Balancing society’s competing needs of development and conservation requires careful consideration of tradeoffs. Renewable energy development and biodiversity conservation are often considered beneficial environmental goals. The direct footprint and disturbance of renewable energy, however, can displace species’ habitat and negatively impact populations and natural communities if sited without ecological consideration. Offsets have emerged as a potentially useful tool to mitigate residual impacts after trying to avoid, minimize, or restore affected sites. Yet the problem of efficiently designing a set of offset sites becomes increasingly complex where many species or many sites are involved. Spatial conservation prioritization tools are designed to handle this problem, but have seen little application to offset siting and analysis. To address this need we designed an offset siting support tool for the Desert Renewable Energy Conservation Plan (DRECP) of California, and present a case study of hypothetical impacts from solar development in the Western Mojave subsection. We compare two offset scenarios designed to mitigate a hypothetical 15,331 ha derived from proposed utility-scale solar energy development (USSED) projects. The first scenario prioritizes offsets based precisely on impacted features, while the second scenario offsets impacts to maximize biodiversity conservation gains in the region. The two methods only agree on 28% of their prioritized sites and differ in meeting species-specific offset goals. Differences between the two scenarios highlight the importance of clearly specifying choices and priorities for offset siting and mitigation in general. Similarly, the effects of background climate and land use change may lessen the durability or effectiveness of offsets if not considered. Our offset siting support tool was designed specifically for the DRECP area, but with minor code modification could work well in other offset analyses, and could provide continuing support for a potentially innovative mitigation solution to environmental impacts. Increasing the amount of renewable energy generation is a primary goal to mitigate emissions contributing to climate change from the energy sector. The direct footprint of utility-scale solar energy development (USSED–facilities with >1MW capacity) is large and has an even greater associated area of disturbance [The impacts of climate and land use change are some of the largest challenges facing biodiversity conservation and natural resource management in the 21turbance , though The mitigation hierarchy of the U.S. National Environmental Policy Act (NEPA) is a commonly used guide in environmental impact assessment for projects such as USSED. It specifies four types of actions to mitigate environmental impacts in decreasing order of preference—avoid, minimize, restore, and offset . Recent Offsets are a valuable tool to protect or enhance habitat for species or natural communities when impacts cannot be avoided, minimized, or restored, by securing habitat or environmental benefits at unaffected sites similar to those impacted , 11. TheApplying spatial conservation planning software to offset siting is a recent, but growing use of conservation planning tools. For example, Kiesecker et al.  describeIn this study we develop an offset siting model that has similarities but largely differs from previous offset siting models , 17 by uWe explore these issues in a case study in the western Mojave Desert of California. The region has many proposed USSED projects, but contains habitat for a number of endemic and endangered species. This combination of factors leads to an important opportunity for conservation and climate change mitigation: renewable energy goals require an increase in solar energy generation, yet how can that increased capacity minimize its impact on biodiversity persistence in a fragile desert ecosystem?www.drecp.org, We developed Mojavset, an offset siting support tool, to evaluate the potential impacts to biodiversity from a solar development site, or set of sites, and to then identify potential biodiversity offset sites for conservation action in the Desert Renewable Energy Conservation Plan area , only conservation features that will be impacted by potential solar sites are used for offset prioritization. For example, if 8 species and 3 ecological systems will be impacted, then only these 11 features out of the entire suite of species and ecological systems will be considered when prioritizing sites for offsets. The distributions of all other conservation features will be ignored and will not add value to a network of offsets sites.To prioritize offsets with the maximum biodiversity conservation gains option (hereafter Max Cons), any mapped conservation feature is considered available for offset prioritization, or a subset of features weighted by importance. Zonation will then prioritize the landscape based on complementarity (core area zonation ), and feTo demonstrate Mojavset, we evaluated proposed solar projects in the Western Mojave region, as defined by Goudey and Smith  Fig 1)..Fig 1). The Western Mojave ecological subsection is a 1.27 million hectare region in the western region of the DRECP boundary . The reghttp://www.energy.ca.gov/33by2020/). We queried this database to determine the location and size of potential facilities for our offset analysis and demonstration. 71 USSED projects are proposed within the Western Mojave ecological subsection, with an estimated total capacity of just less than 3500 MW of power and covering 15,331 ha  solar facilities , and ecological condition  as a proTaxidea taxis) range in this study area. The alkali mariposa lily  is impacted most as a proportion of its total modeled distribution, at 1.3%.The hypothetical impact sites of the Western Mojave are distributed primarily in the western portion of the subsection. These projects occur almost exclusively on private land with minimal overlap with priority areas of the Bureau of Land Management (BLM) and The Nature Conservancy (TNC) . The sitOffsets totaling 32,178 ha are required to meet the stated targets of impacted features . Sites sDifferences between the two offset scenarios are quite apparent, despite prioritizing the same total area of offsets. The Max Cons scenario sited offsets largely within The Nature Conservancy’s priority habitats, and primarily on Department of Defense (DOD) lands . The Dira priori the area required to mitigate unavoidable impacts of multiple projects for many conservation features could potentially assist both energy developers and permit granters to relocate potential site locations to reduce both the potential impact and the cost to offset those impacts.Conservation planning tools like Mojavset are designed to identify efficient solutions to problems with a large number of choices. When considering the collective impacts of multiple projects on multiple species, more efficient solutions will likely arise compared to implementing offsets one species at a time on a project-by-project basis. The degree of efficiency will depend on the extent to which modeled species habitats coincide. Determining In our case study of the Western Mojave, the project sites and impacted area are speculative, but provide a good illustration of our offset siting process. A more nuanced approach would simultaneously site solar facilities to meet solar criteria and to minimize impacts. Any residual impacts could be offset with Mojavset, and would likely result in a much smaller area requiring offset mitigation. Previous work in the Mojave has shown there is room to develop utility scale solar with minimal impacts , 6, thouThe efficiency of locating a multi-species offset solution is apparent by comparing the offset area required, given constraints, to the absolute minimum area needed if species were perfectly collocated. With a 2:1 offset ratio and 15,331 ha of impact, the minimum area required for offsets would be 30,662 ha. Thus, the 32,178 ha of the Direct Offsets solution is only 4.9% more area than the hypothetical minimum needed to meet the targets. In other cases where the range of impacted species are more widely distributed and non-overlapping, the total area required to meet targets may be considerably larger than the obligatory minimum area set by the offset ratio. Minimizing the cost or area required to satisfactorily meet mitigation targets is likely the largest benefit of using tools like Mojavset for offset siting.One interesting result in the case study was the difference between the Direct and Max Cons solutions. The Max Cons solution prioritized the same number of hectares, but only overlapped with the Direct offset solution by 28%, and failed to meet targets for 14 species. This can be explained by the difference in planning method: the Direct scenario had specific species targets to meet, while the Max Cons scenario maximizes overall conservation priority and richness in the region. In practice, all species would require individual weightings and careful consideration of species specific utility functions to ensure the model is parameterized according to users’ goals, and would likely yield a solution more similar to the Direct scenario. This comparison relates to the notion of “strict equivalency” in offset mitigation, or whether offsets can be relaxed to prioritize out of kind targets (targets not directly affected by the actions being offset) , 39. Habwhen species needs in multiple life history stages and across their full range are properly considered [If conservation resources are allocated to offsets, they need to be effective in the long-term to avoid net loss of habitat and increased extinction risk. The literature on design of offsets for climate change is young and emerging, but suggests that offsets could be an effective conservation strategy in response to energy development, nsidered . Researcnsidered .One approach to address offset siting with climate change impacts would give preference to areas that could play an important role in buffering climate change effects , 43, andHigher mitigation offset ratios are also commonly used to cope with uncertainty and to ensure the future durability of offsets , and areA final issue with offsetting as a mitigation tool is ensuring the actions taken are additional to what would have occurred without conservation intervention , 13, parWithin the land use policy realm and in applied use, offsetting the impacts of USSED, and development in general, is likely to increase as a mechanism to mitigate the impacts from development and land use change . Mojavse


