Detailed comparison of reef types reveals that mid-channel reefs in the Florida Keys have the highest net carbonate production (0.84 kg CaCO3 m−2 year−1) and indicates that these reefs may be hold-outs for reef development throughout the region. This study reports that Florida reefs, specifically their physical structure, are in a net erosional state. As these reefs lose structure, the ecosystem services they provide will be diminished, signifying the importance of increased protections and management efforts to offset these trends.Coral reef habitat is created when calcium carbonate production by calcifiers exceeds removal by physical and biological erosion. Carbonate budget surveys provide a means of quantifying the framework-altering actions of diverse assemblages of marine species to determine net carbonate production, a single metric that encapsulates reef habitat persistence. In this study, carbonate budgets were calculated for 723 sites across the Florida Reef Tract (FRT) using benthic cover and parrotfish demographic data from NOAA’s National Coral Reef Monitoring Program, as well as high-resolution LiDAR topobathymetry. Results highlight the erosional state of the majority of the study sites, with a trend towards more vulnerable habitat in the northern FRT, especially in the Southeast Florida region (− 0.51 kg CaCO The Florida Reef Tract (FRT) is one of the most economically important reef environments in the world, with an estimated value of > $8.5 billion USD4. Much of the functionality and ecoservices of coral reefs is dependent on the complex three-dimensional structure of the reef framework5.Coral reefs are one of the most biologically diverse ecosystems3]) and the breakdown, redistribution and dissolution of CaCO3 by physical and biological erosion. Scleractinian corals are often the primary drivers of reef growth and accretion6. For healthy reef systems, the balance between these calcifying and bioeroding forces tends to favor net reef accumulation, and with it, positive reef growth7.Reef framework growth and its persistence through time is a function of the balance between calcification  in the region, but some reefs closer to shore have maintained higher coral cover and exhibited resilience to recurrent bleaching events16.Despite having limited reef growth over the past 3000 years, the FRT maintained the majority of ecosystem functions up until the late twentieth century due to high regional coral coverThese recent trends have led to two key questions. First, how has the loss in live coral throughout the FRT impacted reef framework growth/loss? Second, what are the implications of the region-wide loss of live coral for net carbonate production in the future?ReefBudget17 approach , the highest mean coral cover was in the Dry Tortugas  and the Florida Keys (FLK = 8.57% ± 10.05)  . Despite having lower average coral cover than the FLK, the minimum recorded net carbonate production in DRTO was − 3.9 kg CaCO3 m−2 year−1. Linear regression analysis described a coral cover threshold of 8.2% and 10.5% for DRTO and the FLK, respectively, to maintain positive reef growth   and was highest for the FLK reef sites, with an average biomass of 118.7 ± 160.9 kg ha−1  (Table −1 in SEFL (maximum 851.4 kg ha−1) and 48.4 ± 40.2 kg ha−1 in DRTO (maximum 324.4 kg ha−1).There was a significant negative relationship between parrotfish biomass and net carbonate production (1) Table . This is3 m−2 year−1 (mean ± sem)  and upper Florida Keys (UK =  − 0.395 ± 0.122 kg CaCO3 m−2 year−1) sub-regions were similarly net erosional (Table 3 m−2 year−1) and LK (0.042 ± 0.128 kg CaCO3 m−2 year−1). In comparison to the rest of the FRT, DRTO reefs were found to persist in a state of accretionary stasis, with an average net carbonate production of − 0.023 ± 0.050 kg CaCO3 m−2 year−1.Our results show that reefs in SEFL were almost exclusively net erosional and had the greatest magnitude of erosion of − 0.513 ± 0.063 kg CaCOF = 22.1, P < 0.001 and F = 29.7, P < 0.001, respectively), but gross carbonate erosion did not . The MK showed a clear trend towards increasing net carbonate production further from shore  of reefs surveyed across several Caribbean/Western Atlantic sites  were net erosional19. This shows that the FRT is in a particularly vulnerable and degraded state when compared to the wider Caribbean.Of the 723 reef sites analyzed in this study, 70% were net erosional, indicating reef framework is likely being lost across much of the FRT. These results agree with prior reef carbonate budget studies in the Florida Keys that found that 89%22. These reef systems have been negatively altered over the last 50 years, with one of the most consequential changes being a shift away from the dominant habitat-creating coral species (i.e. Acropora spp. and Orbicella spp.) to opportunistic “weedy” species  with a limited capacity for carbonate production and habitat creation14. The emergence of these novel, minimal-habitat forming benthic communities are unlikely to reverse the current path of habitat loss and signals that a tipping point towards reef degradation may have already been surpassed for much of the FRT. Associated with these community changes is a greater susceptibility to future environmental perturbations, further deterioration of reef framework24, and the depreciation of as much as $823 million in annual economic protection value provided by South Florida reef systems22.These data indicate that coral reef habitat throughout the entirety of the FRT is likely losing structural complexity and undergoing a decline in critical ecosystem functions6. The lack of a sufficient calcifying community has resulted in minimal reef development throughout the region, corroborating past geologic assessments in the area that have estimated a cessation of active upward reef growth 3,700 to 8,000 years ago26.SEFL, the northernmost biogeographic region in South Florida, is the most degraded region of the FRT. SEFL had the highest percentage of net erosional reefs (98% of reefs), as well as the lowest coral cover and gross carbonate production rates of the FRT Table . SEFL re27, but our data show that the mid-channel and offshore sites in the MK have some of the greatest rates of net carbonate production measured. Future work should investigate if regular exposure to the suboptimal Florida Bay waters in this region has stress-hardened these coral communities and allowed them to persist. It is important to note that the greatest regional gross carbonate production rates documented here (~ 5 kg CaCO3 m−2 year−1) are still well below average rates reported from the Caribbean during the Holocene19 (≥ 10 kg CaCO3 m−2 year−1).In comparison to the net erosional state of most SEFL reefs, the mid-channel reefs in the FLK appear to be potential hold-outs for positive net carbonate budgets Table . UK and 24, this evaluation of South Florida reef systems was marginally more optimistic, with a region-wide average coral cover of 8.2%. However, it is important to note that this value still falls below the estimated 10% coral cover threshold required for sustainable reef development19, indicating that South Florida reefs remain in a degraded state.The primary biological drivers of reef development in South Florida were coral cover and parrotfish biomass Fig. . While p28 marked by only small coral colonies29. The lack of complex 3D framework not only limits available habitat necessary to sustain robust parrotfish populations, but is also suboptimal substrate for parrotfish feeding due to their preference for convex, rugose substrata30.SEFL reefs had the lowest parrotfish density and second lowest parrotfish biomass measured along the FRT. Associated with this was the smallest percent contribution by parrotfish to gross carbonate erosion, with SEFL being the only region/sub-region to have virtually equal parrotfish and microbioerosion rates Table . These r18, with the highest parrotfish biomass and erosion rates being measured in the UK and LK. For these sub-regions, inshore and offshore reef types had considerably higher parrotfish biomass compared to the mid-channel systems. This was a main factor driving positive reef development in the mid-channel reefs relative to the net-erosional inshore and offshore counterparts. While previous carbonate budget studies have found significantly higher parrotfish erosion on offshore reef types31, the FLK appear to be unique in having substantial inshore and offshore parrotfish populations, with a potential mid-channel respite from parrotfish erosion. Since parrotfish preferentially feed on dead substrate32, the low coral cover and high availability of bare substrate on inshore and offshore reefs may promote parrotfish erosion compared to the high coral cover, mid-channel reefs.Specific to the FLK, the parrotfish data supports prior assessments in the region33. A comparison of reef types suggest that mid-channel reefs will be potential hold-outs for reef persistence compared to susceptible inshore and offshore reef types, with these trends driven primarily by differences in coral cover and parrotfish biomass. These findings imply that under current conditions, the persistence of FRT habit is in jeopardy unless management strategies intervene to substantially increase carbonate production and coral cover throughout the region.This study represents the most comprehensive assessment to date of carbonate production states for the FRT. Net erosion was observed for 70% of sites surveyed; 98% of reefs adjacent to south Florida’s urban centers are eroding. Spatial trends identified the most prominent net erosional reefs in the northernmost part of the FRT, a result that is consistent with prior carbonate budget studies conducted along the Mesoamerican Reef TractBenthic and fish surveys were conducted at randomly stratified sites throughout the entirety of the FRT by NOAA’s National Coral Reef Monitoring Program (NCRMP). Sites were categorized into three biogeographic regions, including Dry Tortugas , Florida Keys , and Southeast Florida  .Fish and benthic surveys were conducted in accordance with NCRMP methodologies34 Table . The proSurveys were used to quantify the benthic cover at each site. The protocol for these surveys followed a standard line point-intercept sampling design. At each site, a 15 m weighted transect was draped along the reef surface. Surveyors recorded benthic composition at 15 cm intervals along the transect . The benthic composition from these 100 points was then transformed to percent cover of ecologically important functional groups .Planar benthic surveys were adjusted to account for the three-dimensional complexity  of each site using light detection and ranging (LiDAR) data  from topobathymetric mapping surveys of the South Florida eastern coastline conducted by NOAA’s National Geodetic Survey. A 15 m x 15 m region of interest (ROI) was placed around the GPS coordinates of each site using ArcGIS Pro with 3D and Spatial Analyst extensions (ESRI). The ROI was then overlaid with existing multibeam echosounder (MBES) and LiDAR bathymetry data. Within the ROI, LiDAR was extracted using the Clip Raster function from ArcPy (ArcGIS’s python coding interface), and the Surface Volume tool was used to calculate the 3D surface area. Rugosity was calculated by dividing the 3D surface area by the 2D surface area of the