The Shanghai Holstein cattle breed is susceptible to severe mastitis and other diseases due to the hot weather and long-term humidity in Shanghai, which is the main distribution centre for providing Holstein semen to various farms throughout China. Our objective was to determine the genetic mechanisms influencing economically important traits, especially diseases that have huge impact on the yield and quality of milk as well as reproduction.In our study, we detected the structural variations of 1,092 Shanghai Holstein cows by using next-generation sequencing. We used the DELLY software to identify deletions and insertions, cn.MOPS to identify copy-number variants (CNVs). Furthermore, we annotated these structural variations using different bioinformatics tools, such as gene ontology, cattle quantitative trait locus (QTL) database and ingenuity pathway analysis (IPA).The average number of high-quality reads was 3,046,279. After filtering, a total of 16,831 deletions, 12,735 insertions and 490 CNVs were identified. The annotation results showed that these mapped genes were significantly enriched for specific biological functions, such as disease and reproduction. In addition, the enrichment results based on the cattle QTL database showed that the number of variants related to milk and reproduction was higher than the number of variants related to other traits. IPA core analysis found that the structural variations were related to reproduction, lipid metabolism, and inflammation. According to the functional analysis, structural variations were important factors affecting the variation of different traits in Shanghai Holstein cattle. Our results provide meaningful information about structural variations, which may be useful in future assessments of the associations between variations and important phenotypes in Shanghai Holstein cattle.Structural variations identified in this study were extremely different from those of previous studies. Many structural variations were found to be associated with mastitis and reproductive system diseases; these results are in accordance with the characteristics of the environment that Shanghai Holstein cattle experience. Holstein cows are famous for having the highest milk yield in the world; therefore, much attention has shifted to the breeding of Holstein cattle for many years . ChineseThe Holstein breed is categorized as a heat-sensitive cattle breed that originated in Europe. High environmental temperatures have a negative influence on the performance of Chinese Holstein cattle, especially in Shanghai ,3. The lDespite this poor natural habitat, it has been long observed and established that Shanghai Holstein cattle are less susceptible to mastitis. Livestock breeds of Shanghai origin are well adapted to tropical environments because of their ability to thrive under extreme nutritional stress, resistance to diseases, heat tolerance potential and sturdiness ,7,8. TheGenetic variation ranges from the single base pair (bp) to several megabases (Mb) compared to a reference genome . As a tyTherefore, the objective of this study was to identify the structural variants in the Shanghai Holstein population and determine the genetic mechanism of the influence of structural variation on the performance of Shanghai Holstein cattle by annotating the genome for the distribution, density and physical location of the main structural variations on chromosomes.All experimental procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University, and all methods involving cattle were in accordance with the agreement of Institutional Animal Care and Use Committee of Shanghai Jiao Tong University (contract no. 2011-0033).The Holstein population in this study comprised 1,092 cows, the daughters of 17 sires and each family with an average of 64. These cows where born between 2001 and 2012. All the cows where from 24 dairy cattle farms in the Shanghai Bright Dairy and Food Co., Ltd where have been carried out as part of the Dairy Herd Improvement System.AvaII, followed by ligation with a unique adapter-barcode sequence for AvaII . Next, 84 or 108 samples were pooled and enriched by polymerase chain reaction amplification . The quality of the sequencing libraries was evaluated by an Agilent 2100 Bioanalyzer . Lastly, sequencing libraries  were sequenced on an Illumina HiSeq2000  instrument to obtain paired-end (2×150 bp) reads. After sequencing, the sequence reads were filtered for quality using the NGSQC Toolkit v2.3 with the parameter settings referred to in Chen et al  and retinoic acid receptor alpha, core genes related to reproductive system development and function , and eight core genes associated with lipid metabolism , play a key role in DNA and protein phosphorylation, acetylation, and ubiquitination, which are epigenetic processes (http://www.genecards.org). Thus, we can infer that these structural variations were mainly caused by environmental effects. For insertions, a total of 18 significant networks were associated with embryonic , immunological and inflammatory diseases  (etc.) and lipid metabolism (amyloid beta precursor protein)  . Compareprotein) . To sum So far, structural variants in whole genomes have not been reported in the Shanghai Holstein population by using next-generation sequencing (NGS), and relatively few deletions, insertions and CNVs have been detected or confirmed. Shanghai Holstein cattle are the main economic animals providing milk, but due to the unique environment in Shanghai, which is characterize with hot weather