Neisseria gonorrhoeae, the etiological agent of the STD gonorrhea. Here we present the first analysis of N. gonorrhoeae specifically focused on the identification of sRNAs through RNA-seq analysis of the organism cultured under different in vitro growth conditions. Using a new computational program, Rockhopper, to analyze prokaryotic RNA-seq data obtained from N. gonorrhoeae we identified several putative sRNAs and confirmed their expression and size through Northern blot analysis. In addition, RNA was collected from four different growth conditions . Many of the putative sRNAs identified shoed varying expression levels relative to the different growth conditions examine or were detected only under certain conditions but not others. Comparisons of identified sRNAs with the regulatory pattern of putative mRNA targets revealed possible functional roles for these sRNAs. These studies are the first to carry out a global analysis of N. gonorrhoeae specifically focused on sRNAs and show that RNA-mediated regulation may be an important mechanism of gene control in this human pathogen.In the last several years, bacterial gene regulation via small RNAs (sRNAs) has been recognized as an important mechanism controlling expression of essential proteins that are critical to bacterial growth and metabolism. Technologies such as RNA-seq are rapidly expanding the field of sRNAs and are enabling a global view of the “sRNAome” of several bacterial species. While numerous sRNAs have been identified in a variety of both Gram-negative and Gram-positive bacteria, only a very small number have been fully characterized in the human pathogen  Neisseria gonorrhoeae, the causative agent of the STD gonorrhea, is one of the most proliferative bacterial agents in the United States and abroad and is beginning to show an alarming resistance to conventional antibiotics  transcripts.rho-independent terminator (RIT), an inverted repeat which forms an RNA hairpin loop followed by a U-rich sequence that stalls transcription. These characteristics have been used extensively to perform global searches for sRNAs through in silico analysis of bacterial genomes 1-4 , which is regulated by iron availability via the Fur protein   before being split into two cultures. To constitute an iron replete condition, ferric nitrate was then added to one culture at a final concentration of 100 μM. To constitute an iron deplete condition, desferal, an iron chelator, was added to a second culture at a final concentration of 100 μM. RNA was isolated 90 min later using TRIzol according to the manufacturer's instructions. Due to prohibitive costs at the beginning of these studies RNA from high and low iron conditions was combined without barcoding before being sequenced to identify sRNAs. Isolation of RNA for experiments aimed at determining how iron leads to changes in sRNA expression were carried out as above except that high and low iron conditions were maintained for 60 min prior to RNA isolation. In addition, RNA from high and low iron conditions was barcoded prior to sequencing so that iron-mediated regulation could be observed.5 cells/well) and allowed to grow to confluence (106 cells/well) in antibiotic-free KSFM, a process taking 24–48 h. Concurrently, N. gonorrhoeae F62 was plated on GCB plates and grown as above for 17 h. Bacterial cells were removed from plates and resuspended in KSFM. Media was then removed from wells containing End/E6E7 and KSFM containing N. gonorrhoeae was then added to each well at an MOI of 100. An equal amount of N. gonorrhoeae was also incubated with KSFM alone to constitute a negative control. A sample of the original N. gonorrhoeae inoculum was also plated and CFU counted to determine exact MOI. Plates were then incubated at 37° C in 5% CO2 and 2 h later media was removed from End/E6E7 incubated with N. gonorrhoeae and wells were washed 3× with 2.5 mL of PBS to remove all N. gonorrhoeae cells which were not adherent to or within host cells. PBS was removed and TRIzol was then added to each well and plates were agitated gently for 10 min. TRIzol from each well was then transferred to a 2 mL tube and RNA was isolated according to the manufacturer's instructions. For N. gonorrhoeae incubated alone in media the contents of each well were transferred to a 2 mL tube and were centrifuged for 3 min at 3000 rpm. The resulting bacterial pellet was resuspended in 1 mL of TRIzol and RNA was extracted.Epithelial cells End/E6E7  according to the manufacturer's instructions to remove contaminating DNA. Before subsequent sequencing all RNA was visualized on a 1.5% agarose gel to confirm RNA quality and integrity. Identification of sRNAs and analysis of regulation of sRNAs by iron or incubation with epithelial cells was carried out as follows. To identify sRNAs, RNA grown under iron replete or deplete conditions for 90 min was first gel purified to enrich for RNA transcripts 50–250 nucleotides in length. RNA was electrophoresed for 1 h at 200 V through a 15% TBE-Urea gel (Invitrogen) in TBE buffer before being extracted. A cDNA library was then generated from the enriched RNA using Illumina's sRNA Sample Preparation Kit and samples were sequenced on an Illumina GAIIx without barcoding. To analyze the role of iron and epithelial cells on sRNA expression a 3 μg sample of N. gonorrhoeae RNA incubated under iron replete/deplete conditions for 60 min. or with/without epithelial cells for 2 h. was depleted of ribosomal RNA using the MicrobeEXPRESS kit (Ambion) according to manufacturer's instructions. A cDNA library of the resulting mRNA was then prepared using BioChain's Directional mRNA Sample Prep kit according to the manufacturer's instructions. In the experiments described here sRNAs were first identified  and identified sRNAs were then analyzed to determine regulation through additional RNA-seq experiments (under various conditions and with barcoding). Iron regulation and epithelial cell incubation experiments were part of studies examining the response of the entire transcriptome of N. gonorrhoeae to these stimuli and thus RNA was not size selected before sequencing. All libraries analyzed on an Agilent Bioanalyzer before sequencing to confirm library quality and were run using 36–72 basepair reads on an Illumina GAIIX machine. Two biological replicates were performed examining sRNAs specifically or examining iron regulation of sRNAs. One biological replicate was carried out examining sRNAs regulated via incubation with E6/E7 cells.Representative samples of RNA were isolated from N. gonorrhoeae FA 1090 genome. While the F62 genome has been sequenced, it has not yet been fully assembled. Therefore, the FA1090 genome was chosen as a template to align reads to. We have used the FA1090 genome for design of primers and probes that were then used with F62 RNA and DNA and have encountered no problems. Several studies have shown the high degree of similarity between the F62 and FA1090 genomes  for 45–60 min at 37°C with rotation. Simultaneously, ~50 nucleotide oligo probes were labeled with [α-NK Table . Membran32P] ATP radiolabeled oligonucleotide probe at varying temperatures corresponding to the probe's melting temperature. Probes . These results indicate that a size selection step prior to sequencing may increase the amount of sRNAs detected through depletion of much of the coding RNA expressed in the cell.All samples were sequenced on an Illumina GAIIx machine and aligned to the FA1090 genome with Rockhopper, a new program designed to analyze prokaryotic RNA-seq data  or under iron-deplete conditions . As described in the Methods size-selected RNA isolated from these conditions was initially sequenced together without barcoding. Transcripts showing expression were identified within IG regions or antisense to protein coding genes. These parameters generated a list of 280 putative sRNA transcripts that were expressed under either iron replete or deplete conditions  were examined using BLAST to determine if they were unique to Following confirmation of the size and expression of each sRNA we used the transcriptional start sites predicted by Rockhopper in conjunction with Northern blot analysis to determine the genomic locations of the four novel sRNAs that had not been previously characterized. The determination of both sRNA transcriptional start site and size allowed putative genomic coordinates of each sRNA Figure , thus prN. gonorrhoeae utilized RNA isolated from the organism grown under high and low iron conditions. We next set out to determine which sRNAs respond to specific iron conditions. RNA from iron replete and deplete conditions was prepared and sequenced separately in these experiments allowing iron-mediated regulation of sRNAs to be observed. In addition, as these experiments were part of a larger study examining the global transcriptional response of N. gonorrhoeae to iron the entire transcriptome was sequenced without size selection. While fewer sRNAs overall were detected, all of the sRNAs identified and confirmed by Northern blot analysis above were also found under either iron replete or deplete conditions in these experiments and a subset were revealed to show varying abundance as a consequence of growth under variable iron conditions   aligned to the N. gonorrhoeae genome.In addition to the subset of seven sRNAs examined under all conditions  we also examined others sRNAs of on Table . In contN. gonorrhoeae incubated with endocervical cells only ~5% of RNA aligned to the N. gonorrhoeae genome. This is likely due to the large amount of eukaryotic RNA present in the system. We detected a total of 209 sRNAs greater than 30 nucleotides in length expressed either during incubation in cell culture media alone or with endocervical cells  showed increased expression when incubated with epithelial cells compared to media alone. Of the 32 sRNAs found to be expressed under iron replete or deplete conditions 11 (34%) were unique to those conditions and were not found when N. gonorrhoeae was incubated in cell culture media or with endocervical cells. This is despite the fact that more than 4.1-fold more sRNAs were detected under cell incubation conditions compared to iron conditions. Conversely, 188 sRNAs were detected only during incubation in cell culture media or with endocervical cells and not during growth in CDM under iron replete or deplete conditions. These results show that there is large variability in expression patterns of sRNAs in N. gonorrhoeae, however the large number of variables between broth culture and growth in cell culture media or with endocervical cells preclude a definitive identification of environmental conditions stimulating expression of these sRNAs.In contrast, when we analyzed RNA samples obtained from ls Table . Of thesN. gonorrhoeae with endocervical cells detected 148 putative sRNAs and of these 99 were detected only during incubation with endocervical cells and under no other condition. Analysis of N. gonorrhoeae in media alone detected 49 putative sRNAs with only 1 being unique to this condition; there was significant overlap in sRNAs detected with endocervical cells compared to media alone. Growth of N. gonorrhoeae under iron replete conditions induced the expression of 18 sRNAs with 7 being unique to this condition. Growth of N. gonorrhoeae under iron deplete conditions induced expression of 15 sRNAs with 6 being unique to this condition  were all shown to contain regions of homology to smRNA 9 and to be regulated under the same conditions. NGO0825, NGO1183, and NGO1439 were all expressed more highly during incubation with N. gonorrhoeae. This regulatory pattern is identical to smRNA 9 and may suggest that smRNA 9 positive regulates these transcripts. In contrast, pykA exhibited greater expression during incubation with media alone, a regulatory pattern opposite that of smRNA 9, suggesting that smRNA 9 may negatively regulate this transcript.sRNAs regulate mainly mRNA targets, a process carried out through homologous baseparing. To begin to define targets of the sRNA identified under the experimental conditions used in this study we utilized TargetRNA. We reasoned that if a given sRNA/mRNA pair were predicted to form a homologous base paring interaction and both were regulated under the same conditions the possibility exists that the sRNA could function as a regulator of the mRNA. All identified sRNAs showing regulation under at least one condition were examined and several possible targets were found for