ROI.ReefBudget approach by using site-specific rugosity rather than species-specific rugosity17. This was a necessary limitation of this analysis as transect rugosity at 1 m increments was not measured using the NCRMP benthic survey protocol. To ensure that reef topographic complexity was still accounted for, however, rugosity of the entire reef site, calculated from LiDAR bathymetry data, was used in this analysis. While rugosity of the site rather than of each benthic component, specifically for corals, can lead to an under or overestimation of carbonate production rates, we note that site and species rugosity  was low for the vast majority of sites and species surveyed, thereby reducing the probability of an under or overestimation.The methodology for standardizing reef carbonate budgets to topographic complexity  diverged from that of the ReefBudget approach17. Coral carbonate production was derived from species-specific linear extension rates (cm year−1), skeletal density (g cm−3), coral morphology , and percent cover. Carbonate production by CCA and other calcareous encrusters was similarly calculated as a function of surface area, literature reported linear extension rates, and skeletal density17. Gross carbonate production at each survey site was measured as the sum total of carbonate production by all calcareous organisms found at each site and was standardized to site-specific reef rugosity.Reef carbonate budget analysis was performed following a modified version of the ReefBudget methodologies17 (Table −1), volume removed per bite (cm3), and proportion of bites leaving scars to calculate total parrotfish erosion17. The substrate density (1.72 g cm−3) used in these calculations followed that of the ReefBudget protocol17. Microbioerosion was calculated from the percent cover of dead coral substrate, which was multiplied by a literature-derived rate17 of − 0.240 kg CaCO3 m−2 year−1. Macroboring was calculated as the percent cover of clionid sponges multiplied by the average erosion rate of all Caribbean/Atlantic clionid sponges17 (-6.05 kg CaCO3 m−2 year−1). External bioerosion by urchins was calculated using Diadema urchin abundance collected from the benthic surveys. Due to the lack of test size data from the NCRMP benthic surveys, urchin abundance was multiplied by the bioerosion rate of an average test sized36 (66 mm) Caribbean/Atlantic Diadema urchin (-0.003 kg CaCO3 m-2 year−1). While using an average test sized Diadema urchin for this analysis may have led to an under or overestimation of urchin erosion, the abundance of Diadema urchins measured in the surveys was minimal, as they appeared to be functionally irrelevant across the FRT.Gross carbonate erosion for each survey-site was calculated as the sum total of erosion by four bioeroding groups: parrotfish, microborers, macroborers, and urchins. The calculations roughly followed the ReefBudget approach17, we chose to validate our model through a fine scale temporal comparison of annual ReefBudget surveys conducted by NOAA at Cheeca Rocks (UK) to three nearby NCRMP sites used in our analysis. Since the NCRMP surveys were performed in 2014, 2016, and 2018, this study focused exclusively on these three survey years from the NOAA Cheeca Rocks dataset. Temporal trends related to reef growth/erosion were visually compared to see if survey types provided comparable results .As the survey methodologies and data sources employed in this analysis were modified from that of the standard 37 with the R Studio extension38. Generalized linear models (GLMs) were run on response variables involved in habitat production  to evaluate spatial trends related to reef development across sub-regions and reef types. Each GLM was performed with reef type being nested within sub-region. The best fit distribution for each variable was determined using the fitdistrplus R package39. Linear regression analysis was used to evaluate the relationship between net carbonate production and both live coral cover and parrotfish biomass. All plots were created using ggplot2 R package40 and edited for style with Adobe Illustrator41.All model calculations and statistical analyses were performed using RSupplementary Information.


The main strategies for improving RNA-editing tools by increasing their efficiency and specificity are described as well. These data allow us to outline the prospects of base-editing systems for clinical application.Rare genetic diseases reduce quality of life and can significantly shorten the lifespan. There are few effective treatment options for these diseases, and existing therapeutic strategies often represent only supportive or palliative care. Therefore, designing genetic-engineering technologies for the treatment of genetic diseases is urgently needed. Rapid advances in genetic editing technologies based on programmable nucleases and in the engineering of gene delivery systems have made it possible to conduct several dozen successful clinical trials; however, the risk of numerous side effects caused by off-target double-strand breaks limits the use of these technologies in the clinic. Development of adenine-to-inosine (A-to-I) and cytosine-to-uracil (C-to-U) RNA-editing systems based on dCas13 enables editing at the transcriptional level without double-strand breaks in DNA. In this review, we discuss recent progress in the application of these technologies in  All these tools are utilized in clinical trials  for the treatment of various genetic diseases . The moshokB toxin gene that can regulate growth arrest and antibiotic sensitivity   are approved by the FDA , and AAV kbp DNA . Accordi kbp DNA , directe kbp DNA  and chem kbp DNA , reducincapacity . In addicapacity . Deliver4601051) . Moreove4601051) . DisadvaThe complex of the Cas13 nuclease with RNA containing clustered regularly interspaced short palindromic repeats (crRNA) is the foundation of the CRISPR–Cas13 system, which can specifically bind to single-strand RNA and cleave it. In bacteria and archaea, the CRISPR–Cas13 system is a component of the adaptive immune system, which performs programmable RNA-guided degradation of foreign RNAs. Four phylogenetically distinct variants of Cas13 have been identified so far: Cas13a (previously known as C2c2), Cas13b, Cas13c, and Cas13d. All these nucleases have two nucleotide-binding domains (HEPNs), which are required for pre-crRNA processing and for the cleavage of a target single-stranded RNA . The CRIOne of the most promising areas of practical application of CRISPR–dCas13 from the standpoint of medical treatments is the development of the site-directed RNA editing (SDRE) system. This accomplishment has resulted from the fusion of CRISPR–dCas13 with an adenosine or cytosine deaminase (ADAR or APOBEC3A), which is capable of replacing adenosine with inosine (A to I) or cytosine with uracil (C to U) . In thisPrevotella sp. (hereafter referred to as dCas13b) with the deaminase domain of ADAR2 (E488Q) (hereafter referred to as ADAR2DD), they created RNA Editing for Programmable A to I Replacement version 1 (REPAIRv1). This system was successfully utilized for the editing of mutations in cell culture, and editing efficiency reached 35%. A major drawback of this system is a large number of off-target effects. To solve this problem, a modified ADAR2 system with two amino acid substitutions has been created: ADAR2DD (E488Q/T375G). Fusion of dCas13b with ADAR2DD (E488Q/T375G) has been incorporated into REPAIRv2, the use of which in cell culture in vitro has reduced the number of off-target events from 18,385 (REPAIRv1) to 20 (REPAIRv2) according to transcriptomic analysis, confirming significantly higher specificity of the new system , and the resulting system was named RNA Editing for Specific C to U Exchange (RESCUE)  turned out to be lower than that of other systems and did not exceed 15%.Introducing 16 mutations into ADAR2titution . This mu(RESCUE) . NonetheTo improve the RESCUE system, investigators used a Cas13b ortholog: dPspCas13b (instead of dRanCas13b) with a nuclear export sequence (NES) . This syDD with cytidine deaminase activity, in mammals there is a cytidine deaminase APOBEC3A (A3A), which can act on both RNA and DNA substrates  mutation, which is associated with X-linked nephrogenic diabetes, and the 1517G > A mutation (FANCC W506X), which is associated with Fanconi anemia  gene. USH2A is important in the development and homeostasis of the inner ear and retina, whereas the absence of the functional protein leads to sensorineural hearing loss and retinitis pigmentosa. Sequence of exon 60 of USH2A cDNA carrying the c.11864G > A mutation was delivered via a plasmid into HEK293T cells along with the REPAIRv2 construct. The efficiency of target adenosine editing was 43%.One research group  used theDUX4 mRNA translation. Normally, DUX4 is expressed only in the embryonic period, whereas inadequate DUX4 repression in the postnatal period is associated with progressive muscle wasting and weakness . By means of several Cas13b–gRNAs targeted to different parts of DUX4 mRNA, those authors achieved >90% downregulation of the DUX4 protein in experiments on cultured cells and a near 100% reduction of DUX4 expression in tibialis anterior muscles of the TIC-DUX4 mouse model.Thus, successful application of site-directed Cas13-based RNA-editing systems has been demonstrated for RNA editing and for restoration of a protein product’s function . PrelimiDD RNA-editing systems, other RNA-editing systems are currently being actively developed, such as BoxB-λN-ADARDD, SNAP-tag-ADARDD, and MCP-MS2-ADARDD, in which the binding of ADARDD and gRNA is implemented by the λN peptide, SNAP-tag protein, and MS2 bacteriophage coat-binding protein, respectively (DD has manifested greater efficiency of editing of point mutations in Mecp mRNA in vivo (∼50%) and in vitro (∼70%) (Mecp gene are the etiology of Rett syndrome. Treatment of Duchenne muscular dystrophy in the Mdx mouse model via editing of the mutation in the Mdx gene by means of the MCP-MS2-ADARDD system yielded less impressive results (up to 3.6% of edited RNA and up to 2.5% protein restoration)  . Mutatiooration) . SimilarIDUA pre-mRNA both in vitro (efficiency up to 80%) (in vivo (efficiency up to 17%) in Idua-W392X mice (IDUA gene leads to the absence of an enzyme (α-l-iduronidase) and the buildup of large sugar molecules (glycosaminoglycans) in lysosomes, resulting in one form of type 1 mucopolysaccharidosis (Hurler syndrome). Despite relatively low magnitude of RNA editing in the liver of Idua-W392X mice (ash mice (PINK gene (up to 65%) in cultured HEK293T cells; the loss of the functional protein product of this gene is associated with the development of the monogenetic type of Parkinson’s disease   and in v92X mice . A nonse92X mice  (≤17%), ash mice  and succ disease .in vivo can be much different. Furthermore, the percentage of edited RNA is often higher than the percentage of the protein with a restored function. Consequently, new studies are needed to evaluate the effectiveness of CRISPR–Cas13-based RNA-editing systems in vivo.One of the reasons for the scarcity of studies on Cas13-based RNA-editing systems aimed at correcting the effects of genetic mutations may be that these systems were discovered relatively recently. Other approaches to RNA editing have yielded considerable progress in in vivo experiments and therefore offer a wide variety of experimental models for evaluating the effectiveness of CRISPR–Cas13-based systems. The most important characteristics for clinical dissemination of SDRE systems are their effectiveness and specificity (a low number of off-target events). Although it is not yet possible to compare different RNA-editing systems by these parameters, it should be mentioned that the studies in question  were pervia steric hindrance, thereby reducing the off-target effects  that evaluate the effectiveness of editing  . SDRE syIncessant improvement of CRISPR–Cas-based RNA-editing systems and of delivery modalities is step by step bringing about the era when rare mutations will not mean a death sentence for their carriers. On the other hand, at present, there is a large gap between basic research and translation into therapies for rare diseases . One of


