy. The existence of DAZ transcripts in the seminal fluidof non-obstructive azoospermia can be used as a noninvasive molecular marker to predictpresence or absence of adult spermatozoa. From other futuristic studies, transformation ofembryonic stem cells or adult stem cells into germinal cells can be a very valuable startingpoint for solving the infertility problem in an individual whose defect is related to theabsence of stem cells in the testes. Research on the presence of RNAs in differentiatedsperm from stem cells and normal sperm and their role in


Control of gene expression is critical in shaping the pro-and eukaryotic organisms’ genotype and phenotype. The gene expression regulatory pathways solely rely on protein–protein and protein–nucleic acid interactions, which determine the fate of the nucleic acids. RNA–protein interactions play a significant role in co- and post-transcriptional regulation to control gene expression. RNA-binding proteins (RBPs) are a diverse group of macromolecules that bind to RNA and play an essential role in RNA biology by regulating pre-mRNA processing, maturation, nuclear transport, stability, and translation. Hence, the studies aimed at investigating RNA–protein interactions are essential to advance our knowledge in gene expression patterns associated with health and disease. Here we discuss the long-established and current technologies that are widely used to study RNA–protein interactions in vivo. We also present the advantages and disadvantages of each method discussed in the review. The majority of the nascent RNA transcripts are associated with and regulated by several proteins. Almost all RNAs, irrespective of their structure and function, interact with one or more protein partners to function correctly ,2. Both Genome-wide association studies of RNA–protein interactionsRecent advances in molecular biology techniques have expanded into genome-wide studies as “OMICS” dealing with individual genetic components such as genomics for DNA and transcriptomics for RNA. These technologies are often referred to as high-throughput sequencing (HTS) used to screen multiple targets and samples in a given time. Interestingly, when combined with biochemistry and molecular biology assays, HTS provides detailed knowledge of the target genes and delineates the model organism’s biological functions through a network or pathway analysis . CurrentImmunoprecipitation of proteins that form complex with RNA is a commonly used method to detect the unknown RNA components. Based on protein-RNA interaction, separation of protein-RNA complex in a natural condition or under a chemical or UV crosslinking approach requires a strong association between RNA and protein at the intracellular level. The proteins can be subjected to IP using specific antibodies against them, or affinity tagged antibodies against the particular protein can be used to isolate the entire RNA-protein complex. Identification of the RNA molecules is the primary purpose of this strategy, where the untagged proteins or control Immunoglobulin-G (IgG) usually serve as an experimental control for this strategy. Affinity tags used for protein pull-down: Numerous affinity tags, such as c-myc, calmodulin-binding peptide CBP, FLAG, Histidine (His), Glutathione-S Transferase (GST), and the Tandem Affinity Purification (TAP) [Staphylococcus aureus protein A, cloned at either the 5’ or 3’ end of the desired gene in the chromosome. Incorporating the TAP tag in the protein does not alter its expression level and is therefore useful for the purification and characterization of protein-RNA complexes. A modified TAP protocol includes a TEV protease cleavage site replacement with a 3C protease, and CBP is substituted with 6X His [on (TAP) ,12,13, aon (TAP) ,15,16. Ton (TAP) . It was h 6X His ,19. Histh 6X His ,21,22. Tagging RNA molecules: RNA affinity tags (aptamers) like Strepto, Streptavidin S1, and Sephadex D8 are commonly and successfully used to isolate the in vivo RNA-protein complexes. However, due to an added tag to the macromolecules, the RNA native fold distortions may occur. In silico RNA secondary structure must be verified after adding a tag to the RNA by running RNA probing experiments [eriments ,24. The eriments , used wieriments . The Streriments ,16. BothO-methylated RNA oligos have been applied to RNAs for the direct purification of abundantly-expressed RNP complexes such as the spliceosome, telomerase, and long ncRNAs (lncRNAs) from human cells [25 beads following a purification step to capture the particular mRNAs from poly(A) RNAs using 3′-biotinylated 2-O-methylated RNA ASOs are two necessary steps of the TRIP method. The isolated RNAs and proteins can be readily identified by RT-PCR or protein blots; however, global identification of these macromolecules can also be done by RNA sequencing