smRNA 9. Transcripts encoding NGO0825 (putative ferredoxin), NGO1183 (protein synthesis), NGO1439 (ATP-binding transporter), and N. gonorrhoeae. It is possible that sRNAs may play a more prominent role in gene regulation in N. gonorrhoeae as compared to other bacteria due to the relatively low number of global proteomic transcription factors in the gonococcus. Despite this possibility as well as the analysis of more than 100 sRNAs in other organisms such as E. coli, there are only a handful of sRNAs that have been well characterized in N. gonorrhoeae. The analysis presented here sought to identify and characterize sRNAs more broadly in the gonococcus. RNA-seq analysis in N. gonorrhoeae identified several hundred instances of transcription that were outside or antisense to known genes. Interestingly, when using different experimental conditions, most notably a lack of RNA size selection and the inclusion of rRNA depletion, far fewer sRNAs were found. This may be of interest to other investigators focusing on identifying and analyzing classes of RNAs that are of relatively homogenous size. A gel electrophoresis step may increase the number of RNA transcripts detected and possibly allow the detection of transcripts expressed at very low levels. Of the 10 sRNAs that we chose from the 232 that were expressed under all conditions, the expression of seven was confirmed via Northern blot analysis and all seven had a size in the range of known sRNAs. While a number of putative sRNAs detected may represent 5′ or 3′ UTRs, due to the fact that 7/10 of the randomly tested sRNAs fulfill characteristics of sRNAs, it is reasonable to assume that a large number of the remaining 222 candidates indeed correspond to sRNAs. These results greatly increase the number of known sRNAs in the gonococcus and suggest that sRNA mediated gene regulation may be a commonly used mechanism for control of gene expression in the pathogenic Neisseria. Several of the sRNAs tested had a predicted RIT, yet the presence of an RIT was absent in the majority of sRNAs. We would therefore suggest that the presence or absence of a predicted RIT should not be considered a disqualifying characteristic in future analyses which focus on sRNAs in Neisseria and perhaps in other bacteria as well.Bacterial gene regulation via sRNAs is a field of rapidly advancing discovery. New technologies such as global RNA sequencing are making the discovery of novel sRNAs easier, as well as providing the first glimpses of the global sRNAome of bacteria where little is known about sRNA regulation, such as in the pathogen H. pylori found that 50% of genes contained an anti-sense transcript on the opposite strand  was also found in the mtrCDE locus, which encodes for an efflux pump in N. gonorrhoeae. We propose that searches for putative targets of these sRNAs should begin in these regions and that stimuli that regulate genes in these loci may also regulate these putative sRNAs.A subset of sRNAs were found to be antisense to known protein coding genes, such as smRNA 8 and 9. The increasing amount of anti-sense transcription being found in prokaryotic organisms has altered paradigms regarding genetic structure in these organisms. A study of N. gonorrhoeae with human endocervical cells a much larger repertoire of sRNAs was identified despite the fact that input RNA for sequencing was not size selected. This may reflect that fact that regulation by sRNAs is an integral component of successful infection by N. gonorrhoeae. As a strict human pathogen the most commonly encountered set of stimuli for this organism involves human cells. Therefore, it is not surprising that a majority of regulatory pathways would respond to such stimuli. Interestingly, the overwhelming majority of these sRNAs showed increased expression during incubation with endocervical cells compared to media alone.When we examined the expression of sRNA during incubation of N. gonorrhoeae specifically focused on identification of novel sRNAs. We discovered several hundred putative sRNAs and confirm the expression of seven new sRNAs in the gonococcus. It is likely that several of these transcripts act as post-transcriptional regulators and targets of these sRNAs are currently being characterized. The gonococcus contains comparatively few protein regulators and as such may be more likely to use sRNA-mediated mechanisms of control to regulate gene expression. This observation, combined with the fact that many sRNAs known in other organisms are involved in pathogenesis, emphasize the need for continued study of sRNAs in this organism. These experiments begin the work of characterizing sRNAs of N. gonorrhoeae as well as how they may be regulated and open up new avenues of genetic regulatory mechanisms in this human pathogen.In summary, we report on the first RNA-sequencing study of the human pathogen The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


RNA levels are widely thought to be predictive of RNA function. However, the existence of more than a hundred chemically distinct modifications of RNA alone is a major indication that these moieties may impart distinct functions to subgroups of RNA molecules that share a primary sequence but display distinct RNA “epigenetic” marks. RNAs can be modified on many sites, including 5′ and 3′ ends, the sugar phosphate backbone, or internal bases, which collectively provide many opportunities for posttranscriptional regulation through a variety of mechanisms. Here, we will focus on how modifications on messenger and microRNAs may affect the process of RNA interference in mammalian cells. We believe that taking RNA modifications into account will not only advance our understanding of this crucial pathway in disease and cancer but will also open the path to exploiting the enzymes that “write” and “erase” them as targets for therapeutic drug development. In higher multicellular eukaryotes, a crucial gene regulatory step takes place posttranscriptionally through RNA interference using microRNAs (miRNA). miRNAs are 18–24 nt, short, noncoding RNAs that target the RNA interference (RNAi) effector complex RNA-induced silencing complex (RISC) to specific messenger RNAs (mRNAs) through partial base pairing to sequences predominantly found in their 3′ untranslated regions (3′UTR)  [1]. Thihttp://mods.rna.albany.edu) that can be divided into several subcategories depending on their chemical structure, the target site on the RNA polymer, and the functional type of RNA. Here, we will focus on the modifications that have been detected in the two RNAs directly relevant to RNA interference: mRNAs and miRNAs. As in other RNAs, mRNA and miRNA modifications can be added to their 5′ and 3′ ends or internally to specific bases. During transcription by RNAP II, both mRNAs and pri-miRNAs receive a 7-methylguanosine cap (m7G) on their 5′ end and a poly-A tail on their 3′ end  and 3′ uridylation, have been shown to regulate the biogenesis and stability of specific miRNAs in mammalian cells.A key feature of the miRNA biogenesis pathway is the generation of 5′ mono-phosphate (5′P) ends by Drosha and Dicer . The 5′PUridylation consists in the untemplated addition of one or more uridilyl residues on the RNA 3′ end. Uridylation of let-7 family pre-miRNAs by terminal uridylyl transferase enzymes (TUTases) constitutes a beautiful example of how the degree of modification is critical for determining the fate of the modified miRNA precursors. Indeed, the preferred substrate of Dicer is a pre-miRNA with a 2 nt 3′ overhang . Thus, oBase modifications constitute the most diverse body of known RNA modifications . They haA to I editing corresponds to the conversion of adenosine to inosine by removal of the amino group at carbon 6 of the adenine ring . On mRNAm6A is the most abundant base modification in mRNA  but has m5C is the second-most abundant base modification in mRNA, around 20 times less abundant than m6A . This moThe recent discovery of new writers, erasers, and readers of RNA modifications with important cellular phenotypes has triggered the rebirth of the RNA modification field . DespiteThe challenges mentioned above are applicable to all modified RNAs. However, another challenge specific to the miRNA pathway is the lack of reliable methods to amplify and sequence precursor miRNAs. While this may be a consequence of their supposed short half-life as intermediates, it is also possible that it is the combination of their stable hairpin structure and the presence of RNA modifications that makes them poor substrates for cDNA synthesis and thus deep sequencing analysis. A similar problem is encountered with highly modified tRNAs, despite their abundance . To resoAccurately detecting RNAs and their modifications may prove to be important not only from a basic scientist’s perspective, but also from a biomarker development point of view. Indeed, the absence or presence of specific RNA modifications in disease setting may be more informative than simply measuring RNA levels. This is very likely given the involvement of the writers, erasers, and readers of RNA modifications in cellular processes highly relevant to human disease . All metAs highlighted throughout this review, an increasing number of observations point to the important role played by RNA modifications in the miRNA biogenesis pathway and RNA interference. Given the importance of miRNAs in numerous cellular processes and their deregulation in many diseases, including cancer, miRNA inhibitors and miRNA mimics are being considered for therapeutic purposes . While n


The crystal structure of the complex reveals the molecular architecture of the heterohexamer as two rhomboid-shaped ring structures of Pnkp1–Rnl–Hen1 heterotrimer fused at the Pnkp1 dimer interface. The four active sites required for RNA repair are located on the inner rim of each ring. The architecture and the locations of the active sites of the Pnkp1–Rnl–Hen1 heterohexamer suggest an ordered series of repair reactions at the broken RNA ends that confer immunity to recurrent damage.Ribotoxins cleave essential RNAs for cell killing, and RNA repair neutralizes the damage inflicted by ribotoxins for cell survival. Here we report a new bacterial RNA repair complex that performs RNA repair linked to immunity. This new RNA repair complex is a 270-kDa heterohexamer composed of three proteins—Pnkp1, Rnl and Hen1—that are required to repair ribotoxin-cleaved RNA  Bacterial ribotoxins target and cleave RNAs involved in translation, with target organism resistance being mediated by RNA repair systems. Here, Wang et al. identify, report the crystal structure, and characterize the activity of Pnkp1–Rnl–Hen1, a novel RNA repair complex from Capnocytophaga gingivalis. Competition between organisms, microbes in particular, for a limited resource of nutrition require adaptation at many levels; some organisms employ a variety of toxins to eliminate competition, while others use molecular defence systems to neutralize toxins for survival. Among many cellular targets of toxins, the protein translation apparatus is among the more frequently targeted systems due to its conservation and essential role in all organisms. A majority of toxins targeting protein synthesis appear to be ribotoxins, which cleave essential RNAs  and messenger RNA) involved in protein translation. With the exception of several ribotoxins generated in eukaryotic organisms123410With the exception of ricin and some metal-dependent ribotoxins2131415161718in vitroO-methylation, the RNA repaired by Pnkp–Hen1 resists future RNA