Advances in RNA biology such as RNAi, CRISPR, and the first mRNA vaccine represent the enormous potential of RNA research to address current problems. Additionally, plants are a diverse and undeniably essential resource for life threatened by climate change, loss of arable land, and pollution. Different aspects of RNA such as its processing, modification and structure are intertwined with plant development, physiology and stress response. This report details the findings of researchers around the world during the 23rd Penn State Symposium in Plant Biology with a focus in RNA biology. The advent of the first mRNA vaccines for COVID‐19 shows how invaluable RNA research can be to providing solutions for modern problems. The 23rd Penn State Symposium in Plant Biology was held May 18–20, 2022, at the University Park campus of The Pennsylvania State University. It gathered RNA aficionados to share their exciting, new discoveries in plant RNA biology. This conference featured over 100 participants, and research spanning 20 countries, as well as 24 states within the United States.trans‐kingdom communication utilizing small RNAs (sRNAs).In this meeting report, we describe research that was presented on RNA processing that occurs within the cell, such as alternative splicing, noncanonical RNA caps and RNA editing, RNA turnover, and RNA‐directed regulation of transcription and translation. We delve into new insights regarding short and long noncoding RNAs (ncRNAs) in epigenetic reprograming. Finally, we discuss the RNA‐based tool CRISPR, RNA sensing of environmental responses via RNA modification and structure, and RNA interactions outside the cell through its role in 1.1Arabidopsis thaliana. The two isoforms of SR45, SR45.1 (long) and SR45.2 (short), have been shown to function in different developmental processes including flowering and root growth, respectively  or ABA‐related genes. This suggests the long isoform of SR45 is critical to regulate splicing in salt stress‐related pathways  is a processing step that expands the transcriptome of eukaryotes through the formation of multiple isoforms of a gene, each capable of independent functions. This process has been shown to primarily occur cotranscriptionally in the spliceosome and through recruitment of additional RNA‐binding proteins Reddy, . Dr. AniIn addition to mRNA processing occurring cotranscriptionally, evidence presented by Dr. Artur Jarmolowski  suggested that pri‐miRNAs (the primary transcripts that give rise to microRNAs) are processed cotranscriptionally in Arabidopsis. Dr. Jarmolowski's work utilized “plant native elongating transcripts sequencing” (plaNET‐seq) to show that cotranscriptional processing of pri‐miRNAs occurs within Arabidopsis and that the process relies heavily on R‐loop formation at the transcriptional start site of the miRNA loci. Interestingly, growth conditions may impact whether a transcript is cotranscriptionally or posttranscriptionally processed, potentially due to regulation of R‐loop formation . C‐to‐U conversions are widespread in plant mitochondria and chloroplasts. Dr. Stephane Bentolila  demonstrated the involvement of the RanBP2 zinc finger (RanBP2 Znf) domain in plant organelle RNA intron editing and splicing , have been identified in plants and other eukaryotes  is a eukaryotic mRNA surveillance system that shapes the transcriptome by eliminating mRNAs that contain premature stop codon. Dr. Misato Ohtani  discovered that deficiency of NMD alters tissue‐specific cell redifferentiation. NMD‐deficient mutants formed adventitious roots instead of adventitious shoots, under the shoot induction condition of tissue culture  and exonuclease digestion by EXORIBONUCLEASE (XRN4) or 3′ to 5′ degradation via SUPPRESSOR OF VARICOSE (SOV) or the exosome pathway  were solely regulated by decay which supports the idea of transcriptional balance between synthesis and decay. Dr. Sieburth also found a subset of sov mRNAs with shorter half‐lives, or high flux, but moderately increased abundance in comparison with WT. This indicates transcription plays a significant role in determining mRNA abundance. This phenomenon where significant changes in half‐life were measured but no significant changes in abundance were detected is an RNA decay defect feedback pathway known as RNA buffering. One key similarity for transcripts exhibiting RNA buffering is degradation involving mRNA decapping by VCS. These VCS‐dependent, high‐flux RNAs included genes known to function as environmental and developmental signals, which indicates probable regulatory function.mRNA abundance is controlled by the balance of two factors: its synthesis from transcription and its decay. mRNA turnover is also critical in cellular homeostasis. A major pathway in plants known as the cytoplasmic mRNA decay pathway consists of mRNA deadenylation followed by either 5′ to 3′ degradation via the decapping enzyme DCP1‐ASSOCIATED NYN ENDORIBONUCLEASE 1 (DNE1) targets. DNE1 is a cytoplasmic mRNA decay factor known to be involved in mRNA decapping and NMD  presented her group's research on the elucidation of Arabidopsis RNS2, the major RNase in Arabidopsis, autophagy‐related (ATG) genes, and the RNA helicase SKI2. Dr. MacIntosh's group has shown through a rns2‐2 null mutant that more total RNA is present in the cell comparison to WT, RNA accumulates in the vacuoles, and rRNA has an extended half‐life , that ski2‐5 null mutant plants have reduced ability to transport fluorescent RNA to vacuoles  presented his work on the vacuolar RNA salvage pathway through 1.4Eukaryotic organisms have evolved several mechanisms to alter the expression of genetic loci in developmental or stress‐specific manners through sRNA. The symposium featured talks on two major pathways of sRNA‐mediated control: sRNA silencing transposons through the deposition of chromatin modification marks from Dr. Julie Law  and Dr. Zofia Szweykowska‐Kulinska's  work understanding microRNA‐mediated response to abiotic stress in barley.Global DNA methylation patterning has emerged as a critical layer of genetic regulation with the establishment, removal, and regulation of these marks indicative of a complex and highly regulated evolved mechanism in Arabidopsis and other eukaryotes , as a major class of gene expression regulators in the context of drought stress in barley. Her group's tissue‐specific identification of novel miRNAs and their targets through sRNA‐seq and degradome seq target suggest an important involvement of microRNAs in the development and function of floral organs as well as abiotic stress  tackled how plastid DNA is structurally organized  are open reading frames that have start codons contained within the 5′ untranslated region  presented his research on conserved peptide uORFs (CPuORFs), specifically bZIP11's CPuORF function in ribosome stalling in high sucrose concentration leading to lower translation for bZIP11's main ORF  discussed that abundant 24‐nt siRNAs are produced from a small number of “siren” loci in ovules . Dr. Mary Gehring  reported the effects of parental Pol IV activity and the resulting developmental impacts on seed fitness and gene expression within the endosperm of Arabidopsis. Pol IV functions in the RdDM pathway by producing short, noncoding transcripts that are eventually processed into 24‐nt sRNAs. Dr. Gehring's work focused on NRPD1, a subunit of Pol IV, which previous studies have shown to play a major role in endosperm gene expression. Using a series of homozygous and heterozygous 1.8Since the initial discovery of the CRISPR‐Cas9 system, significant advances have been made in manipulating this system for the purposes of genetic engineering. The Cas9 nuclease will bind a single‐guide RNA (sgR) that it uses to scan the genome for the complement in the organism in which it is expressed  reported that the fat mass and obesity‐associated protein FTO, an RNA methylase, is responsible for demethylating m6A long‐interspersed element‐1 (LINE1) ultimately leading to chromatin state alterations and changes in gene expression that affect mouse oocyte/embryonic development  transcript is an example of a riboSNitch possessing a G to A SNP that alters its secondary structure  (riboSNitches) and climatic factors in Arabidopsis. The 1.11trans‐species miRNAs in a plant–plant interaction, and Dr. Hailing Jin  presented findings detailing the mobility and function of sRNAs traveling between organisms of differing taxonomic kingdoms in a plant–pathogen model.Presentations on the role of RNA in plant interactions with other organisms were focused on sRNAs generated in one organism and functioning in another. This phenomenon has been studied in the context of sRNAs traveling between interacting organisms with the outcome of inducing transgene silencing of pathogen and parasite mRNAs and suppressing host immune response  and RDR6‐ (RNA‐DEPENDENT RNA POLYMERASE/SGS2), they measured differential abundance of each siRNA to confirm that parasite Cuscuta‐derived miRNA activity is dependent on host machinery. Target mRNAs included TIR1 (TRANSPORT INHIBITOR RESPONSE 1) and AFB2 (AUXIN SIGNALING F‐BOX 2) involved in bacterial pathogenesis and defense signaling  involved in reducing sap loss after wounding  (Cai et al., 2Contributions from this conference highlight the importance and necessity to further understand RNA properties due to RNA's expansive and diverse impacts within the cell, ability for communication outside the cell and between organisms, and potential for crop improvement. Novel RNA discoveries continue to deepen our understanding of both established and newly discovered pathways and processes. RNA phenomena in plants provide a unique perspective as parallel pathways in animals can differ to varying degrees, as evidenced by the research presented in this report. In turn, the continued development of tools such as CRISPR as illustrated by Dr. Yiping Qi's dual base editing and gene activation system, and RNA‐based crop improvement methods, shown by Dr. Zofia Szweykowska‐Kulinska's work in barley miRNA and Dr. Chuan He′s work in potato and rice through RNA modifications, can be used to address current biological and agricultural problems. The field of RNA biology, especially in plants, remains a research area full of potential discoveries to improve crop systems, reveal novel aspects of established pathways, and identify noncanonical processes.The authors declare no conflict of interest associated with the work described in this manuscript.