condition and high humidity particularly in the summer with most days having temperature of above 35°C (95°F) and thereby subjecting them to many diseases as well as influencing the yield and quality of milk. Therefore, it is urgent to determine the mechanisms of structural variants related to these traits. In our study, we performed gene sequencing and structural variant calling in a large population, which was composed of 1,092 dairy cows from the Shanghai Bright Dairy and Food Co., Ltd. and the Dairy Cattle Breeding Centre born between 2001 and 2012. Furthermore the variants identified were annotated using bioinformatics tools.A total of 1,092 samples were genotyped according to the GGRS protocol in our study. The average depth and coverage of the sequence data were 4.0% and 1.8%, respectively. A total of 16,831 deletions, 12,735 insertions and 490 CNVs from the sequence data of the Shanghai Holstein population were detected by using DELLY and cn.MOPS; among these CNVs, there were 102 CNVRs found in only one sample. Jiang et al  used theSNP chip (Illumina Bovine SNP Beadchip) and high-throughput sequencing are both widely used for DNA sequencing of different animals. So far, the chips applied to bovines are the Illumina BovineSNP50 Beadchip with approximately 54K SNPs, BovineLD with approximately 7K SNPs and BovineHD with approximately 780,000 SNPs. In addition, SNP probes which cannot cover the whole genome on the chip are neither dense enough nor uniformly distributed to achieve an unbiased and high-resolution cattle CNV map. In addition, the price is relative expensive, and the reference population used to design the chips was composed of Europe and America cattle. Therefore, it is not completely appropriate for Asian populations. The other technology to study genetic variants is NGS, and since the advent of this technology, it has been rapidly evolving, with increasingly widespread adoption of several platforms and decreases in the cost of DNA sequencing, which allows for CNV reconstruction at a higher effective resolution and sensitivity and systematic identification CNVs at a genome-wide level . CurrentDue to the importance of CNVs and other structural variations in the genome, advanced technologies have been created, including microarrays, that permit high-throughput methods that are now relatively common . For ideFor deletions and insertions, at present, after quality control and recalibration, sequencing data are subsequently processed by different software such as DELLY, Breakdancer, SVseq2, CNNdel, MetaSV, and Pindel for calling SVs ,40–44. GRANBP10, which acts a novel coactivator for the androgen receptor [CREB1 being associating with T-cell activation and interleukin-2 production [HERC2 was overlapped with the most significant core haplotypes in A. Gurgul’s identification of genome-wide selection signatures in the Limousin beef cattle breed [RMND5A which identified a critical role of miRNAs in regulation of host immune responses and homeostasis of PMECs mediated by affecting pathways including cytokine binding [SRPK2 has been recognized as a differentially expressed gene between the endometrial tissue collected from day 7 of the estrous cycle of animals with high and low fertility by Aideen P Killeen’s microarray analysis [EPAS1 associated with pulmonary hypertension in Angus cattle. Genetic variations of NR3C2 could explain the alterations in animals to adapt to challenges, and therefore, their influence on production traits [LRIF1 is a nuclear protein that is known to be involved in the inactivation of the human X chromosome; however, its function in immunity is unknown [By annotating the structural variants using GO terms and KEGG pathways, we found that many terms are significantly related to diseases and one of the reasons for this could be that the hot and high humid environment in Shanghai. For deletions, bta05200:Pathways in cancer, GO:0032845~negative regulation of homeostatic process, bta04142:Lysosome, GO: 0045321~leukocyte activation and bta05412:Arrhythmogenic right ventricular cardiomyopathy are all significant pathways, and among them, bta04142:Lysosome responds to foreign substances such as bacteria, viruses and other antigens. GO: 0045321~leukocyte activation is associated with mastitis . For insreceptor . Sallam receptor  performeoduction . HERC2 wle breed . Jaeger le breed  provides binding . In addi binding . Genes oanalysis . Heaton analysis  found thn traits . LRIF1 i unknown . TherefoGroenen et al  reportedSo far, many studies have been conducted to discover CNVs; however, the association between CNVs and phenotype has not been analysed. Using genome-wide genotyping to discover the genetic variations is the only precondition for many studies, such as those investigating the molecular mechanisms of livestock breeding and conservation, especially for genome-wide association studies, genomic selection, and genomic conservation. In this study, we systematically identified structural variation, including deletions, insertions and CNVs and also determined the potential variations that may result in mastitis and affect reproduction. Therefore, for further studies, we will make full use of our findings in this study to estimate the genetic effect of structural variation for economic traits, gain a more particular knowledge of the population structure, and make decisions about the genomic selection programme. In addition, we aim to research on genomic predictions, thus laying a foundation for forming a reference population in Shanghai.