damage by the same ribotoxin. Thus, the bacterial Pnkp–Hen1 complex performs RNA repair with immunity.Building on the study by Martin and ShumanO-methylation during RNA repair. Recent studies carried out in the Shuman laboratory as well as ours revealed the molecular mechanism for achieving the first condition23et al.O-methylation during RNA repair.Because methylated RNA repair is superior, there is great incentive for the Pnkp–Hen1 repair complex to have evolved to maximize the production of methylated RNA repair products. In theory, this can be achieved if the RNA repair complex adopts a molecular arrangement and architecture that (i) does not permit RNA repair to occur without Hen1, and (ii) is conducive for efficient 2′-in vitro. The new RNA repair complex is distinct from the Pnkp–Hen1 RNA repair complex, but also shares several features  to AvaHen1. However, unlike the genes encoding Pnkp and Hen1, which are always present in the same operon  into an overexpression vector and purified all three recombinant proteins individually . Cleaved tRNAArg-ΔT has previously been shown to be an effective substrate of RNA repair carried out by the Pnkp–Hen1 heterotetramerTo evaluate relative rates of four enzymatic activities of the Pnkp1–Rnl–Hen1 heterohexamer, we performed time-dependent reactions focusing on individual enzymatic activity . For theO-methylation on RNA ligation, the RNA substrates with the processed ends  were subjected to RNA ligation by the Rnl–Hen1 heterodimer in the absence or presence of S-adenosylmethionine (SAM) (Arg-ΔT at a slower rate than the one carried out by the Rnl–Hen1 heterodimer (compare To assess the effect of 2′-ne (SAM) . The prene (SAM) . Surpris compare . The mecO-methylation, the RNA repair products shown in O-methylation by Hen1 during RNA repair results in immunity of the repaired RNA to the ribotoxin. On the basis of the difference of cleavage shown in O-methylated at the junction of repair. The incomplete cleavage of the repaired RNA without 2′-O-methylation might be due to partial misfolding of tRNAArg-ΔT , which is presumably responsible for the ligase activation in Rnl by Hen1 based on our previous study of the Pnkp–Hen1 RNA repair system2 buried surface areas, respectively.To assess the relative contribution of each protein–protein interaction to the stability of the overall complex, we calculated the surface area buried at each protein interface. The most extensive interaction was found at the interface of the Pnkp1 homodimer, with ∼6,800 ÅFormation of the Pnkp1–Rnl–Hen1 heterohexamer requires recognition between the kinase and phosphatase domains of Pnkp1, Pnkp1 and Rnl, Rnl and Hen1, and Pnkp1 and Hen1. The crystal structure of the Pnkp1–Rnl–Hen1 heterohexamer revealed the molecular basis for these recognitions as described below.T4Pnkp2728T4Pnkp structuresThe formation of the kinase module of Pnkp1 is via interactions between two kinase domains with antiparallel orientations . The intT4Pnkp2728T4Pnkp structuresT4Pnkp ablate the phosphatase activityUnlike the kinase domains, formation of the phosphatase module results from interactions between two phosphatase domains oriented in parallel . FormatiEach Rnl only makes contact with one kinase domain of the Pnkp1 homodimer, and the interaction is mainly electrostatic. The main interactions are the salt bridges formed between the positively charged residues in Pnkp1  and the negatively charged residues in Rnl (E369 and D379) . An addiAs was previously observed in the structure of the ligase module of the Pnkp–Hen1 RNA repair complexRecognition of Pnkp1 by Hen1, which was not observed in solution with the binary mixture of Pnkp1 and Hen1 , panel 2in vivo. As shown in C. gingivalis (with Rnl being synthesized first), whereas Hen1 is synthesized separately. Therefore, Rnl and Pnkp1 should form a complex first, followed by association with Hen1. Thus, the copy of Rnl and Pnkp1 that make physical contact, together with the copy of Hen1 that interacts with the Rnl, should be assigned to the same unit of the Pnkp1–Rnl–Hen1 heterotrimer. Labelling of the six proteins of the Pnkp1–Rnl–Hen1 heterohexamer shown in in vivo sequential events.To understand why the RNA repair complex requires two copies of each protein, the two units of the Pnkp1–Rnl–Hen1 heterotrimer must first be defined based on the expected sequential events of protein synthesis and heterohexamer assembly To provide insight into the mechanism of RNA repair by the Pnkp1–Rnl–Hen1 heterohexamer, we soaked crystals with adenosine triphosphate (ATP) before data collection, which resulted in a crystal structure with cofactors occupying all active sites . Guided 2830O-methylation before joining the 5′ end in the ligase site. With the presence of both processed ends in the ligase site, RNA ligation occurs, which results in repaired RNA with 2′-O-methylation.The damaged RNA might approach the four active sites for repair from the side of each ring, as indicated by arrows shown in O-methylation of the repaired RNA is desired due to its immunity to future damage. RNA repair performed by the four active sites shown in Because RNA repair requires four different enzymatic activities, we suggest a possibility of a single RNA repair event performed by the four active sites located in the same inner rim. Utilizing four active sites contributed from both rings for a repair event would demand that an RNA substrate accesses active sites far away from each other, which would likely require the RNA substrate to associate with and dissociate from the RNA repair complex more than once. As described previously, efficient 2′-If our mechanistic hypothesis described above stands, the structure of the Pnkp1–Rnl–Hen1 heterohexamer provides an explanation for the requirement for two copies of each active site in the RNA repair complex. Among the four active sites located on the inner rim of the same ring structure, the kinase and phosphatase active sites are from one unit of the Pnkp1–Rnl–Hen1 heterotrimer, and the methyltransferase and ligase active sites are contributed from the other unit  and 6. AO-methylation during RNA repair. Our structure revealed that the methyltransferase site is located between the phosphatase and ligase sites, providing the 3′ end of the damaged RNA maximal opportunity to be 2′-O-methylated after dephosphorylation but before ligation. It is possible that the 3′ end of the damaged RNA might skip the methyltransferase site and be transferred directly from the phosphatase to ligase sites. Such an event might occur occasionally, as we have observed that 2′-O-methylation is not complete for all repaired RNAThe relative locations of the four active sites and the size of the vacant space surrounded by the four active sites could be responsible for efficient 2′-In this study, we described the discovery of a new RNA repair system in bacteria, which was subsequently biochemically and structurally characterized. In addition to providing mechanistic insight into RNA repair by the Pnkp1–Rnl–Hen1 complex, the study may have broader implications discussed below.The study of the Pnkp1–Rnl–Hen1 RNA repair complex may provide insight into how RNA is repaired by the bacterial Pnkp–Hen1 RNA repair complex, whose structure is unknown. As illustrated in in vivo RNA substrates and the nature of RNA repair carried out by the complex. Despite extensive in vitro studies of the Pnkp–Hen1 as well as the new Pnkp1–Rnl–Hen1 RNA repair complexes, the in vivo RNA substrates still remain unknown. The difficulties of revealing in vivo RNA substrates stem from the assumption that RNA damage and repair most likely occur when organisms are in the wilderness, where they are exposed to ribotoxins released by nearby species. Therefore, the events of RNA damage and repair occurring in the wilderness are difficult to reproduce in a laboratory setting. The crystal structure of the Pnkp1–Rnl–Hen1 heterohexamer described here indicates that the four active sites required for repair appear to be readily accessible to a variety of damaged RNAs. This is consistent with our previous in vitro biochemical studies that the Pnkp–Hen1 RNA repair complex exhibits broad substrate specificityin vivo.The study described here may also shed some light on the likely in vivo biological functions of RNA repair carried out by the Pnkp1–Rnl–Hen1 heterohexamer, we were able to identify 10 bacterial species that possess the Pnkp1–Rnl–Hen1 RNA repair complex. Furthermore, based on the analyses of the data released by the Human Microbiome Project (HMP), we were able to locate an ecosystem for many of them. Among 10 bacterial species, six were isolated from human, one from the mouth of a dog and three were from environmental samples with unknown sources. Metagenomic analyses using the data released by HMP revealed that, of five locations in humans where samples were collected, bacteria possessing the RNA repair complex were only found in the mouth  strain individually, and the proteins were expressed at 18 °C for 20 h after induction with 0.5 mM isopropyl-b-D-thiogalactoside (IPTG). Cells were harvested by centrifugation and stored at −80 °C. Thawed cell pellets were resuspended in lysis buffer ) and lysed using the French press. The cell lysate was centrifuged and the proteins were purified from the supernatant by a fast protein liquid chromatography (FPLC) system. Pnkp1 was purified to homogeneity using diethylaminoethanol (DEAE) ion exchange, heparin affinity, Mono Q ion exchange and Superdex 200 size-exclusion chromatography. Rnl was purified the same as Pnkp1, except the Mono Q ion exchange step was omitted. Hen1 was purified similarly as Pnkp1, except heparin affinity chromatography was omitted. All purified proteins were stored in the gel filtration buffer  for further study.DNA corresponding to Pnkp1, Rnl and Hen1 were amplified from the E. coli Rosetta strain was used for expression and methionine pathway inhibition was used for cell growth. The protein was purified the same as the wild-type Pnkp1 described above.To produce selenomethione-incorporated Pnkp1, the The purified recombinant proteins were analysed individually using size-exclusion chromatography. To assess protein–protein interaction, two different proteins were combined in equimolar and incubated at 4 °C for an hour before size-exclusion chromatography. To assemble the entire RNA repair complex, all three proteins were mixed in equimolar concentrations, incubated at 4 °C for an hour and analysed by size-exclusion chromatography.33P-internally radiolabelled and colicin D-cleaved tRNAArg was prepared as described previously2, 0.5 mM MnCl2, 0.05 mM EDTA, 5 mM DTT and 2.5% glycerol) in the presence of 0.2 mM ATP and 0.05 mM AdoMet at 37 °C for 45 min. After the reaction, the sample was processed with phenol extraction followed by ethanol precipitation to recover RNA. RNA was dissolved in DPAGE loading buffer, heated at 95 °C for 3 min to denature RNA and the sample was analysed by 15% DPAGE. The radioactivity of the repaired and unrepaired tRNAs was visualized using a PhosphorImager system (Molecular Dynamics).To carry out RNA repair, Arg-ΔT was employed, which allowed 5′-half (38 nt) and 3′-half (22 nt) RNAs to be purified after cleavage by colicin D. The 5′-phosphoylation reaction was carried out in a 30-μl reaction volume containing 10 μM 3′-half RNA, 0.025 μM enzyme (CgiPnkp1 homodimer or CgiPnkp1–Rnl–Hen1 heterohexamer) and 0.2 mM 33P-α-ATP in Repair buffer at 37 °C. Five μl of reaction solution was taken out at the time points of 0, 5, 15, 30 and 60 