During infection, the influenza A virus RNA polymerase produces both full-length and aberrant RNA molecules, such as defective viral genomes (DVGs) and mini viral RNAs (mvRNAs). Subsequent innate immune activation involves the binding of host pathogen receptor retinoic acid–inducible gene I (RIG-I) to viral RNAs. However, it is not clear what factors determine which influenza A virus RNAs are RIG-I agonists. Here, we provide evidence that RNA structures, called template loops (t-loops), stall the viral RNA polymerase and contribute to innate immune activation by mvRNAs during influenza A virus infection. Impairment of replication by t-loops depends on the formation of an RNA duplex near the template entry and exit channels of the RNA polymerase, and this effect is enhanced by mutation of the template exit path from the RNA polymerase active site. Overall, these findings are suggestive of a mechanism involving polymerase stalling that links aberrant viral replication to the activation of the innate immune response. Inefficient replication of short RNA molecules produced during influenza A virus infection leads to innate immune activation. Influenza A viruses (IAVs) are important human pathogens that generally cause a mild to moderately severe respiratory disease. A range of viral, host, and bacterial factors can influence the outcome of infections with IAV  into the host cell nucleus. These RNPs consist of oligomeric viral nucleoprotein (NP), a copy of the viral RNA-dependent RNA polymerase, and one of the eight segments of single-stranded negative-sense viral RNA (vRNA) that make up the viral genome (In addition to full-length vRNA and complementary RNA (cRNA) molecules, the RNA polymerase can produce aberrant RNAs that are shorter than the vRNA or cRNA template from which they derive. These aberrant RNAs include defective viral genomes (DVGs) .mvRNAs bind RIG-I and activate the MAVS signaling cascade . These oWe subsequently validated the IFN-β promoter activation by the NP47, NP56, and NP76 mvRNA templates during replication by the IAV RNA polymerase. To this end, we transfected plasmids expressing the RNA polymerase subunits PB1, PB2, and PA; a plasmid expressing NP; and a plasmid expressing the NP76 template mvRNA into HEK293T cells. Primer extension analysis showed efficient amplification of NP47, NP56, and NP76 and the production of several smaller aberrant RNA products that were shorter than the mvRNA template in the case of NP56 and NP76 , while the other two (NP71.2 and NP71.3) were not . Among tRenilla reporter plasmids into HEK293T cells. Retransfection of NP71.1 to NP71.5 showed an inverse pattern of IFN-β promoter activation in comparison to To exclude that a differential recognition of the engineered mvRNAs by host pathogen receptors of the host cell was responsible for the observed increased IFN-β promoter activity on the NP71.2 and NP71.3 mvRNAs, we isolated total RNA from HEK293T transfections and retransfected equal amounts of these RNA extracts together with IFN-β and MAVS−/− IFN::luc HEK293 cells (MAVS−/− cells do not express endogenous MAVS (fig. S4A), blocking any RIG-I–mediated innate immune signaling, but overexpression of a MAVS-FLAG plasmid still triggers IFN-β promoter activity, indicating that the IFN-β reporter is still functional (fig. S4B). Expression of NP71.1 to NP71.5 in the MAVS−/− cells did not induce IFN-β promoter activity (fig. S4C). Subsequent primer extension analysis showed that the differences in replication between NP71.1 to NP71.5 had been maintained in the MAVS−/− cells (fig. S4C), demonstrating that the differential replication efficiency is not dependent on the innate immune response.To verify that the different replication efficiencies had not been the result of the effect of NP71.2 and NP71.3 on the innate immune response, we also expressed these mvRNAs and the WSN RNA polymerase in To investigate whether the effect of the NP71.3 and NP71.4 mvRNAs was specific to the WSN polymerase, we expressed these mvRNAs alongside the pandemic H1N1 A/Brevig Mission/1/18 (abbreviated as BM18) or the highly pathogenic avian H5N1 A/duck/Fujian/01/02 (abbreviated as FJ02) RNA polymerases. We found that the BM18 and FJ02 RNA polymerases were impaired on the NP71.2 and NP71.3 mvRNA templates and triggered a stronger IFN-β promoter activity on the NP71.3 template relative to the NP71.1 template (fig. S5). We also observed that the BM18 RNA polymerase produced short aberrant RNA products, while the FJ02 RNA polymerase did not despite inducing IFN-β promoter activity (fig. S5). Together, these results suggest that the mvRNA template is the innate immune agonist, rather than the aberrant RNA products derived from the mvRNA template, and that innate immune activation is dependent on a sequence-specific interaction between the active IAV RNA polymerase and the mvRNA template.G) for every putative t-loop and every putative secondary RNA structure upstream and downstream of the RNA polymerase . For each position analyzed, we excluded 20 nt from the folding analysis for the footprint of the IAV RNA polymerase (G) between the computed ΔG values for the individual structures .ructures .To confirm that t-loops affect RNA polymerase processivity and IFN-β promoter activity, we replaced two A-U base pairs of the NP71.2 t-loop duplex with two G-U base pairs, creating NP71.6 . FollowiThe mvRNAs NP71.2 and NP71.3 contain a t-loop in the first half of the positive-sense mvRNA template. To confirm that t-loops also affect RNA polymerase activity in the negative sense, we engineered three additional 71-nt-long mvRNA templates with t-loops in different locations of the template (NP71.10 to NP71.12) . In lineTo investigate whether t-loops affect RNA polymerase processivity in vitro, we purified the WSN RNA polymerase from HEK293T cells using tandem-affinity purification (TAP) . By contTo investigate whether t-loop containing templates remained stably bound to the RNA polymerase or triggered template dissociation, we immobilized mOrange-tagged RNA polymerase on magnetic RFP-trap beads and incubated these immobilized complexes with radiolabeled template. After removal of unbound template by three washes with binding buffer, adenosine-guanine dinucleotide (ApG) and nucleoside triphosphates (NTP) were added to initiate RNA synthesis and complexes incubated at 30°C for 15 min. Next, the immobilized complexes were washed three times to remove dissociated RNA, and the reactions stopped with formaldehyde/EDTA loading dye. Analysis of the bound and unbound radiolabeled RNA levels by dot blot and autoradiography showed no difference among the NP71.1, NP71.10, and NP71.11 templates (fig. S8A). To rule out that released template was rebound upon dissociation from the RNA polymerase, we added excess unlabeled NP71.1 template as RNA polymerase trap at the start of the reaction. Again, no difference between the templates was observed (fig. S8A).To confirm that the immobilized RNA polymerases were active, we immobilized RNA polymerase bound to unlabeled template mvRNA on magnetic beads as described above. Next, we added ApG, NTPs, and radiolabeled guanosine 5′-triphosphate and incubated the immobilized complexes at 30°C for 15 min. Following the removal of unincorporated NTPs by three washes with binding buffer, the nascent RNA in solution and associated with the immobilized complexes was analyzed by denaturing PAGE and autoradiography. As shown in fig. S8B, partially extended and full-length nascent RNAs remained associated with the immobilized RNA polymerases. Partially extended nascent RNAs were also found in the unbound fraction. Addition of inactive RNA polymerase (PB1a) to serve as encapsidating polymerase in RNA polymerase dimers increased the release of partially extended nascent RNAs but not the release of full-length RNAs. Together, these results suggest that t-loops do not induce template release upon RNA polymerase stalling and that partially extended nascent strands can be separated by the RNA polymerase from the template strand and released.In the IAV RNA polymerase elongation complex, the 3′ terminus of the template is guided out of the template exit channel via an exit groove on the outside of the thumb subdomain. This groove consists of PB1 and PB2 residues and leads to promoter binding site B  and 4A  and 4A, To test whether dysregulation of the exit groove leads to more IFN-β promoter activation, we mutated PB1 lysine-669, which resides at the start of the exit groove , to alanmvRNAs are produced during IAV infection in vitro and in vivo .G values for these mvRNAs revealed potential t-loops in the first half of the sequence for mvRNAs C, D, H, and J and potential t-loops in the second half of the sequence for mvRNAs E, F, and G  into pPolI plasmids . Analysis of the ΔΔF, and G . SubsequF, and G . These dF, and G  and thatG of the first half of the template  from the mvRNA sequences identified during infection into pPol expression plasmids and transfected these plasmids into HEK293T cells. As shown in fig. S12, PA and hemagglutinin (HA) mvRNAs induced both high and low levels of IFN-β promoter activity compared with our NP71.1 control. Together, these results indicate that viral infections produce mvRNAs with different potentials to induce IFN-β promoter activity and that t-loops play a key role in the potential of mvRNAs to induce IFN-β promoter