The 2C method allows the rapid and straightforward isolation of nucleic acid–protein complexes, greatly simplifying downstream applications for the study of DNA– and RNA–protein interactions. Proteins interact with nucleic acids to regulate cellular functions. The study of these regulatory interactions is often hampered by the limited efficiency of current protocols to isolate the relevant nucleic acid–protein complexes. In this report, we describe a rapid and simple procedure to highly enrich cross-linked nucleic acid–bound proteins, referred to as “2C” for “complex capture.” This method is based on the observation that silica matrix–based columns used for nucleic acid purification also effectively retain UV cross-linked nucleic acid–protein complexes. As a proof of principle, 2C was used to isolate RNA-bound proteins from yeast and mammalian Huh7 cells. The 2C method makes RNA labelling redundant, and specific RNA–protein interactions can be observed and validated by Western blotting. RNA–protein complexes isolated by 2C can subsequently be immunoprecipitated, showing that 2C is in principle compatible with sensitive downstream applications. We suggest that 2C can dramatically simplify the study of nucleic acid–protein interactions and benefit researchers in the fields of DNA and RNA biology. From storage and transmission of genetic information, in the form of DNA and RNA, to the regulation of gene expression by silencing mechanisms and effector functions like ribozymes, nucleic acids play central roles in cellular life. For decades, intense research efforts were directed to the development of simple methods to isolate pure nucleic acids from diverse biological origins. Key discoveries for the implementation of simple nucleic acid isolation protocols were the strong protein denaturing properties of chaotropic salts , the difBiologically, the cellular roles of nucleic acids can only be understood in the context of proteins that assist and accompany them throughout their existence . DiffereThe study of RNA–protein complexes, or RNPs, faces additional technical challenges derived from both the lability of RNA and the more dynamic nature of RNA-based processes within cells . One of Here, we report that a silica-based solid-phase extraction, developed for the purification of nucleic acids, can be used to capture individual proteins cross-linked to nucleic acids or complete nucleic acid–bound proteomes. We refer to this simple and robust approach as “complex capture,” or 2C. The 2C method dramatically simplifies the isolation of nucleic acid–protein complexes, does not require the use of radioactivity for the detection of specific nucleic acid–binding proteins, and simplifies downstream applications.Because silica columns are used to retain and purify DNA and RNA based on charge, we wondered whether they might also retain nucleic acid–binding proteins when covalently cross-linked to nucleic acids. The presence of strong denaturing agents would prevent the retention of non–cross-linked proteins and only cross-linked proteins or peptides would be retained indirectly via their bound nucleic acid. These nucleic acid–binding proteins could later be co-eluted with pure DNA, RNA, or both .Saccharomyces cerevisiae cells were cross-linked by irradiation with 3 J/cm2 UV light at a wavelength of 254 nm. Equivalent samples of non-irradiated cells were used in parallel as negative controls. Cell lysates were treated with a guanidinium thiocyanate–containing buffer, which is commonly used for RNA purification. Under these conditions, proteins are fully denatured, and the binding of RNA to the silica matrix relative to DNA is favored by the addition of ethanol  has been identified as an RBP in multiple interactome capture studies, we selected it as our next candidate. As shown in Of note, comparison of input signals with the 2C eluates allows assessment of the RNA binding capacity of different RBPs, although the amount of RNA-bound protein, compared with its overall abundance, is also influenced by its ability to be cross-linked to RNA. As expected, Pab1 behaves as a potent RNA binder and/or displays the features of a protein with high RNA cross-linking efficiency. Conversely, a much smaller fraction of GAPDH appears to be bound to RNA for either or both of the reasons discussed above. Analysis of the DNA-binding histone H3, used here as a negative control, demonstrates the limits of possible DNA contamination in our RNA purifications .Similarly, the yeast glycolytic enzymes hexokinase (Hxk) and triose phosphate isomerase (Tpi), which were previously identified as RBPs , were alWe next evaluated the performance of 2C in mammalian cells. To this end, we used human liver carcinoma-derived Huh7 cells. Similar to the yeast methodology, Huh7 cells were cross-linked in vivo and the cell lysates were subjected to the 2C protocol. The proteins hnRNPC1/C2 were analyzed as examples of strong RBPs . Similar2C involves protein denaturation for stringent removal of non–cross-linked polypeptides. As a consequence, proteins in the eluate may lack their native conformation hindering the subsequent immunoprecipitation of specific native or tagged RBPs and their bound RNAs. Therefore, we tested tagged RBPs in affinity purification experiments after 2C capture.Tdh3, corresponding to isoform 3 of yeast GAPDH, and Pab1 were tagged with the protein A or the TAP tag, respectively. 2C eluates were used as inputs for affinity purification experiments, and aliquots from input, supernatant, wash, and eluate fractions were tested by Western blotting. As expected, little or no protein was detected in the lanes corresponding to non–cross-linked samples . Both PaThe main finding of this study is that silica matrices are not only capable of retaining nucleic acids but also polypeptides that are cross-linked to them. Here, we used RNA and RBPs to demonstrate the validity of the 2C technique. This seemingly trivial finding has profound potential to advance experimentation in RNA biology, especially of RNA–protein interactions. Because silica binding of DNA follows the same fundamental principle, we expect that 2C is equally applicable to DNA-binding proteins.One of the popular methods to identify and study RNA–protein interactions is UV cross-linking, which, however, suffers from the low efficiency of covalent bond formation between nucleic acids and proteins . As a coIn contrast, the 2C method is based on a simple, well-established principle: the inherent property of silica matrices to strongly and specifically retain nucleic acids . This inAn additional advantage of 2C is the simplicity of detection and validation of specific RBPs. The conventional protocol to validate RBPs involves immunoprecipitation, stringent washing, and subsequent radiolabeling of cross-linked RNA with T4 polynucleotide kinase assay. The RBP is then eluted, resolved by SDS–PAGE, and detected by autoradiography. Sensitivity of the signal to RNase treatment supports the conclusion that the immunoprecipitated protein is an RBP . By contWe envisage the use of 2C to reach far beyond the detection and validation of RBPs shown here and to simplify several downstream applications to study RNA–protein interactions , both foFrom an RNA perspective, cross-link and immunoprecipitation protocols are frequently challenging for non-canonical RBPs. Because 2C enriches cross-linked proteins away from the “free” proteins, immunoprecipitation and RNA sequence analysis from 2C eluates could putatively benefit from sequencing libraries of higher quality. However, because 2C involves protein denaturation before RNP binding and elution, insufficient renaturation can interfere with immunoprecipitation by antibodies that recognize folded epitopes.Moreover, 2C eluates could be used directly in protein–RNA footprinting experiments. Treatment of 2C eluates with RNase will preferentially degrade RNA that is not protected by cross-linked RBPs. After RNA digestion, the RBPs are degraded by proteinase K treatment, and the short RNA fragments that were protected from RNase treatment can be sequenced, generating a general map of RNA sequences bound by RBPs across the entire transcriptome.Overall, the simplicity of the 2C method should also facilitate comparative analyses of multiple samples or conditions for the vast majority of the downstream applications discussed above.Although this report focuses on RBPs and UV cross-linking as a means to induce covalent bonds between RNAs and RBPs, silica matrices can efficiently bind both DNA and RNA. Optimization of cross-linking procedures, lysis conditions, and buffers used for binding of the cross-linked nucleic acids to the silica matrices should further expand the utility to DNA-binding proteins and respective applications.MATa, his3Δ1, leu2Δ0, met15Δ0, and ura3Δ0) and PAB1-TAP  strains were purchased from Dharmacon. GAPDH-ProtA  strain was generated in this study. A fragment of the TAP tag comprising the tobacco etch virus (TEV) cleavage target sequence, the two protein A tandem sequences, the ADH1 terminator, and the His3MX6 selection marker were amplified by PCR. Sequences