min, and the samples were processed and analysed analogous to the ones in the repair assay described above.For kinetic studies, tRNACgiPnkp1–Rnl–Hen1 heterohexamer in the presence of 0.2 mM 33P-γ-ATP (to introduce the radiolabelled phosphate at the junction of the repaired RNA), followed by re-cleavage with colicin D. The 3′-dephosphoylation reaction was carried out analogous to one of the kinase reactions, except that the RNA substrate was 1.0 μM, 5′-half RNA was radiolabelled at the 2′,3′-cyclic phosphate, the concentration of the enzyme was 0.25 μM and no ATP was added.To carry out the 3′-dephosphorylation reaction, the 5′-half RNA was prepared with RNA repair by The published protocol employed for the studies of the Pnkp–Hen1 RNA repair complex was used for RNA ligation reaction without and with SAM, except that the processed RNA substrates were used in this study−1 and mixed with a reservoir solution containing 8% PEG 6000, 0.2 M NaCl, 15 mM MgCl2 and 100 mM MES (pH 6.2). Full-size crystals of the Pnkp1–Rnl–Hen1 heterohexamer grew using the hanging drop vapour diffusion method at 4 °C in 2 weeks. Crystals were soaked in a series of cryoprotecting solutions containing all the components of the reservoir solution supplemented with increasing percentage of glycerol to a final concentration of 30%. For the data set that produced the structure of the Pnkp1–Rnl–Hen1 heterohexamer in complex with ATP, 5 mM ATP was also included in crystal soaking. The cryoprotected crystals were mounted in nylon loops and flash-frozen in liquid nitrogen. Data were collected at 21-ID beamline at the Advanced Photon Source and processed by HKL2000  data from a crystal of the heterohexamer with the selenium-containing Pnkp1. Of 18 selenomethionine residues present in Pnkp1 homodimer, 17 of them were found with AutoSol of the Phenix softwareectively .CgiHen1 of the Pnkp1–Rnl–Hen1 RNA repair complex were employed for a BLASTP search against metagenomic data released by the HMP (https://img.jgi.doe.gov/cgi-bin/imgm_hmp/main.cgi). The E-value for the search was 1e−50. Because of significant difference between Hen1 from the Pnkp1–Rnl–Hen1 RNA repair complex and the one from the Pnkp–Hen1 system, the E-value of 1e−50 ensures that a positive result from a search is an indication of the presence of the Pnkp1–Rnl–Hen1 RNA repair system, not the Pnkp–Hen1 system, in the data set used for the search.Amino-acid sequences of CgiHen1 was carried out against data sets of each of the five groups . Second, the search was carried out against data sets of each of the sub-groups of the human mouth. Third, BLASTP search using the sequences of Hen1 from six human-hosted bacteria was carried out one at a time against the data sets of supragingival plaque, subgingival plaque and throat. For the third search, the positive result of the search also required the sequence to be at least 97% identical to distinguish different Hen1 within these six bacterial species.The entire metagenomic analyses consist of three stages. First, BLASTP search using in vitro reconstitution experiments. P.W. carried out crystallization. P.W., K.S. and R.H.H. carried out data collection. P.W. and R.H.H. carried out structural determination and model refinement. All authors contributed to the writing of the manuscript.P.W. carried out cloning, overexpression and purification of proteins with the help from K.S. P.W. performed Accession codes. Coordinates and structure factors have been deposited in the Protein Data Bank with accession code 4XRP for the Pnkp1–Rnl–Hen1 heterohexamer alone, and 4XRU for the Pnkp1–Rnl–Hen1 heterohexamer in complex with cofactors.How to cite this article: Wang, P. et al. Reconstitution and structure of a bacterial Pnkp1–Rnl–Hen1 RNA repair complex. Nat. Commun. 6:6876 doi: 10.1038/ncomms7876 (2015).Supplementary Figures 1-12, Supplementary Table 1 and Supplementary References


IGH;BCL3 rearrangement [t] that juxtaposes a proto-oncogenic gene BCL3 and an immunoglobulin heavy chain, a translocation that may be associated with shorter survival. In addition to the IGH;BCL3 rearrangement, other translocations involving 14q32 locus are involved in various lymphoproliferative pathologies pointing toward the significance of IGH locus in oncogenic progression. Significantly, in the majority of B-cell neoplasms that carry an IGH;BCL3 rearrangement, it is a sole translocation involving an IGH locus.Chronic Lymphocytic Leukemia (CLL) is a lymphoproliferative disease characterized by multiple recurring clonal cytogenetic anomalies and is the most common leukemia in adults. Chromosomal abnormalities associated with CLL include trisomy 12 and IGH;BCL3 translocation and an additional clonal IGH;BCL2 translocation involving IGH and another proto-oncogene BCL2, t, commonly found in follicular lymphoma. Further single nucleotide polymorphism (SNP) array-based analysis detected a duplication of the 58.8 kb region at 19q13.32 adjacent to the BCL3 translocation junction on chromosome 19q13. Interestingly, the duplicated region contained ERCC2 gene, which encodes a DNA excision repair protein involved in the cancer-prone syndrome, xeroderma pigmentosum.We report a patient who, in addition to trisomy 12, carried a rare double-hit translocation characterized by the IGH loci and proto-oncogenes BCL2 and BCL3. Importantly, the IGH;BCL3 translocation was characterized by the duplication of the genomic region adjacent to BCL3, containing a major DNA repair factor, ERCC2.Taken together our findings indicate the existence of double-translocation driven oncogenic events involving both The online version of this article (doi:10.1186/s13039-015-0203-y) contains supplementary material, which is available to authorized users. ATM gene, loss of 13q14.3 and 6q, loss of 17p13 containing TP53 gene, and others  which juxtaposes the immunoglobulin heavy chain locus   and diffmas (FL) –8.IGH;BCL3 translocation or both, t IGH;BCL3 and t IGH;BCL2 translocations. Whereas single translocation events involving IGH and BCL3 or BCL2 loci can be detected in lymphoid cancers, double-hit translocations involving both IGH;BCL2 and IGH;BCL3 rearrangements in the same patient are exceptionally rare. Additionally, using SNP array analysis we detected a local microduplication event at the BCL3 translocation junction on chromosome 19 involving a DNA repair factor ERCC2. An additional file describing experimental procedures is available  Our patient was an 82-year-old African-American female who presented to her oncologist for leukocytosis. Complete blood count (CBC) data showed a white blood cell (WBC) count of 24.5 K/ MicroL with relative and absolute lymphocytosis of 70 % and 17.2K/ MicroL respectively. A bone marrow biopsy and aspirate was performed and the specimen was sent to the hematopathologist for evaluation. Flow cytometric analysis showed 40 % monoclonal B-cells with lambda light chain restriction of moderate intensity with dim CD5 co-expression and the following immunophenotype: CD10-, CD19+, CD20+, CD200-, CD23 +/- (dim), FMC7 +/- (dim), CD38-, CD25-, CD103- and CD11c-.By histology the bone marrow showed marked involvement by lymphocytes with interstitial pattern of distribution estimated at 80–90 % of the total marrow cellularity. H&E staining, CD20 and Pax5 immunohistochemistry confirmed the nature of the lymphocytes consistent with B-cell line of differentiation  was considered a strong possibility.BCL3 and IGH break-apart probes confirmed BCL3 and IGH translocations in the first clone  which, in combination with karyotype data, was indicative of the presence of eight IGH derivative translocation products. This suggests that all IGH loci are rearranged in the second clone in addition to the near duplication of the chromosome content. BCL2 probe picked up six BCL2 signals  indicating on the presence of four translocated BCL2 loci and two intact BCL2 loci from the non-translocated chromosome 18. Importantly, IGH and BCL2 signals formed four fusions , six cells, contained a translocation between the long (q) arm of chromosomes 14 and 19 Fig. , arrows, 19 Fig.  and seco 19 Fig. .Fig. 2ax2[cp2]/46,XX[14].nuc ishx2[198],[78/200],[117/200],(IGHx2)(5'IGH sep 3'IGHx1)[153/300]/(IGHx4)(5'IGH sep 3'IGHx4)[1/300],[123/300]/(IGH con BCL2x4)[1/300],x2[189],(BCL3x2)(5'BCL3 sep 3'BCL3x1)[99/200].The patient’s ISCN was 47,XX,+12,t[6]/89 ~ 90,slx2,tBCL3 translocation junction (19q13.1) containing a major nucleotide excision repair gene ERCC2 (ISCN arr[hg19](12)X3,19q13.32x3), , Fig. -d. Polymunction 1q13.1 conIGH loci and both BCL3 and BCL2 gene loci. Additionally, we detected a microduplication event in the genomic locus adjacent to BCL3 gene involving a nucleotide excision repair protein ERCC2. Our karyotype analysis identified two abnormal clones in this patient. The first clone contained, in addition to trisomy 12, a single cytogenetically defined translocation t involving IGH;BCL3 loci. The second clone represented a near duplication of the chromosome content of the first clone and the presence of two copies of an additional translocation involving IGH locus and BCL2 gene t. Therefore, all IGH loci located on the chromosome 14 were rearranged in the second clone which was confirmed by our FISH studies.In this report we describe a case of a monoclonal B-cell lymphoma/leukemia with extensive bone marrow involvement and CLL-like immunophenotype showing CD5 expression by flow cytometric analysis and furthermore displaying a double translocation event between IGH and BCL3 loci, which results in altered BCL3 expression, has been described in aggressive forms of lymphomas and atypical CLLs [BCL2 on 18q21 within the immunoglobulin heavy chain locus on 14q32 and therefore deregulates the BCL2 function. Juxtaposition of both BCL3 and BCL2 genes next to the active IGH locus leads to their altered expression, decreased apoptosis, and leukemia progression [BCL2(18q21) and BCL3(19q13) have been reported in atypical chronic lymphocytic leukemia at Richter's transformation [BCL3 and BCL2 are proto-oncogenes, however mechanistically, BCL2 functions as a key regulator of apoptosis through the mitochondrial pathways [IGH;BCL3 clone followed by the genome near duplication event and the appearance of the second clone containing both IGH;BCL3 and IGH;BCL2 rearrangements in our patient might, therefore, have an additive effect on the development of this neoplasm.These results are consistent with a neoplastic process in this patient’s bone marrow specimen. Importantly, the translocation tq32;q13) involving 2;q13 invcal CLLs . The tragression , 11. Conormation . Both BCERCC2 duplication in this patient is an intriguing finding, because of the role of ERCC2 in DNA nucleotide excision repair process. Specific ERCC2 polymorphisms are implicated in susceptibility to melanoma [http://dgv.tcag.ca/dgv/app/home), duplications of ERCC2 genomic region are also found in healthy individuals. Therefore, the significance of ERCC2 duplication in context of IGH;BCL3 and IGH;BCL2 rearrangements requires further investigation. Additionally, the existence of the microduplication event at the major translocation junction supports the previously reported genomic instability events at the translocation breakpoints and emphasizes the significance of the molecular analysis of sequences adjacent to the cytogenetically defined translocation junctions including balanced rearrangements [melanoma  as well melanoma . Howeverngements .Written informed consent was obtained from the patient for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.