activity by affecting the ability of the RNA polymerase to efficiently replicate them.Two factors important for inducing an innate immune response in IAV infections are active viral replication and the binding of vRNA molecules to RIG-I . We suspIt is unclear how RIG-I gains access to the t-loop containing mvRNA once the polymerase has stalled . We obseIt is possible that t-loops affect influenza RNA polymerase activity on full-length vRNAs or DVGs. However, it is more likely that t-loops form only on partially formed RNPs or NP-less templates, because NP may modulate the presence and location of secondary RNA structures. During vRNA synthesis, NP dissociates and binds vRNA in a manner that is coordinated by the vRNA polymerase. When NP levels are reduced, aberrant RNPs or NP-less RNA products may form in which secondary RNA structures that are absent in the presence of NP contribute to t-loop formation and RNA polymerase stalling . We prevDuring viral infection, mvRNA molecules of various lengths and abundancies are produced (pcDNA3-based plasmids expressing influenza A/WSN/33 (H1N1) proteins PB1, PB2, PA, NP, PB2-TAP, and the active site mutant PB1a (D445/D446A) have been described previously (IFNB promoter [pIFΔ(−116)lucter] and constitutively expressing Renilla luciferase plasmid (pcDNA3-Renilla) were described previously  (Firefly luciferase reporter plasmid under the control of the MAVS−/− cells expressing luciferase under the control of the IFNB promoter were a gift from J. Rehwinkel and were described previously (l-glutamine (Sigma-Aldrich). Transfections of HEK293T or HEK293 cell suspensions were performed using Lipofectamine 2000 (Invitrogen) and Opti-MEM (Invitrogen) following the manufacturer’s instructions, and transfection of confluent, adherent HEK293T cells were performed using polyethylenimine (PEI) (Sigma-Aldrich) and Opti-MEM. Infections were performed at MOI 3 as described previously , anti-PB2 , and anti-NP  diluted 1:1000 in TBSTM [tris-buffered saline (TBS)/0.1% Tween 20 (Sigma-Aldrich)/5% milk]. Cellular proteins were detected using the rabbit polyclonal antibodies anti–glyceraldehyde-3-phosphate dehydrogenase  diluted 1:4000 in TBSTM and anti-RNA Pol II  diluted 1:100 in TBSTM; the mouse monoclonal antibodies anti-MAVS E-3  diluted 1:200 in TMSTM and MitoTracker [113-1]  diluted 1:1000 TBSTM; and the rat monoclonal antibody anti-tubulin  diluted 1:1000 in TBSTM. Mouse monoclonal antibody anti-FLAG M2  diluted at 1:2000 was used to detect MAVS-FLAG. Secondary antibodies IRDye 800 donkey anti-rabbit , IRDye 800 goat anti-mouse , IRDye 680 goat anti-mouse , and IRDye 680 goat anti-rat  were used to detect Western signals with a LI-COR Odyssey scanner.G of duplex formation between a stretch of 10-nt upstream of the footprint and 10-nt downstream of the footprint. The formation of upstream and downstream structures was computed for 24-nt windows (the footprint of NP) upstream and downstream of the moving footprint. The ΔΔG was computed by subtracting The ViennaRNA package commands duplexfold and cofold were used to compute the ΔG values  and resuspended in an equal volume of Dual-Glo reagent (Promega), followed by Dual-Glo Stop & Glo reagent (Promega). Firefly and Renilla luminescence were measured after 10-min incubation with each reagent respectively as per the manufacturer’s instructions for the Dual-Glo Luciferase Assay System  using the GloMax luminometer (Promega).To measure IFN expression in RNP reconstituted HEK293T or HEK293 cells, luciferase assays were carried out 24 hours after transfection. RNP reconstitutions were carried out in a 24-well format by transfecting 0.25 μg of the plasmids pcDNA3-PB1, pcDNA3-PB2, pcDNA3-PA, pcDNA3-NP, and a pPolI plasmid expressing a mvRNA template. HEK293T and HEK293 cells were additionally cotransfected with 100 ng of the plasmid pIFΔ(−116)lucter and 10 ng of the plasmid pcDNA3-g for 5 min at 4°C, diluted in 2 ml of NaCl (Sigma-Aldrich), and bound to prewashed immunoglobulin G Sepharose beads (Cytiva) for 2 hours at 4°C. Beads were prewashed 3× in binding buffer . After binding, beads were washed 3× in binding buffer and 1× in cleavage buffer . Beads were cleaved with AcTEV protease (Invitrogen) overnight at 4°C and cleared by centrifugation at 1000g for 1 min as described previously (Influenza virus A/WSN/33 (H1N1) recombinant polymerases were purified from HEK293T cells. Ten-centimeter plates of adherent cells were transfected with 3 μg of pcDNA3-PB1, pcDNA3-PB2-TAP, and pcDNA3-PA with PEI (Sigma-Aldrich). Forty-eight hours after transfection, cells were harvested in PBS and lysed on ice for 10 min in 500 μl of lysis buffer . Lysates were cleared by centrifugation at 17,000Fractionation of transfected cells into cytoplasmic, mitochondrial, and nuclear components was carried out using the Abcam Cell Fractionation Kit (Abcam) following the manufacturer’s instructions, with volumes adjusted on the basis of the number of cells. Samples of unfractionated whole cells in buffer A were retained as input controls. Whole cells and subcellular fractions were dissolved in TRIzol for RNA extraction and analyzed as described above or in 10% SDS protein-loading buffer for protein expression analysis by SDS-PAGE and Western blot.MAVS−/−).Statistical testing was carried out using GraphPad Prism 9 software. Error bars represent SDs, and either individual data or group mean values are plotted. One-way analysis of variance (ANOVA) with Dunnett’s test for multiple comparisons was used to compare multiple-group means to a normalized mean . Two-way ANOVA with Sidak’s test for multiple comparisons was used to compare multiple pairs of group means (e.g., between two cell types, HEK293 WT to HEK293


T. cruzi and T. brucei are protozoan parasites that present efficient mechanisms of immune system evasion, leading to disease chronification. Currently, there is no vaccine, and chemotherapy is effective only in the absence of severe clinical manifestations. Nevertheless, resistant phenotypes to chemotherapy have been described in protozoan parasites, associated with cross-resistance to other chemically unrelated drugs. Multidrug resistance is multifactorial, involving: (i) drug entry, (ii) activation, (iii) metabolism and (iv) efflux pathways. In this context, ABC transporters, initially discovered in resistant tumor cells, have drawn attention in protozoan parasites, owing to their ability to decrease drug accumulation, thus mitigating their toxic effects. The discovery of these transporters in the Trypanosomatidae family started in the 1990s; however, few members were described and functionally characterized. This review contains a brief history of the main ABC transporters involved in resistance that propelled their investigation in Trypanosoma species, the main efflux modulators, as well as ABC genes described in T. cruzi and T. brucei according to the nomenclature HUGO. We hope to convey the importance that ABC transporters play in parasite physiology and chemotherapy resistance.Human trypanosomiasis affects nearly eight million people worldwide, causing great economic and social impact, mainly in endemic areas.  Trypanosoma genus comprises several species of protozoan parasites of vertebrates that have the following characteristics: a region containing mitochondrial DNA, called the kinetoplast (Kinetoplastida class); a single flagellum (Trypanosomatidae family); and a heteroxenic evolutionary cycle in which it presents itself in one of the evolutionary forms as a trypomastigote in the vertebrate host , or GSH-conjugated compounds. In trypanosomatids, the main non-protein thiol is T(SH)2 that has two GSH molecules linked to a polyamine chain, usually spermidine ..TbABC1, T. brucei [ABCE1 gene encodes a protein with 631 amino acids and 71 kDa with an RLI metal binding domain, a 4Fe-4S binding domain and two NBDs, and a predicted cytosolic localization  found the ribonuclease L inhibitor (RLI) protein, now recognized as ABCE1 in . brucei . The ABClization . It appeGAT1-3 in pro-cyclic forms of T. b. brucei [GAT1-3 encodes half-size transporters with a TMD–NBD arrangement  sequences, named . brucei . Glycoso. brucei . GAT1-3 angement  with lowxokinase . Accordixokinase . ABCD2 axokinase .Atm1) and multidrug resistance-like (Mdl) genes from S. cerevisiae as the query, Harakova and colleagues (2015) identified homologous genes in the T. brucei genome, which were designated TbAtm and TbMdl [TbAtm encodes a protein of approximately 79 kDa and shares about a 40% amino acid identity with the homolog genes Atm1 from S. cerevisiae, Atm3 from A. thaliana and human ABCB7. TbMdl encodes a protein of a predicted molecular weight of 76 kDa that shows almost a 40% amino acid sequence similarity to the homolog genes Mdl1 and Mdl2 of S. cerevisiae and human ABCB10. Both are mitochondrial ABC transporters: Atm is involved in the Fe-S cluster in yeast [Mdl interacts with Fe-S-containing ferrochelatase, an enzyme of the heme biosynthesis pathway in erythroid cells [TbAtm and TbMdl correspond to TbABCB3 and TbABCB1  drug entry, (ii) activation, (iii) metabolism and (iv) efflux. Among the factors involved, the overexpression of ABC transporters is directly related to increased drug efflux and reduced toxic effects within cells. Several protozoan parasites exhibit an MDR phenotype, causing a reduction in the efficacy of chemotherapy.