and mass spectrometry, respectively.Although the above-mentioned RNA-aptamer approaches are relatively adaptable and signify an efficient tool for detecting the RNA-protein complexes, a significant disadvantage is the indirect nature of the interaction. These interactions may result from the tagged RNA’s expression in trans that cross-reacts with native RNAs and RNPs. A few other techniques besides aptamers have also been used to determine the in vivo RNP complexes. Antisense oligonucleotide (ASO) probes like biotinylated 2-an cells ,27. Addian cells . PrimariIsolation of cellular RNA-protein complexes: The plasmids carrying the tagged protein or RNA are introduced into the cells for pull-down, for which empty vectors are used as a control. Pull-down experiments are performed after the cell lysis, after which lysate is passed through an affinity column resin or matrix containing an antibody against the tag or protein or RNA of interest. Finally, the native complex can be eluted using specific chelating agents, like imidazole, mostly used for His-tagged proteins or streptavidin for biotin tags on RNA. After purification, the components of the complex are characterized by liquid chromatography-assisted mass spectrometry (LC/MS). The LC/MS is commonly used to identify proteins; still, with advanced technology, MS offers several advantages for RNA analysis, evolving from its ability to provide mass and sequence information from limited amounts of the sample [e sample ,29. Oncee sample .In the study of RNA-protein interaction in vivo, it is critical to use methods that rapidly halt the RNA-protein complexes and prevent their rearrangement during cell lysis. Multiple crosslinking agents are used, but reversible crosslinking agents are the most useful because they facilitate the subsequent characterization of the interacting partner molecules. Cross-linking can stabilize RNA and protein complexes and can be a valuable tool for analyzing sequence-specific interactions. This can be achieved using UV light or chemical crosslinkers ,31,32. SIn vivo UV crosslinking has the benefit of analyzing RNA–protein interactions in their native states. However, it requires prolonged exposure, thus permitting the redistribution of the components and crosslinking of the UV-damaged molecules. The in vivo UV crosslinking technique is based on UV light’s ability to crosslink proteins to RNA in living cells covalently. Photoreactive crosslinking is commonly used in cell lines that express tagged proteins. However, the cells containing endogenous RNA-protein complexes, for which an antibody against the protein is required, can be analyzed by this method. In proteins, the amino acids cysteine, tyrosine, phenylalanine, arginine, lysine, and tryptophan most readily crosslink to poly-U.Additionally, it is often noticed that pyrimidines have higher efficiency to be crosslinked than purines and RNA, with uridine residues compared to thymidine in DNA, shows greater reactivity when comparing addition to cysteine ,35. BesiThe UV-irradiated cells are harvested with lysis buffer containing sodium dodecyl sulfate (SDS) to liberate genomic DNA and subsequent heating and passing through a spin column to remove the DNA, which in turn increases the RNA yield. This step can be substituted with sonication, which may lead to significant RNA degradation. Next, the lysate is cleared by centrifugation for further analysis with a specific antibody bound to beads (agarose or magnetic) to detect the proteins ,38,39,40This procedure does not require any chemical agent, thus avoiding disturbance of the RNA-protein interface.UV irradiation is a zero-length crosslinker, which creates the RNA-protein crosslinks at their point of contact, thus displaying a direct RNA-protein interaction.Advantages:RNA-protein interfaces must be compatible with the chemistry of the photocrosslinking reaction. In addition to potential amino acid biases, there is a preference for uridine to act as a crosslinking base .The efficiency of these crosslinks is often less than that of chemical crosslinking.The covalent bonds created by crosslinking are irreversible; therefore, small adducts persist after proteinase K removal of the bound proteins, which inhibit the reverse transcriptase elongation process. Thus, RT-PCR may not be performed to analyze the RNA.Disadvantages:Formaldehyde crosslinking is widely used for chromatin immunoprecipitation ChIP  and RNP Based on chromatin immunoprecipitation ChIP protocols ,43, chemThe crosslinks are reversible. Therefore, specificity can be achieved by detecting the bound RNA by RT-PCR.This assay preferentially works for nucleophilic lysine residues, which are readily crosslinked by formaldehyde .Advantages:The failure to detect the RT-PCR signal can be due to incomplete reversal of the crosslinks or crosslinking