complementary to the end of the TDH3 gene were included by a second PCR, and the corresponding cassette was used to transform a WT strain for genomic integration of the TEV-Protein A cassette in frame with the TDH3 gene.Standard methods were used for yeast culture, transformation, and manipulation . WT BY47600 nm≈ 0.8–1.0), collected by centrifugation, resuspended in ice-cold buffer A , and transferred to 15-cm petri dishes on ice. Living cells were irradiated with 3 J/cm2 of UV light at 254 nm in a Spectrolinker XL-1500 (Spectronics Corporation) cross-linker. The cells were collected, concentrated by centrifugation, and the pellets were frozen until further use. Non-irradiated cells were processed in parallel as negative controls.Yeast cells were grown on YPD medium to mid-log phase  supplemented with protease inhibitors . The cells were broken by vortexing with 0.5-mm-diameter acid-washed glass beads  five times for 80 s, alternating with 1-min incubations on ice to avoid overheating of the samples. Lysates were clarified by centrifugation at 20,000 g for 20 min and the supernatants were transferred to new tubes. Protein concentration was quantified by colorimetric assay , and the samples were stored at −80°C until further use.For cell lysis, pellets were thawed on ice and resuspended in ice-cold buffer B  supplemented with protease and RNase inhibitors. The cells were lysed on ice using a tip sonicator (Branson Sonifier Cell Disruptor B15) with the following settings: two cycles of 10 shots each at 50% duty cycle and output 4, with 10-s gap between the cycles. Cell debris and intact cells were spun down at 10,000 rpm, 4°C, for 10 min, and the clarified cell lysate was flash-frozen until further use.Huh7 cells at 80%–90% confluency were washed twice with 10 ml chilled PBS on ice after media removal. Next, 20 ml chilled PBS was added on top of the cells, creating a thin liquid layer. The plates were placed on ice and irradiated with 150 mJ/cmg in a table-top microcentrifuge. The samples were consecutively treated with 400 μl of RNA prewash buffer and twice with RNA wash buffer. The column was then placed on top of a new microcentrifuge collection tube, and purified RNA and RNPs were eluted with 300 μl of water by centrifugation. The RNA concentration was measured using NanoDrop 1000 (Thermo Fisher Scientific) and RNA integrity was assessed with Bioanalyzer RNA 6000 Nano Chips   .Between 20 and 30 μg of RNA from yeast or Huh7 cells were treated with or without 500 U of RNase I  for 30 min at 30°C in a final volume of 50 μl. Laemmli loading buffer 4×  supplemented with 2-mercaptoethanol  was added to each sample and boiled at 95°C for 5 min, and 25 μl were loaded onto 4%–15% Criterion SDS–PAGE gels . Silver staining was performed according to the method of 2; 0.5 mM DTT; and 0.01% NP-40), and another time with buffer B. Pab1-TAP was eluted by incubating the beads in buffer D  with 2.5 μg of TEV protease at 34°C for 1 h and GAPDH-ProtA was eluted by resuspending the beads in buffer D plus loading buffer and incubating them at 95°C for 5 min.PAB1-TAP and GAPDH-ProtA strains were cross-linked as described and RBPs were purified by 2C. Non-irradiated samples from each strain were used as negative controls. Protein G–coupled Dynabeads  were incubated with rabbit IgG  for 30 min at room temperature. Unbound IgG was eliminated by washing the beads three times with buffer B, and fractions from the 2C eluates equivalent to 100 μg of RNA were incubated with the beads with gentle rotation at 4°C for 2 h. Unbound material was removed, and the beads were washed once with buffer B, three times with buffer C (25 mM Tris–HCl, pH 7.5; 1 M NaCl; 1.8 mM MgCl


C. elegans, providing new mechanistic insights into small RNA biogenesis. They also identified a second essential function for PETISCO in embryonic development mediated by TOST-1.In this study, Cordeiro Rodrigues et al. identified a novel protein complex, PETISCO, acting in 21U RNA production in  Caenorhabditis elegans. In this organism, even though genetic screens have uncovered 21U RNA biogenesis factors, little is known about how these factors interact or what they do. Based on the previously identified 21U biogenesis factor PID-1 (piRNA-induced silencing-defective 1), we here define a novel protein complex, PETISCO , that is required for 21U RNA biogenesis. PETISCO contains both potential 5′ cap and 5′ phosphate RNA-binding domains and interacts with capped 21U precursor RNA. We resolved the architecture of PETISCO and revealed a second function for PETISCO in embryonic