Here, we measured the in vivo termination efficiencies of 72 Rho-dependent terminators in Escherichia coli by systematically performing qRT-PCR analyses of cDNA prepared from mid-log phase bacterial cultures. We found that these terminators exhibited a wide range of efficiencies, and many behaved differently in vivo compared to the predicted or experimentally determined efficiencies in vitro. Rho-utilization sites (rut sites) present in the RNA terminator sequences are characterized by the presence of C-rich/G-poor sequences or C > G bubbles. We found that weaker terminators exhibited a robust correlation with the properties  of these C > G bubbles of their respective rut sites, while stronger terminators lack this correlation, suggesting a limited role of rut sequences in controlling in vivo termination efficiencies. We also found that in vivo termination efficiencies are dependent on the rates of ATP hydrolysis as well as Rho-translocation on the nascent RNA. We demonstrate that weaker terminators, in addition to having rut sites with diminished C > G bubble sizes, are dependent on the Rho-auxiliary factor, NusG, in vivo. From these results, we concluded that in vivo Rho-dependent termination follows a nascent RNA-dependent pathway, where Rho-translocation along the RNA is essential and rut sequences may recruit Rho in vivo, but Rho-rut binding strengths do not regulate termination efficiencies.Bacterial Rho is a RNA-dependent ATPase that functions in the termination of transcription. The  The Rhlex (EC) , 4, 5, 6lex (EC) , 8, 9. Tlex (EC) . Recentlvia its c-terminal domain (NusG-CTD) (in vitro (in vivo (The Rho-dependent termination is facilitated by the transcription elongation factor NusG  that dirusG-CTD) , 13, 14,in vitro , 16, 17. to interact with Rho, presence of different cis-acting elements like RNA-hairpins and riboswitches, Rho-antagonists, etc. It is also important to know how the nature and pattern of the C > G bubbles  are corin vivo termination efficiencies of these terminators using E. coli strains expressing termination-defective Rho mutants and Rho-binding defective NusG mutants. These terminators exhibited a wide range of efficiencies and many of them behaved differently in vivo compared to that predicted or experimentally shown in vitro  of the C > G bubble stretches (described as “descriptors”) of the rut sites. This correlation disappeared as the terminator strength increased suggesting a limited role of the nature of rut sequences in controlling the termination efficiencies in vivo, even though the binding affinities of Rho for the rut sites were dependent on the nature of the C > G bubbles. The termination efficiencies of the majority of the terminators showed good positive correlations with their respective rates of ATPase activities of Rho on the RNA templates having the terminator-zone sequences. This indicates the Rho-translocase activity-dependence of the majority of the terminators in vivo. Consistent with the earlier proposals . Multiple primers probes were used to experimentally validate the presence of Rho-dependent termination zones upstream of these probes sites (A–C). The length of the RNA transcripts of different genes that were analyzed in these assays varied from 91 to 5914 nt , the fold-change values of each gene give a semiquantitative measure of the in vivo termination efficiencies of the terminators present in these terminator zones. The higher fold change values indicate the higher termination efficiency and the presence of a stronger terminator.Several genomics studies revealed the existence of genes as well as operons, expressions of which are under the control of Rho-dependent termination in vivo , 30, 31.mination . We primmination . Later, mination . This Rhmination , whereasmination . And henmination A. Multipes sites , A–C. Thse genes , A–C, thrut sites within a terminator zone are characterized by C > G sequence bubbles in the RNA sequences (sequence of non-template DNA)  . We scanIn vitro termination efficiencies of many Rho-dependent terminators have been measured, and an algorithm developed from these experimental data was used to predict their strength (in vivo termination efficiencies of these genes should be quite different as Rho-dependent termination faces many competing processes inside the cell (see Introductory paragraphs). We measured the upregulation (in terms of fold-change) of 72 genes in a strain expressing the Rho N340S mutant relative to that expressing the WT variant by RT-qPCR (A). Among them, some regions were previously identified to have Rho-dependent terminators in an in vivo 3′-end mapping study , moderate (2–10 fold), strong (10–30 fold), and very strong (>30) (A). Out of the 54 terminators that belong to the very strong to the moderately strong category, 31 were predicted to be strong from the in vitro data  A. Out oftro data , whereasykfG, yafZ, ykfI, yagM, and yagL; Qin prophage: ydfU; CP 4-57: yfjI) are the strongest Rho-dependent terminators, which are essential to suppress the toxic prophage genes (thrW and selC) also belonged to the very strong/strong category. They were not categorized under any group in Nadiras et al. 2018. An RNA folding analysis of these two genes revealed the presence of extensive secondary structures, which could have affected theirs in vivo characters. It has been reported that tRNA genes are highly regulated by Rho-dependent termination (thiC and thiM (A).We observed that terminators present upstream of the prophage genes . We calculated the length of the RNA from the transcription start site  to the sites of the reverse primer (RP) probe of the gene  for their duplex formation did not correlate with the measured fold changes obtained in Figure 2A (B and C). This indicated that the high RT-qPCR values did not arise from longer and more stable RNA. Next, we experimentally determined the in vivo stabilities of the 12 RNA transcripts transcribed from the selected genes that showed a wide range of fold change values in Figure 2A. We isolated RNA at different time points from the mid-log phase culture of an MG1655 strain expressing Rho N340S mutant and performed RT-PCR to quantitate the time-dependent decay of the RNA (A and B) and calculated their half-lives (D). A plot of half-lives against their corresponding fold change values  did not show any correlation. These data strongly indicated that the fold change values described above were not influenced by the in vivo RNA stability and they truly represent the in vivo termination efficiencies of the 72 terminators. It should also be noted that the general stability of RNA transcripts in the Rho N340S strain is less than its WT counterpart , which rut sites as well as the terminator zones upstream of the 72 genes described above, we screened for C > G bubble stretches (C-rich and G-poor sequences) using 70 nt sliding windows from 5′ to 3′ direction of the RNA . However, when the fold change values less than 10-fold were plotted against these descriptor values, significantly high positive correlations were observed with the following descriptors: area of longest C > G bubbles (r2 = 0.6), cumulative area of all the C > G bubbles (r2 = 0.54), maximal difference (r2 = 0.54) and average difference between %C and %G (r2 = 0.51), and density of C > G bubbles (r2 = 0.47) (B). Other descriptors also showed moderate positive correlations (B and S8B). These correlations were not at all observed when > 10-fold fold change values were plotted against these descriptor values . The values of descriptors did not observe to increase proportionally with the fold change values beyond a certain point. A larger C > G bubble determined by the above-mentioned descriptors offers long stretches of C-rich sequences that improve the strength of the rut sites for Rho-binding. And hence, these data indicated that the strength of the rut sites could regulate only the termination efficiencies of the weaker terminators in vivo. The factors other than the rut sequence are more important for achieving high termination efficiencies in vivo.A scattered plot of all the fold change values against different descriptor values appeared to lack any such correlation Figs. 3 = 0.47) B. Other elations B and S8B = 0.47 Brut sites with higher C > G descriptor values have higher binding constants (Kd) for the Rho protein (A–D). We synthesized radiolabeled RNAs having the terminator zone sequences of selected genes, termination efficiencies  of which were measured in Figure 2A (A and B). We have synthesized >200 nt RNA sequences so that all the putative rut sites of a terminator zone are accommodated. These terminators have a wide range of fold change values (0.7–78 fold). We performed gel-shift assays using WT Rho to calculate the Kd values of each of the Rho–RNA transcript interactions . The sigmoidal nature of the binding isotherms indicated the presence of multiple Rho-binding sites in these RNA transcripts , which indicated that the Rho-interactions with the high-affinity rut sites are specific. However, it should be noted that the difference between specific and nonspecific binding at the weaker rut sites would be much less. Upon plotting the Kd values against the descriptor values of the rut sites present in these synthesized RNA transcripts, we observed a moderate correlation (r2 = 0.44) with the length of the C > G bubbles (C and D). This indicates that the higher descriptor value of a rut site could reflect higher binding affinity (lower Kd) toward Rho. An increase in the length of the C > G bubble increases the length of C-rich sequences, and hence the binding constant increases. Interestingly, these measured Kd values did not show any correlation with the fold change values of the respective terminators that these rut sites belong to (E). This indicates that the binding strength of the rut sites for Rho does not influence the termination efficiency, even though these sites are required to recruit Rho to the elongation complex at some stage. Post-RNA recruitment steps such as Rho-isomerization . These templates were able to induce ATPase activity of WT Rho with a wide range of rates in the in vitro assays . The template with the