involving the antibody’s epitope, thus making it inaccessible to the antibody used during the IP.Additionally, besides protein–RNA interactions, protein–protein interactions can also result from formaldehyde crosslinking, making the detection of the direct RNA-protein complexes from the protein–protein RNA complexes difficult.Disadvantages:HITS-CLIP  can be aThe method produces a large number of high-throughput sequencing reads for analysis.Thymidine deletion or mutation during RT of the crosslinked site can help determine the nucleotides with high accuracy, and adapter adenylation is not needed for this method.Advantages:UV cross-linking efficiency may be low.A large number of reads can often produce high noise levels, resulting in difficulty in data interpretation.The on-bead ligation can be less efficient for small RNAs, including miRNAs.The reverse transcriptase may stall at the crosslinked site, resulting in a truncated cDNA that does not contain the 5’-adapter and ultimately fails to produce a PCR product.Disadvantages:Although both photoactivatable ribonucleoside-enhanced CLIP (PAR-CLIP)  and HITST to C or G to A conversion during RT of the crosslinked site can help determine the nucleotides with high accuracy and separate the labeled RNAs from the unlabeled RNAs.Advantages:4-SU analogs are expensive. Thus, this procedure is not cost-effective.The 4-SU analogs are not always tolerated by the cells.UV cross-linking may be insufficient.Disadvantages:Similar to HITS-CLIP, iCLIP , or indiThe method is viable under most experimental conditions and often in cases where PAR-CLIP is not feasible, e.g., animal tissues.HITS-CLIP, thymidine deletion, or mutation during the crosslinking of the sites can help determine the nucleotides with high accuracy.Circularization of the cDNA, instead of 5’ adapter ligation, might prove to be more efficient.Advantages:This method does not have many disadvantages. However, the protocol is exceptionally specialized and long, requiring several enzymatic and precipitation steps. Also, the circularization step of iCLIP can be an unreliable reaction at times.Disadvantages:The eCLIP  maps theAvoids the circularization step of iCLIP, which can be an unreliable reaction at times. This strategy also avoids the usage of radioactive markers.High-throughput mapping of protein-RNA binding sites.Ligation efficiency is better than the other methods.Advantages:Extensive protocol and UV crosslinking may be poor.A single-stranded DNA adaptor must be ligated to single-stranded cDNA.Disadvantages:By using simple phenotypic assays, the three-hybrid system detects RNA–protein interactions in yeast . This syEscherichia coli LexA protein. HIS3 and lacZ are the most widely used reporters in this assay. Control experiments are used to show that each component of the system is required for the assayed interaction. Reporter gene expression is assayed either by β-galactosidase activity or by determining the level of resistance to 3-aminotriazole 3-AT, which monitors His3 activity.The general strategy of the three-hybrid system is shown in This method can be used successfully for RNAs of different sizes 21–1600 nucleotides .There is no strict rule for the bait  position in the hybrid RNA.The three-hybrid system allows for differential identification of the RNP complexes. This strategy helps to select proteins that bind to RNA at different steps in a functional pathway.Advantages:RNAs with post-transcriptional modifications, necessary for interaction with their protein binding partners, cannot be studied using this method as the hybrid RNAs used here may be partially modified or unmodified.Many proteins require cofactors to bind to their RNA partners, and these cofactors might not be available in the nucleus because they are located in other cellular compartments.Some of the proteins and their target RNAs interact in a transient manner and with low affinity, thus preventing complexes’ detection with the three-hybrid strategy.Regarding the three-hybrid strategy, the RNA-protein interaction occurs in vivo, where many cellular parameters can influence it. These influences can lead to false-positive results, which additional time-consuming screening tests must eliminate.This strategy requires an effective fusion of the proteins and RNA, which renders a potential risk in altering the structures of the macromolecules, especially the RNA structure in the fusion.Disadvantages:The approaches to characterize RNA complexes associated with specific RNA-binding proteins depend on immuno- or affinity purification procedures and subsequent identification of the co-purifying RNA or protein molecules. Understanding these experiments is usually based on the assumption that the in vivo assembled RNA-protein complexes’ integrity is preserved during the investigation. However, the validity of this