development. This essential function of PETISCO is mediated not by PID-1 but by the novel protein TOST-1 (twenty-one U pathway antagonist). In contrast, TOST-1 is not essential for 21U RNA biogenesis. Both PID-1 and TOST-1 interact directly with ERH-2 using a conserved sequence motif. Finally, our data suggest a role for TOST-1:PETISCO in SL1 homeostasis in the early embryo. Our work describes a key complex for 21U RNA processing in C. elegans and strengthens the view that 21U RNA biogenesis is built on an snRNA-related pathway.Piwi proteins are important for germ cell development in most animals. These proteins are guided to specific targets by small guide RNAs, referred to as piRNAs or 21U RNAs in  Drosophila, for example, a piRNA amplification loop driven by two Piwi paralogs is coupled to the activity of a third nuclear Piwi protein that drives transcriptional silencing , and the small RNA cofactors bound by PRG-1 are known as 21U RNAs . Mutants for tost-1 produce 21U RNAs, display enhanced 21U-driven silencing, and have a Mel phenotype. Both PID-1 and TOST-1 interact with ERH-2 using a conserved motif, strongly suggesting a mutually exclusive mode of binding to PETISCO. PID-1-bound PETISCO has a role in 21U RNA biogenesis, and the TOST-1-bound version functions in another pathway, which is essential for embryonic viability. Our data suggest that the latter might be linked to SL1 splice leader RNA homeostasis.Here, we follow up on our previous identification of PID-1 (piRNA-induced silencing-defective 1) as a protein essential for 21U RNA production . Mutants complex . Since wpid-1 loss-of-function strains. Both experiments identified a rather restricted set of proteins . TOFU-6 and PID-3 were identified as the most prominent PID-1 interactors, and, for the latter, we further validated the interaction with PID-1 through immunoprecipitation-Western blotting . Given that these two proteins were identified in an RNAi screen for 21U biogenesis factors . Two other proteins were consistently identified as PID-1 interactors: IFE-3 and ERH-2 . IFE-3 is one of the five C. elegans homologs of eIF4E. Previous work demonstrated that IFE-3 binds to the 7-methylguanylate (m7G) cap  cap . Interes-spliced , implyinescribed , its molSupplemental Table S1). All transgenes were able to rescue mutant phenotypes , indicating that the expressed proteins are functional. All four interactors show a characteristic perinuclear punctate localization in the germline syncytium, overlapping partially, but not fully, with the P-granule marker PGL-1 , whereas the remaining interactors show a dispersed cytoplasmic distribution across the entire early embryo . These results show that all of the identified PID-1 interactors are expressed concomitantly in early embryos, and the germline, where they are found in close proximity to P granules.We next probed the subcellular localization of PID-1 and its four identified interactors by expressing them from transgenes generated through the so-called miniMos approach  and had very little effect on IFE-3 partners, resulting only in the loss of PID-1 . PID-3 IP-MS in the absence of PID-1 demonstrated that all interactions were maintained . Finally, in size exclusion chromatography, PID-1, TOFU-6, and ERH-2 displayed similar elution profiles, with an apparent molecular weight of ∼400 kDa , implying that IFE-3 may not play a role in initiating translation. We did detect a number of additional proteins bound to IFE-3 . This resulted in the following interactions that are most likely direct: IFE-3 interacts only with TOFU-6. TOFU-6 in turn interacts with PID-3, which in turn also interacts with ERH-2. Finally, both PID-1 and TOST-1 were found to interact only with ERH-2. Self-interaction was detected for ERH-2, although under lower-stringency conditions, indicating that ERH-2 may be present as a dimer in PETISCO.Having established the components of PETISCO, we next dissected the molecular interactions within this complex. Using the yeast two-hybrid (Y2H) system, we scored interactions between each possible pair of individual PETISCO subunits  mutation that is rescued by transgenic expression of PID-1. In this strain, the sensor is silenced (Since at least two components of PETISCO (PID-1 and TOFU-6) play a major role in 21U RNA biogenesis, we hypothesized that the other components are also part of this pathway. We first tested whether they affect the silencing of a GFP sensor construct that reports on the activity of the 21U pathway (21U sensor) . We usedsilenced A,B, althpid-1 and prde-1 activated the