Arabidopsis thaliana) is important for influencing gene expression and the inhibition of transposons by the deposition of repressive chromatin marks such as histone modifications and DNA methylation. A key event in de novo methylation of DNA by RdDM is the production of long non-coding RNA (lncRNA) by RNA polymerase V (Pol V). Little is known about the events that connect Pol V transcription to the establishment of repressive chromatin modifications. Using RNA immunoprecipitation, we elucidated the order of events downstream of lncRNA production and discovered interdependency between lncRNA-associated proteins. We found that the effector protein ARGONAUTE4 (AGO4) binds lncRNA independent of the RNA-binding protein INVOLVED IN DE NOVO2 (IDN2). In contrast, IDN2 binds lncRNA in an AGO4-dependent manner. We further found that the de novo DNA methyltransferase DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2) also associates with lncRNA produced by Pol V and that this event depends on AGO4 and IDN2. We propose a model where the silencing proteins AGO4, IDN2 and DRM2 bind to lncRNA in a stepwise manner, resulting in DNA methylation of RdDM target loci.Ribonucleic acid-mediated transcriptional gene silencing (known as RNA-directed DNA methylation, or RdDM, in  In RdDM, the DNA-dependent RNA polymerase IV (Pol IV) produces a transcript that is converted into double-stranded RNA (dsRNA) by the RNA-dependent RNA Polymerase RDR2  is recruited and deposits DNA methyl marks . The triple mutant idn2/idnl1/idnl2 had lower levels of CHH methylation than the single idn2 mutant. However, there was still a substantial number of loci with DNA methylation in contrast to the nrpe1, ago4 and drm1/drm2 mutants  experiments with polyclonal antibodies against AGO4, IDN2 and DRM2. This method allows us to detect RNA bound to the pulled-down proteins after immunoprecipitation. By using appropriate mutant plants, we can determine if an interacting RNA is produced by Pol V and if other proteins are also required for this association. For our assays, we selected four RdDM targets with IDN2/IDNL1/IDNL2-dependent CHH methylation.idn2 mutant as well as nrpe1, ago4 and rdr2 mutants. Non-specifically immunoprecipitated ACTIN2 RNA was used to monitor for comparable levels of total RNA in the samples  and strongly reduced in the idn2-1 mutant (Col-0 background), confirming antibody specificity. The IDN2 protein levels were not significantly changed in any of the other RdDM mutants tested when compared with the wild type  and ago4 [ago4-1  was obtained from INRA. drm2-2 (SAIL_70_E12) was described previously  and unchanged (>75% of Col-0) DMR categories. These categories were intersected with TAIR10 annotated genomic features, counted and plotted.AGO4 peaks with a nrpe1 DMR categories (idn2/idnl1/idnl2 changed and unchanged) were counted and plotted as boxplots with the ratio of siRNA of each mutant to Col-0. The mutants examined for siRNA were nrpd1, nrpe1  according to manufacturer's recommendations. In addition to the on-column DNase I digestion, the RNA was digested with Turbo DNase as described . The means and standard deviations were obtained from three biological replicates. The number of test loci was determined by the amount of RNA recovered in RIP and the requirement to perform all assays in multiple biological repeats. The following primer pairs were used: IGN29 JA227 CGTTTGTTTATGTAGGGCGAAAG and JA228 TAAAACTTTTCCCGCCAACCA  was used for production of a polyclonal antibody in rabbits. The antibody was purified as described in Zhu et al. . α-IDN2 http://www.scbt.com/).Total protein extracts were isolated from 100 mg of approximately 3-week-old Arabidopsis seedlings and separated on 12% polyacrylamide gels. Western blots were performed using α-IDN2 (1:333) and α-DRM2 (1:500) antibodies. Donkey anti-rabbit secondary antibody (1:2000) conjugated with horseradish peroxidase was used  according to the manufacturer's recommendations. One hundred nanograms of DNA was digested with 10 U of NlaIII or DdeI, respectively, and PCRs were performed as described in Zheng et al. (AT5G58510 JR529 AGAGATCCGCTTCGGGAAAG and JR530 AGAAACCATTGATAGAGATGGTCTTAG, IGN34 JR883 ATTGCCCGACGACTCCAAAT and JR884 AACTTTTGTAAGTCATGGTGTGTGTT, IGN35 JR885 GGGCCGGGCTTAGAGGATAG and JR886 CACATCTTCTACACGTGTCTTTAGGC, DdeI control JR535 TCCAAGATTGAGGCCAAATTA and JR536 AAAAGGAGTGGCCAAGTTGGAA, IGN36 JR887 GATTTTGATATTGTTACAGCATTGTT and JR888 TCCATATTCAGTACTTTTTAACCTACC, NlaIII control JR889 ACCGTTTGTTTATGTAGGGCGAAA and JR890 AAGATAACAGAAAAGACGATGATGACG.Genomic DNA was isolated from 100 mg of approximately 3-week-old seedlings using a DNeasy Plant kit (Qiagen, g et al. . One rep


Sweet potato virus 2 (SPV2) from sweet potato in Australia and East Timor, and compare these with five complete SPV2 genome sequences from South Korea and one each from Spain and the United States. Both were closely related to SPV2 genomes from South Korea, Spain, and the United States.We present here the first complete genome sequences of  Sweet potato virus 2  is a single-stranded RNA virus within the genus Potyvirus, family Potyviridae . The total RNA extracts were treated with RNase-free DNase (Invitrogen) and measured using Qubit (Invitrogen). RNA integrity was confirmed using RNA screen tape . Libraries were prepared from total RNA using a TruSeq stranded Total RNA sample preparation Ribo-Zero plant kit . The final size and concentration of each library were verified using Qubit and D1000 ScreenTape . Sequencing was performed by HiSeq 2500 using a TruSeq SBS kit version 4 (Illumina) with 151 cycles of paired-end reads. The reads were assembled and genomes annotated using CLC Genomics Workbench 6.5 (CLC bio) and Geneious 8.1.7 (Biomatters)  cards  were senDe novo assembly generated 1,307 contigs and 13,415 reads mapped to the contig of interest, with a coverage of 298×. Sample AusCan yielded 6,260,728 reads, and, after trimming, 5,405,928 reads remained. De novo assembly generated 832 contigs, with 964,917 reads mapping to the contig of interest, giving a coverage of 1,460×. Both Tm37 and AusCan SPV2 sequences coded for 10 proteins, as occurs with other potyviruses  and KX017448 (AusCan).