assumption has not been tested experimentally. The cell mixing strategy shows that an RNA-binding protein expressed in one cell population can associate with a target RNA expressed in a different cell population after cell lysis ,53,54. TSix different cultures, A to F, are used for this assay . CultureThe procedure is straightforward, without any crosslinking or stripping of the RNA from proteins. And cells are lysed by using relatively milder cell lysis procedures, which helps decrease RNA degradation and does not interfere with other downstream analyses.Advantages:The major disadvantage of the cell mixing protocol is that it can rarely be applied to cellular RNA–protein interactions.Disadvantages:In contrast to the UV crosslinking discussed in method, one cannot determine whether an observed interaction between an RNA and protein is direct or indirect in the cell mixing assay.Trimolecular fluorescence complementation TriFC is used to detect RNA–protein interactions in live cells . In thisIn this method, a portion of the Venus fluorescent protein  is attacThis method enables the simple detection and localization of the interacting RNA and protein components in living cells where RNAs and proteins are expressed in their native locations, making this method both physically and biologically relevant.Advantages:Similar to most other in vivo systems, this method can also be nonspecific at times. Because the complexes can form heterogeneous in natural environments, the endogenous proteins or RNAs can bring the TriFC components into proximity. In turn, this proximity enables the fluorescent signal to be detected in cases where the TriFC components do not directly interact but instead associate via a bridging RNA or protein. This problem can be resolved by analyzing purified components in vitro or by RNAi knockdown of the candidate bridging molecules.Although this method detects the RNA–protein interactions in situ, its other drawback is the irreversible crosslinking between the studied RNA-binding protein, MS2–protein, and likely the target RNA.BiFC protein’s maturation takes a long time after the formation of the protein–protein complex . TherefoDisadvantages:Fluorescence resonance energy transfer (FRET) is a photophysical phenomenon in which energy is transported between two suitable fluorophores, a donor and an acceptor, appropriately oriented and located within a specific range usually, <10 nm. In cell biology, fluorophores are often used as tags on proteins, and their proximity is achieved through an interaction with the proteins. However, in FRET, the tagged proteins do not need to interact directly, and it is sufficient when FRET partners are part of the same complex. For example, FRET has successfully been used to measure protein–protein interactions between splicing factors in the spliceosome ,59.FRET was first used by Lorenz  to detecThis method has overcome some of the disadvantages of the TriFC assay. This experiment’s strategy is shown in FRET signals between the ECFP-protein of interest and MS2-EYFP pairs are measured by the acceptor photobleaching method, which relies on detecting de-quenched donor fluorophores in the presence of acceptors  Figure . FurtherAdvantages:This strategy is specifically useful for studying RNA-binding proteins with known RNA target sequences.As the studied RNAs and proteins can be expressed within a living cell, both the physical and biological properties of these complexes can be authentically examined, limiting in vitro assays.The PAIR approach helps identify RBPs that interact with a specific target m/RNA sequence in vivo . This apankylosis ank RNA, a pan-neuronal dendritically localized RNA, to develop PAIR. As shown in p-benzoylphenylalanine (BPA)-PNA dissociates from CPP after entering into the cell  The RNP-interaction trap assay is modified from the yeast three-hybrid assay, and it allows in vivo re-formation of tripartite protein-RNA-protein ribonucleoproteins RNPs. This technique has been used explicitly in the yeast system , in whicIn the field of RNA-protein complexes, IP techniques are developed to identify and characterize the components of small RNPs in mammalian cells . HoweverO-adducts at conformationally flexible RNA nucleotides, both in vitro and in vivo [Z > 0 (more than 1.96 standard deviations when comparing the two conditions) are considered to undergo significant changes in SHAPE reactivity.SHAPE-MaP  uses a highly validated SHAPE RNA structure probing method followed by selective sequencing to achieve high-throughput examination of RNA flexibility at single-nucleotide resolution ,75. In S in vivo . In brieThe studies described in this report have successfully proven efficient for analyzing RNAs of different sizes and their interactions with target proteins. Each assay has its advantages and disadvantages . To date