sequence of terminator zones of gfcA yielded the highest rate that was still 3-times lower than that we achieved with the best synthetic substrate of Rho, polyC RNA. This data indicates that the Rho most likely translocates with a wide range of rates on the different nascent RNAs from different operons in vivo, which could have variable effects on its termination efficiencies.Rho is an efficient molecular motor that can translocate along the RNA driven by its RNA-dependent ATP hydrolysis activity, and this translocation activity has been implicated in its transcription termination function , 4, 6. WA) from which these RNA transcripts were prepared. We observed a positive correlation (r2 = 0.62) between these two parameters for most of the terminators, except those having very high in vivo termination efficiencies . Among them, the thiC terminator is part of the thymine pyrophosphate–activated riboswitch  with the descriptor values that describe the area and length of the C > G bubbles (A and B). This correlation was observed for the ATPase rates < 50 pmol/min. The highest correlation was observed with the descriptor that defines the length of the longest C > G bubble. The descriptors defining other parameters of the C > G bubbles did not show good correlations  in the C > G bubble sequences of these terminators. We chose MG1655 strains that express the Rho-binding defective NusG mutants, G146D, V160N, and L158Q . We calculated the average fold change values from the fold change data of all the three NusG-mutants (A).A subset of Rho-dependent terminators is dependent on NusG . We expl-mutants A.Figure A, A and B). However, the values were less compared to that obtained in the presence of the Rho mutants, N340S , and also the NusG- and Rho-mutant data sets did not follow a linear correlation. In general, for the strong terminators, the fold change values obtained in the Rho mutant are much higher than that observed in the NusG mutants . But in cases of weak or moderately strong terminators, these differences in the upregulation (fold change) were observed to be much less. As NusG-dependent terminators are a subset of Rho-dependent terminators and the NusG dependency of all the Rho-dependent terminators varies considerably . The plot showed a biphasic nature, where the ratio of fold-change values > 0.5 was considered to be highly NusG-dependent (21 genes). Values within 0.2 to 0.5 were classified as moderately NusG-dependent (31 genes) and the values < 0.2 were classified as NusG-independent (20 genes). Most of the terminators that are categorized as none or weak terminators in Figure 2A were also observed to be highly NusG dependent in vivo. Hence, the nature of in vivo NusG dependency of these 72 terminators was consistent with the mechanism of NusG action proposed from the in vitro studies  or NusG mutants  and established their correlation or lack of it with the C > G bubble patterns of their respective rut sites, binding constants of Rho–rut site interactions, the Rho translocase activity on the nascent RNA, and in vivo NusG-dependence of these terminators. We made the following observations. (1) These terminators exhibited a wide range of terminator strengths in vivo where the prophage terminators exhibited to be the strongest ones. Many of the terminators behaved differently in vivo compared to that predicted or experimentally shown in vitro  of the C > G bubble stretches of the rut sites. This correlation disappeared as the terminator strength increased suggesting a limited role of the nature of rut sequences in controlling the termination efficiencies in vivo (rut sites were dependent on the nature of the C > G bubbles (in vivo termination efficiencies of the majority of the terminators showed good correlations with the rates of ATPase activities of Rho on the RNA templates made of the sequence of the respective terminator-zones (in vivo. (4) Consistent with the earlier propositions  Onl in vivo , and S8. bubbles . (3) Theor-zones  and S11 ositions , 19, the on NusG  and S13. on NusG  and S14.in vitro Rho-dependent transcription termination assays, two types of termination pathways have been proposed: RNA-dependent and RNAP-dependent pathways  and [α-32P] UTP (3000 Ci/mmol) were from Jonaki, BRIT. Antibiotics, lysozyme, DTT, and bovine serum albumin were from USB. Primers for PCR and dC34 were obtained from Eurofins. Restriction endonucleases, T4 polynucleotide kinase, and T4 DNA ligase were from New England Biolabs. Taq DNA polymerase was from Roche Applied Science. RNAlater used for storing RNA samples used in microarray experiments were from Ambion. RNAeasy kit and RNAprotect Bacteria used for RNA isolation were from Qiagen. DNase I amplification grade, Superscript III-RT, RNase out, EDTA, DTT, and random hexamers were purchased from Invitrogen. Twenty millimolar MgCl2 and ten millimolar dNTP solutions were from Thermo Scientific. dATP and poly(C) RNA were obtained from Amersham. TB Green Premix Ex Taq II (Tli RNase H Plus) from Takara was used for RT-qPCR. Sodium-deoxycholate and Phenol were from USB. AmpliScribe T7 High Yield Transcription Kit was purchased from Lucigen. Malachite green (MG), ammonium molybdate, and polyvinyl alcohol were obtained from Sigma. All the bacterial growth media were from Difco. – [Ct of internal control]; and ΔΔCt = [ΔCt of mutant] – [ΔCt of WT]. The level of rpoC mRNA was used as an internal control. Primer pairs were designed to probe various regions of an operon so that the PCR products were within 200 nt in size. For the terminators, trac, trpt’, and λtR1, the primer probes were in lacZ gene fused downstream to them.The RS862 was transformed with pCL1920 expressing WT and the mutant derivatives of Rho and NusG followed by deletion of chromosomal copies of these two genes. RNA was isolated from mid-log phase , area of the longest C > G bubble, cumulated area of all C > G bubbles, area density of C > G bubbles, maximum %C in a longest C > G bubble, maximal %C - %G in a longest C > G bubble, average %C - %G in a longest C > G bubble, number of [(YC)N9→13]1 motif (YC dimers) in longest C > G bubble, number of [(YC)N9→13]2 motifs in longest C > G bubble, number of [(YC)N9→13] 3 motifs in longest C > G bubble, and the density of [(YC)N9→13]1 motif (%YC) (see t).The descriptor values were calculated using the python script “MakeDescriptors” as described in Nadiras C and %G  and S6. %YC) see  was dete0 + ax). The goodness of fit for the linear regression (r2) values was also calculated using Sigmaplot 13 and the probability of a hypothesis  was calculated using GraphPad Prism. The plots with higher correlation coefficients (r2 > 0.3) had p-values in the range of > 0.0001 to 0.041. Linear regression plots with lower r2 values were found to have higher p-values (>0.05) and hence considered to be not significant.In all the correlation graphs, scattered points were plotted using SigmaPlot 13 and they were fitted to straight lines by linear regression analyses using the formula  are represented as dotted symbols.rut site sequences were synthesized by PCR in such a way that in all the cases, transcription starts from T7 φ10 promoter. This promoter sequence was incorporated in the forward primers. The reverse primer was designed in such a way that all the PCR products were ∼300 bp in length. The PCR amplifications were performed using high fidelity Deep Vent polymerase from NEB and the chromosomal DNA was used as a template for the PCR reactions. These templates were transcribed using the AmpliScribe T7 High Yield Transcription Kit following the manufacturer’s protocol. For the Rho-RNA binding assays, the RNAs were labeled with [α-32P] UTP during the synthesis. For the ATPase assays, the RNAs were synthesized in the same way, except that radioactive NTP was not used. The poly (dC34) was labeled with [γ-32P] ATP using T4 polynucleotide kinase.Different linear DNA templates for RNA synthesis containing the predicted 2, 50 mM KCl, 1 mM DTT and 0.1 mg/ml of bovine serum albumin) with the increasing concentrations of the WT Rho (0–50 nM). The binding reactions were performed at 37 °C for 5 min before loading onto a running 4% (w/v) native acrylamide gel under cold (4 °C). Gels were scanned in phosphor-imager Typhoon 9200 and fractions of unbound species were quantified by ImageQuant software. Gel-shift assays with radio-labeled polydC34 were also performed in the same way. The dissociation constant (Kd) values were calculated by fitting the binding curves to either sigmoidal or hyperbolic equations using the SigmaPlot 13.WT Rho-RNA binding constants were measured by gel-shift assays using radio-labeled RNA. In each assays, 5 nM of RNA was used in the transcription buffer (25 mM Tris–HCl (pH 8.0), 5 mM MgClin vitro ATPase study . Two milliliters of culture was harvested (time point 0) and immediately RNAprotect Bacteria (Qiagen) were added to stabilize the RNA in vivo. At that point, transcription was stopped by treatment with 50 μg/ml rifampicin and the cells were incubated for an additional 2, 4, 6, 8, and 10 min, respectively, following which cells were harvested and were resuspended in RNAprotect Bacteria, and RNA was isolated using the Qiagen RNeasy Kits. Residual genomic DNA in the RNA preparations was eliminated by DNase I treatment. One microgram RNA was used for the synthesis of cDNA using Superscript III Reverse Transcriptase following the standard procedures. The cDNA was used for RT-PCR amplification for estimating the amount of RNA left at different time points. PCR products were run on 1.5% agarose gels that were visualized on Alpha Imager Gel Imaging System. The decrease of DNA band intensities relative to the control (time point 0) was estimated by ImageQuant software. Band intensities were plotted and the curves were fitted to an exponential decay equation to calculate the half-life and degradation constants of the RNAs in vivo.et al., 2018 and are available upon request.All the raw datasets are available upon request. Computer codes (in python script) are written based on that described in Nadiras This article contains The authors do not declare any competing interests. P. I. R. C. is a DBT Senior Research fellow.