RNA in cells is always associated with RNA-binding proteins that regulate all aspects of RNA metabolism including RNA splicing, export from the nucleus, RNA localization, mRNA turn-over as well as translation. Given their diverse functions, cells express a variety of RNA-binding proteins, which play important roles in the pathologies of a number of diseases. In this review we focus on the effect of alcohol on different RNA-binding proteins and their possible contribution to alcohol-related disorders, and discuss the role of these proteins in the development of neurological diseases and cancer. We further discuss the conventional methods and newer techniques that are employed to identify RNA-binding proteins. In this review we provide a succinct overview of the role of RNA-binding proteins (RBPs) in regulating gene expression. Our summary is by no means exhaustive but is meant to give a flavor of the repertoire of RBPs recognized as important players in different disease processes. We have divided the review into subheadings under which we discuss the effect of alcohol on RNA-binding proteins, RNA-binding proteins in neurological diseases and cancer, and finally the methods employed to identify RBPs and/or ligands of a RBP.Gene expression is comprised of a series of well-coordinated events both at the transcriptional and post-transcriptional level. While we have a fairly good understanding of transcriptional events, post-transcriptional processes are comparatively far less understood. Transcription takes place in the nucleus where genes are transcribed from DNA, the blueprint of the cell. The transcribed RNA now represents information required to direct cellular function to maintain cell homeostasis. Before this information can be relayed to the cytoplasm, the nascent RNA undergoes extensive post-transcriptional processing before arriving at its final destination in cells. For this process the RNA is associated with nuclear proteins that aid in splicing and nuclear export . As mRNAFrom the diversity of their function it is apparent that these proteins have an important role to play in most if not all post-transcriptional events. With the advent of high throughput screening techniques and quantitative proteomics, several hundred potential RBPs have been identified ,3. Givenet al. have compiled a census of human RBP [et al. set out to systematically analyze RNA motifs recognized by a diverse group of RBPs. The results of their study provide an important resource for post-transcriptional regulatory processes [In an attempt to organize the expanding data on RBPs, Gerstberger uman RBP . They iduman RBP . Becauserocesses .http://www.mirbase.org) has been created for all published miRNA sequences [http://starbase.sysu.edu.cn/) have been developed to identify mRNA-miRNA (+ other non-coding RNAs) and mRNA-protein-RNA interaction networks [MicroRNAs (miRNAs) have emerged recently in the landscape of post-transcriptional regulation. They are considered both friends and foes due to their mode of action. A brief introduction to microRNAs and their mode of action is provided here. MicroRNAs are short (~22 nucleotides long) non-coding RNAs that are expressed in a tissue specific manner and bind specifically to target mRNAs. If RBPs are considered guardians of genetic messengers, then microRNAs may be referred to as “micro” guards of mRNAs. MicroRNAs control gene expression by the down regulating and fine tuning of transcripts by translational repression and/or degradation of respective mRNAs ,13,14. Requences . Since mequences . As bothequences ,18. Datanetworks . SimultaAlcohol abuse poses a large disease with a high economic and social burden nationwide. In 2006 alcohol-related costs in the United States incurred the country $223.5 billion. Globally, 3.3 million deaths have been attributed to alcohol use . AlcoholN-methyl-D-aspartate receptors (NMDA) belong to the family of glutamatergic receptors and are a well-established target of alcohol. They have been implicated in the development of alcohol dependence, tolerance, and withdrawal syndrome [in vitro and in vivo [cis-acting region in the 3'-UTR of GluN1 mRNA, and three RBPs bind to this region. Two of these RBPs are identified as annexin A2, a known RBP [The glutamatergic neurotransmitter system is one major neurotransmitter system that is affected by alcohol exposure . N-methysyndrome . NMDA resyndrome . The Glusyndrome . This susyndrome . Chronic in vivo ,32,33 wi in vivo . Our lab in vivo . However in vivo . This ob in vivo , our labnown RBP , and thenown RBP ,39. The nown RBP . GII enznown RBP ,41. The nown RBP . The notnown RBP . For exanown RBP . GAPDH inown RBP . A micronown RBP . Multifuc-myc and its own mRNA occurred via a putative RNA-binding domain consisting of five nucleotides 5'-AA(C/G)(A/U) [The second GluN1 mRNA-binding protein, annexin A2 is a known RBP. Annexin A2 belongs to a large family of proteins that bind anionic phospholipids in a calcium dependent manner and are important in membrane organization and cytoskeletal interactions . First, /G)(A/U) . This RN/G)(A/U) . It appe/G)(A/U) . Indeed −/−) mice, levels of inflammatory molecules such as TNFα and IL-1β are markedly attenuated as compared to wild type mice following alcohol consumption. Thus, both in vivo and in vitro studies assign a new function to CIRP of being a proinflammatory molecule that mediates inflammation in the brain following alcohol consumption [Binge drinking refers to consumption of 4–5 drinks* (as ≥4 drinks for a woman and ≥5 drinks for a man on a single occasion) within 2 h as per NIH-NIAAA guidelines, and this type of drinking brings blood alcohol levels to 0.08 g/dL. According to NIH-NIAAA, a standard drink in the US is considered as 12 ounces of regular beer  or 5 ounces of wine  or 1.5 ounces of 80-proof distilled spirits  . Binge dsumption . Recent sumption . A normasumption .et al. observed a reduction in Napor expression in forebrain, midbrain and hindbrain of fetal rats exposed to alcohol during development [Alcohol does not only affect the mature brain but also has profound effects on the developing brain. Maternal alcohol exposure during pregnancy results in a number of abnormalities collectively termed fetal alcohol syndrome (FAS). At the cellular level, FAS is characterized by neurodegeneration mainly as a result of apoptosis . In the elopment . ConsideDrosophila as an essential protein for neural development [Alcohol affects almost all tissues in our body. The liver is another organ that has been examined for changes in expression of RBPs, as altered expression of RBPs disrupts molecular pathways in different cells and tissues. The RBP HuR is a member of the embryonic lethal abnormal vision (ELAV) family of RBPs originally identified in elopment . HuR is elopment . Tumor nelopment . TNFα exelopment . HuR is elopment . The AU elopment . The binelopment . An increlopment . The maielopment . It is aelopment .Liver disease is a major and debilitating consequence of chronic alcohol consumption. Excessive alcohol exposure can lead to hepatosteatosis and to more severe consequences such as liver fibrosis and cirrhosis. How alcohol brings about this effect has been the focus of much investigation. Galβ1,4GlcNAc-α2,6-sialyltransferase  mediates the addition of α2,6-linked sialic acid to glycoproteins in the Golgi apparatus. It was observed that the mRNA levels of ST6Gal1 are reduced in post-mortem human livers of moderate and heavy alcohol drinkers. Subsequent studies involving laboratory rodents showed a significant decline in ST6Gal1 activity accompanied by reduction in ST6Gal1 mRNA levels following chronic alcohol exposure ,74. The de novo protein synthesis [RBPs are ubiquitous partners of RNA and control all aspects of RNA metabolism. Given their omnipresent nature, dysregulation of RNA processing in a number of pathologies ranging from neurologic diseases to muscular dystrophies and cancer places them on a higher pedestal. Thus, the pathologies have become a target of novel therapeutic drugs ,77,78. Aynthesis . These fA [Paraneoplastic neurologic disorders (PND) are neurological manifestations resulting from autoantibodies against two well-known families of RBPs, the Nova and the Hu ,82. FamiA . GephyriA . As a coA . The NovcFos and granulocyte-macrophage stimulating factor, results in stabilization of the respective mRNA [The Hu family members  are a second set of RBPs with diverse functions and mediate antigen-associated immune disorders. Two Hu family members, HuC and HuD are specifically expressed in the CNS. The Hu family of RBPs regulates half-lives of several mRNAs by binding to ARE regions in their 3'-UTRs. The binding of Hu family members to mRNAs, such as epidermal growth factor, ive mRNA . Becauseive mRNA . In contive mRNA . Diverseive mRNA . Moleculive mRNA . The majive mRNA .In addition to modulating splicing events and regulating mRNA half-lives, RBPs play important roles in translational control during embryonic development, cell-cycle progression , learninCancer is a heterogeneous disease, and we have no clear understanding of the etiology of different tumors. Traditionally, it is believed that the development of cancer is associated with mutations, transcriptional dysfunction and signal pathways gone awry. However, as we gain a greater understanding of post-transcriptional regulation of gene expression, we realize that post-transcriptional events contribute to cell proliferation, differentiation, apoptosis and angiogenesis—all processes that modulate the development and progression of cancer . RNA-binThe Hu family of RBPs is another example involved in the development of cancer. Of the Hu family of proteins, HuR is the one that is most frequently associated with the development of different cancers including breast, pancreatic, prostate, lung and ovarian cancer . In a reTranslation control is a critical constituent of cancer development and progression. During the multistep process of protein translation, the translation factor elF4E binds to the 7-methyl guanosine cap structure at the end of mRNAs, providing a critical interface for the recruitment of the 40S ribosomal subunit and other translation factors . Expressin vitro methods have been utilized to detect RNA-protein interactions, and these methods remain in vogue. EMSA has been employed extensively in past years and has yielded invaluable information. In this assay, radiolabeled RNA was incubated with the source of RBP (purified protein or cell lysate or microsomes or membrane free cytosol), and the resulting complex was electrophoresed on a non-denaturing polyacrylamide gel and radiolabeled bands were visualized by exposure to X-ray film or PhosphorImager. Non-specific RNA-protein interaction was prevented by the incubation of RNA and protein source in the presence of tRNA and/or heparin. The principle behind this technique is the observation that naked RNA has certain mobility on gels, but its mobility is retarded upon binding to proteins. To confirm the specificity of the RNA protein interactions, an excess of unlabeled competitor RNA is added to the reaction mix, and this obliterates the formation of RNA-protein complex. To confirm the presence of a specific protein in the RNA protein complex, gel super shift assays can be performed. These assays were performed as in EMSA, except for the addition of specific antibody to the protein of interest. Formation of RNA-protein-antibody complex further slows down the migration of complex on a gel. Once the interaction between a specific mRNA and a RBP is established, immune-RT/PCR can be employed to confirm the RNA-protein interactions [cis-acting region. The size of RBPs binding to RNA can be determined by northwestern analysis. In this technique, protein lysates are separated on SDS-PAGE and proteins are re-folded and then transferred to a support membrane such as nitrocellulose membrane. Proteins transferred to membrane were probed with radiolabeled RNA. Non-specifically bound RNA was washed off and the membrane was exposed to X-ray film to reveal the number of proteins highlighted by RNA. The molecular size of proteins was extrapolated from the standard molecular weight marker proteins separated along with protein samples on the gel.Traditionally, ractions . Brieflyractions ,115. In in vitro techniques continue to be powerful tools in the RNA field. To detect