RNA molecules can form secondary and tertiary structures that can regulate their localization and function. Using enzymatic or chemical probing together with high-throughput sequencing, secondary structure can be mapped across the entire transcriptome. However, a limiting factor is that only population averages can be obtained since each read is an independent measurement. Although long-read sequencing has recently been used to determine RNA structure, these methods still used aggregate signals across the strands to detect structure. Averaging across the population also means that only limited information about structural heterogeneity across molecules or dependencies within each molecule can be obtained. Here, we present Single-Molecule Structure sequencing (SMS-seq) that combines structural probing with native RNA sequencing to provide non-amplified, structural profiles of individual molecules with novel analysis methods. Our new approach using mutual information enabled single molecule structural interrogation. Each RNA is probed at numerous bases enabling the discovery of dependencies and heterogeneity of structural features. We also show that SMS-seq can capture tertiary interactions, dynamics of riboswitch ligand binding, and mRNA structural features. Chemical probing combined with sequencing is broadly used for RNA structural profiling . Most ofDirect sequencing of long native RNA molecules after chemical probing can reveal insights into the complete structural profiles of longer RNA molecules ,10. ThisIn vitro synthesis of RNA. Custom sense and antisense hairpin oligos with T7 promoter sequence were purchased from Sigma (Sigma-Aldrich) and in vitro transcribed using T7 RNA polymerase (NEB). F. nucleatum FMN riboswitch RNA was synthesized in vitro using T7 RNA polymerase from a pUT7 vector that carries 112-nucleotides FMN sensing domain with hammerhead ribozyme at the end to obtain identical 3′ ends (in vitro using T7 RNA polymerase. TPP template was prepared using sense and antisense TPP oligos with T7 promoter sequence (sequence  purchaseSaccharomyces cerevisiae) strain Y187 was grown in YPD medium at 30°C. Saturated cultures were diluted to OD600 of ∼0.1 and grown to a final OD600 of 0.7 followed by RNA extraction using hot phenol–chloroform method. Poly(A) and non-poly(A) separation was performed using Poly(A)Purist MAG Kit (Invitrogen) following manufacturer protocol. The remaining non-poly(A) RNA was precipitated overnight at −80°C following the RNA ethanol precipitation protocol.Cell culture. Yeast (2 for 20 min at 20°C. For (+)DEPC, RNA was treated with 10%  DEPC for 45 min at 20°C. DEPC reaction was performed in the absence and presence of 6-fold molar excess of riboflavin 5′-monophosphate and thiamine pyrophosphate (Sigma-Aldrich) for FMN and TPP riboswitch, respectively. The reactions were immediately purified using RNA Clean & Concentrator (Zymo Research) or RNA XP bead (Beckman Coulter).Diethyl pyrocarbonate (DEPC) treatment of RNA. All structural probing was carried out using DEPC (Sigma), which carbethoxylates unpaired adenosine at N-6 or N-7 by opening the imidazole ring  Figure . 1μg RNAhttps://github.com/hyeshik/poreplex). After renaturation and treatment with DEPC, in vitro synthesized and yeast non-poly(A) RNA were polyadenylated using Escherichia coli polyA polymerase (NEB) at 37°C for 15 min. To obtain full-length reads and a molecule long enough to be processed, the hairpin and riboswitches were ligated to the 3′ ends of a longer RNA molecule  by replacing the 3′ adapters (RTA) with custom-made barcodes for multiplexing  (https:/molecule . RTA adamolecule . The firhttps://github.com/hyeshik/poreplex) and resquiggled using Tombo v1.5 (ONT) (Read mapping: Reads were demultiplexed using poreplex (v 0.5) (.5 (ONT)  by mappi.5 (ONT) .Chemical probing of adenine residues with DEPC. Several studies indicate that DEPC can be used for detecting single-stranded adenosine in RNA molecules . Chemica4 (prepared fresh) and incubated on ice for 30 min in the dark followed by ethanol precipitation. The pellet was resuspended in 100 μl of 1 M aniline-acetate buffer  and incubated at 60°C for 15 min in the dark followed by ethanol precipitation. The resulting fragments of RNA were end-labeled by 32P-ATP using T4 polynucleotide kinase and run on 15% denaturing PAGE.End labeling: The ring opening and the percentage of modifications in RNA by DEPC were validated using cleavage reaction and NMR. The cleavage reaction was performed on DEPC treated and untreated hairpin RNA as follows: 50 μl 1 M Tris, pH 8.0, and 25 μl of 0.4 M NaBHNMR: The abundance of adenosine in the DEPC treated and control hairpin RNAs sequence was quantified by proton NMR spectrometry using a Bruker BioSpin NEO600 spectrometer equipped with a cryogenic probe operating at 298 K for all measurements. We have standard pulse sequence noesygppr1d with a delay of 60 s to allow complete relaxation of nuclei between individual scans. Assignment of proton scans was confirmed by literature and by supplemental HSQC scans.https://git.app.uib.no/valenlab/sms-seq-pipeline). Due to Tombo working on the consensus level  we had to identify a threshold for the Tombo statistic which will allow us to work at the read level. We identified the false discovery rate (FDR) as a function of threshold using a synthetic RNA, which has adenine bases in a large number of different 5-kmer configurations DEPC and (−)DEPC data sets and derived the final DEPC modification for adenosine using Tombo  method murations . This waurations . To evalurations , resultiurations  to be 0.urations . This inurations . Next, wurations . For deturations . Additioz-score (dAMI). Therefore when multiple distances have high dAMI a cluster will become visible on the grid, indicating the mutual information.U and V which are represented by vectors of modified/unmodified states (corresponding to 1 and 0) for each of the reads that span both positions U and V. We required at least 10 reads between positions U and V for calculations.Adjusted mutual information is a variation of mutual information correcting for agreement due to chance . The number of reads resulting in the same structure was counted. Yeast non-coding RNAs were folded – based on sequence minimum free energy (MFE), maximum expected accuracy (MEA), and centroid – and reads were clustered to them using the binary distance metric, i.e. the proportion of reads in which both of the two positions are modified/open relative to the number of reads where at least one is modified/open. The structures of the riboswitches were obtained from previous studies . Th. Thin viobed RNA  at high obed RNA . This isobed RNA .To assess how well this approach could capture structural information, we synthesized and probed a hairpin RNA with a known, stable structure . We firsRecently, a nanopore sequencing-based method demonstrated consensus calling of RNA structure ,10. Consz-score normalization (see Methods), this provided us with a distance-normalized AMI (dAMI). Applying this metric to the hairpin revealed that bases that are part of the same structural unit share a high dAMI  between all pairs of bases . Mutual information measures how much information the state of one base informs of the state of another base. Since calling the modification state of a base using nanopore sequencing is influenced by its neighboring bases, we scaled the AMI according to the distance between the pair of bases considered. Using I Figure . In parte Figure . These mF. nucleatum Flavin mononucleotide  and folded these in vitro in the presence and absence of DEPC. We first aligned the reads to in silico folded structures and to known structures from RNACentral . We similarly obtained thousands of reads for rRNAs showing modification patterns in agreement with sub-structures of this long molecule  RNA libraries from yeast  RNA from yeast and probed these plicates  and simiplicates . We compplicates , and obsplicates . We coulplicates  in line plicates . Globallplicates  ), dimethyl sulfate (DMS) and nanopore sequencing resulted in low quality, truncated and unmappable sequences , 1-methyl-7-nitroisatoic anhydride (1M7) and equences ,10. In cAnother shortcoming of DEPC is its lack of ability to modify other bases than adenosine. Although a new probing reagent that could target all 4 nucleotides, acetylimidazole, has been synthesized to use in nanopore-based structure determination, it is shown that at the higher concentrations necessary to obtain extensive modification rate the quality and fraction of full-length reads were reduced . FurtherBuilding on our and other long-read-based approaches with increased accuracy and structure prediction, we expect full-length single-molecule structural probing of native RNA by direct RNA sequencing will eventually become a standard tool in predicting secondary structure and inferring structural dependencies and tertiary interactions.https://git.app.uib.no/valenlab/sms-seq.The sequencing files are available on the European Nucleotide Archive website, under accession code PRJEB36658. Scripts used to process the data are available on gkac775_Supplemental_FileClick here for additional data file.