RNA-protein interactions in vivo, Darnell et al. developed the cross-linking and immunoprecipitation (CLIP) method [in vitro assays. Another advantage of this technique is that one can identify target RNAs of one RBP. The CLIP procedure was comprised of a number of steps that we have briefly summarized here. For a detailed protocol, the reader is referred to the original publication [et al. to identify 34 targets of NOVA in the brain [The aforementioned ) method . A distilication . In shorhe brain .et al. replaced the conventional Sanger sequencing method employed in CLIP with high-throughput sequencing (HITS-CLIP) [With the advent of high-throughput sequencing, it was then possible to map RNA-binding sites even more comprehensively. Using such a platform, millions of unique RNA tags were able to be identified and mapped back to the genome yielding transcriptome-wide RNA-protein binding maps. To further refine the CLIP protocol, Darnell TS-CLIP) . This mehttp://rbpdb.ccbr.utoronto.ca/) [Caenorhabditis elegans, as many as 887 genes may encode for RBPs [Saccharomyces cerevisiae, approximately 600 transcripts encode for putative RBPs [in vitro or in vivo cross-linking of RNA and proteins, digestion of unbound RNA, immunoprecipitation using an antibody to a RBP of interest, reverse transcription, PCR amplification, and high-throughput sequencing. High-throughput sequencing data is typically uploaded onto public databases such as the Gene Expression Omnibus (GEO from NCBI) and ArrayExpress (from EBI), and three recently developed databases for CLIP-seq: CLIPZ, doRiNA, and starbase. As a next step, using computational methods, such as Bowtie, RMAP, Novoalign, and TopHat, the “short reads” (sequencing data sets) are aligned to a reference sequence (transcriptome and genome) while eliminating technical sequencing errors and true genetic variations. Once the “short reads” are aligned to the reference sequence, several restrictions are applied to filter the noise in processing the data further to detect a cluster of reads. A cluster of reads is a group of reads that has some sequence overlap. Usually, reads with a length above a certain threshold are considered for further analysis to detect RBP binding sites. RBP binding site detection is a challenging process. Several computational methods such as WavClusteR, PARalyzer, Piranha, PIPE-CLIP, dCLIP, and GraphProt have been developed and utilized to detect RBP binding sites while minimizing the detection of false positives and background. Computational methods such Piranha and RIP-seeker have been developed specifically for peak calling [et al. [RBPs interact with mRNA via a number of RNA recognition motifs or RNA-binding domains . A largento.ca/) . In Caenfor RBPs  and, in ive RBPs . As our ive RBPs . Computaive RBPs . The utiive RBPs . In gene calling . Peak ca calling . PARalyz [et al. . In addi [et al. . Becausecis acting elements”. When efficient gene delivery systems become a reality RNA-binding proteins may become the “drugs” of choice for customized therapies.The search for better and more selective drugs for the treatment of cancer, infections and many other pathologies is an ongoing and challenging effort. New discoveries in the RNA field open up opportunities for RNA-binding proteins to serve as therapeutic agents. When we have a better understanding of the targets of RNA-binding proteins, it will be possible to modulate gene expression at several post-transcriptional levels. Tailored RNA-binding proteins can be used to alter expression of single genes or even complete gene families whose transcripts share common “


RNA-Seq was used to examine the microbial, eukaryotic, and viral communities in water catchments (‘tanks’) formed by tropical bromeliads from Costa Rica. In total, transcripts with taxonomic affiliation to a wide array of bacteria, archaea, and eukaryotes, were observed, as well as RNA-viruses that appeared related to the specific presence of eukaryotes. Bacteria from 25 phyla appeared to comprise the majority of transcripts in one tank (Wg24), compared to only 14 phyla in the other (Wg25). Conversely, eukaryotes from only 16 classes comprised the majority of transcripts in Wg24, compared to 24 classes in the Wg25, revealing a greater eukaryote diversity in the latter. Given that these bromeliads had tanks of similar size , and were neighboring with presumed similar light regime and acquisition of leaf litter through-fall, it is possible that pH was the factor governing these differences in bacterial and eukaryotic communities (Wg24 had a tank pH of 3.6 and Wg25 had a tank pH of 6.2). Archaeal diversity was similar in both tanks, represented by 7 orders, with the exception of Methanocellales transcripts uniquely recovered from Wg25. Based on measures of FPKG (fragments mapped per kilobase of gene length), genes involved in methanogenesis, in addition to a spirochaete flagellin gene, were among those most highly expressed in Wg25. Conversely, aldehyde dehydrogenase and monosaccharide-binding protein were among genes most highly expressed in Wg24. The ability to observe specific presence of insect, plant, and fungi-associated RNA-viruses was unexpected. As with other techniques, there are inherent biases in the use of RNA-Seq, however, these data suggest the possibility of understanding the entire community, including ecological interactions, via simultaneous analysis of microbial, eukaryotic, and viral transcripts. Because epiphytic bromeliads virtually lack absorptive roots, these tanks provide the plants with water and nutrients released by heterotrophic decomposition of impounded debris Due to their small size, defined boundaries, and relatively simple faunal communities, bromeliad catchments are ideal for the investigation of whole ecosystem complexity, a notoriously difficult task for lakes and other large, more complex freshwater habitats With a new appreciation for the intertwined worlds of metazoans and microbes, it is an exciting prospect to explore the possibility of examining the entire community (from viruses to eukaryotes) with a single technique. The current state of knowledge with regard to plant-associated aquatic habitats is based on the counting of animals or protozoa or, in the case of bacteria and archaea, based on 16S rRNA ribosomal surveys ex. . There h22.1Werauhia gladioliflora specimens in La Selva Biological station, a wet  lowland neotropical reserve located in northeastern Costa Rica . Samples were collected in June 2012, from two plants, neither of which was in bloom, growing at heights of ~ 2.0 m from the ground. The bromeliads had tanks of ~ 10 cm depth , and were neighboring with presumed similar light regime and acquisition of leaf litter through-fall. Since previous studies revealed strong correlations between bacterial diversity and bromeliad tank pH, which can range from pH 3 to 7 Bromeliad tank debris and water were collected from two 2.2Zap Wipes (Ambion). The RNA PowerSoil Total RNA Isolation Kit (MoBio Laboratories) was used for bulk extraction of RNA, with slight protocol modifications to extract sufficient quantities of nucleic acid for metatranscriptomic analysis (~ 90–120 ng μl− 1). Modifications included the use of freshly prepared low-pH phenol:chloroform:isoamyl alcohol  for nucleic acid extraction (PowerSoil Step 5), a room temperature incubation for secondary debris precipitation (PowerSoil Step 9), gentle encouragement of fluid flow through the RNA capture column using a syringe (PowerSoil Steps 16–19), and an overnight nucleic acid precipitation step (PowerSoil Step 20). Successful nucleic acid extraction was verified by NanoDrop UV spectrophotometry, ensuring sufficient RNA concentrations and purity. RNA extraction was immediately followed by removal of contaminant DNA from the extracted RNA, using TURBO DNA-free DNase Treatment and Removal Reagents (Ambion). A treatment of 4 Units TURBO DNase per 100 μl RNA was applied, with a 20 min 37 °C incubation for sufficient DNA contaminant removal.Nucleic acid extraction was performed at Occidental College, using RNase-free materials (tubes and pipette tips) on workstations treated with RNase2.3Total extracted RNA was submitted to Otogenetics Corporation  for RNA-Seq analysis. Briefly, 9–12 μg of total RNA was subjected to rRNA depletion using the RiboZero Meta-Bacteria kit (Epicentre Biotechnologies) and cDNA was generated from the depleted RNA using the NEBNext mRNA Sample Prep kit (New England Biolabs). Illumina libraries were created from the cDNA using NEBNext reagents (New England Biolabs). The quality, quantity and size distribution of the Illumina library products were determined using an Agilent Bioanalyzer 2100. The libraries were then submitted for Illumina HiSeq2000 sequencing according to standard operations.2.4− 5 against the Integrated Microbial Genomes (IMG) system v.4.0 http://github.com/minillinim/sam2fpkg; Paired-end 100 nucleotide (nt) reads were generated and checked for data quality using FASTQC (Babraham Institute). For removal of ribosomal RNA sequences  that were not subtracted via RiboZero depletion, SortMeRNA software 3Poplar biomass reactors, enrichment cultures), and associated with eukaryotes  was performed on the water catchments formed by tropical bromeliads from a Costa Rican rainforest. The community associated with Costa Rican tropical bromeliads was deduced through affiliation of non-ribosomal transcript sequences with particular taxa in public databases. Overall results from two bromeliad tanks indicate the presence of genes from 25 bacterial phyla (including 4 classes within the Proteobacteria), 7 archaeal orders, 21 metazoan classes (including 6 orders of insect), 8 fungal classes, as well as viruses and transcripts related to those recovered from environments, including plant biomass reactors, freshwater lakes, and the rhizosphere , Table 3Oophaga pumilio, which commonly uses bromeliad tanks as nurseries Bromeliad eukaryote communities are abundant and diverse, typically dominated by annelids and larvae of aquatic insects, including many species of Coleoptera and Diptera Transcripts from numerous bacterial phyla (including 4 classes within the Proteobacteria), were recovered from the bromeliad tanks, comprising 25 phyla in Wg24 and a subset of these (~ 14 phyla) from Wg25 . Both taThe metatranscriptome data for archaeal presence and diversity also followed the same pattern as recovered from previous DNA-based studies. Transcripts with archaeal taxonomic assignments were present at similar levels in both bromeliad tanks, comprising ~ 7% of the transcript reads. Archaeal diversity was similar in both tanks, represented by 7 orders, with the exception of Methanocellales transcripts uniquely recovered from Wg25 , Table 242−) and hydrogen peroxide metabolites, and small heat shock proteins, were almost exclusively detected in Wg24. Together, such differences in genes relevant to cellular defense and response mechanisms perhaps reflect the high-stress, low-pH environment in this particular bromeliad catchment.Bacterial communities play essential roles in energy and matter flow in aquatic microcosms, through detrital mineralization and nutrient release. Of particular interest is the microbial role in nitrogen and carbon-cycling in bromeliad tanks, due to the abundance of carbon-based leaf litter, animal carcasses, and feces that constitute the organic detritus. Despite their status as one of the most abundant aquatic ecosystems in the world, and their classification as true microcosms , remarkably little is known about the function of bromeliad-associated microbial communities 52 on the planet could result in the loss of biodiversity within bromeliad tanks over time. In order to confirm this hypothesis, it will be necessary to conduct a more thorough analysis of the structure and function of organisms in these habitats.Freshwater tropical ecosystems, including bromeliad tanks, support disproportionate levels of biodiversity, especially microorganisms and invertebrates, relative to their spatial coverage, and are expected to be highly vulnerable to habitat perturbations resulting from climate change Number of bacterial species represented in metatranscriptomic libraries from two bromeliad tanks. For Wg25, with a pH of 6.2, species number for each phylum was multiplied by 1.3 from  to accouThe following is the supplementary data related to this article.