sensor. Likewise, RNAi against ife-3, pid-3, tofu-6, and erh-2 activated the sensor. These data show that PETISCO subunits, like the PETISCO interactor PID-1, are required for a fully functional 21U RNA silencing pathway.Animals from this sensitized sensor strain were subjected to RNAi targeting the various PETISCO subunits, and sensor activity was evaluated by microscopy and RT-qPCR A,B. As epid-3, erh-2, and ife-3  and isolated and sequenced small RNAs. We did not make mutants for tofu-6, since RNAi on tofu-6 was already shown to significantly reduce 21U RNA production . First, miRNAs were found to be largely unaffected . Next, we analyzed 21U RNA abundance and found that all tested PETISCO subunits significantly affected 21U RNA abundance . The effect of ife-3(xf102) mutation on 21U RNA accumulation is less pronounced . Redundancy between IFE-3 and other eIF4E homologs could be a reason for this less pronounced effect on 21U RNA levels, as we found IFE-1, a nonselective TMG/m7G binder . As described previously for pid-1 mutants .To extend the above observation, we sequenced small RNAs from mutants. To enrich for 21URNAs, we did not pretreat RNA with 5′ pyrophosphatase, preventing analysis of the highly abundant 22G RNAs. We created mutant alleles using CRISPR–Cas9 for each of the PETISCO subunit genes. We were able to derive deletion alleles for oduction . All exponounced C, but, oG binder , enriche mutants , the 21USupplemental Fig. S6E). The 26G RNAs are similarly weakly expressed and surprisingly also appear to be mildly affected by loss of PID-3, ERH-2, or IFE-3 . This finding may reflect a currently unknown mechanistic link between 26G RNA and 21U RNA biogenesis but may also result from indirect effects.The so-called type II 21U RNAs, which come from loci lacking the canonical Ruby motif , are onlSupplemental Fig. S2C; Supplemental Table S4). This phenotype has already been described for tofu-6, which is also known as mel-47 .Besides the defects in 21U biogenesis, the PETISCO mutants also display a so-called Mel phenotype: Homozygous mutant offspring from a heterozygous animal develop into fertile adults, but the embryos display a fully penetrant arrest before gastrulation and never hatch . Furthermore, tost-1(rnai) displays enhanced rather than disrupted silencing of the sensitized 21U sensor , when aligned with PID-1 and TOST-1 homologs from other nematode species, a conserved motif emerges {[+][+]Ψ(T/S)L(N/S)[−]RFxΨxxxG(Y/F)}  } A. Strikif (R61C) . When inf (R61C) B. These tost-1 using CRISPR–Cas9 . Loss of TOST-1 did not significantly affect 21U RNA biogenesis  but did result in a fully penetrant Mel phenotype . Among the alleles that we generated, we found one allele, tost-1(xf196), with a 58-bp deletion that removes the splice acceptor site of its third exon , resulting in either a frameshift or a C-terminal truncation. tost-1(xf196) displays a temperature-sensitive (TS) effect: At 25°C, the animals are fully Mel, while, at 15°C, the animals are viable. The fact that this allele is viable at 15°C suggests that the N-terminal part of TOST-1 is critical. The interaction motif that we identified is still intact in TOST-1(xf196), consistent with the idea that it is essential for TOST-1 function. This TS allele allowed us to probe the Mel phenotype in more detail through temperature shift experiments . First, animals grown at the restrictive temperature were able to produce viable offspring after shifting them to the permissive temperature. This shows that there are no structural or developmental defects in the germline of these animals that prevent them from producing live offspring. Second, the TS allele allows us to probe when TOST-1 function is required. The time required for animals shifted to the permissive temperature to produce viable embryos (8 h) is markedly longer than the time it takes from fertilization to egg-laying (∼200 min at 15°C), implying that the embryos laid after 8 h were fertilized and raised at the permissive temperature. Despite functional TOST-1 in these embryos, they still arrest; thus, TOST-1 activity in the germline is essential for embryonic development of the subsequent generation. Nevertheless, TOST-1 function is still required in early embryos because, when animals are shifted from permissive to restrictive temperature, the first arrested embryos are laid already after 2 h. This is very close to the time of residency within the uterus (∼150 min at 25°C), and hence these embryos had been fertilized very close to the time of the temperature shift in a gonad that still had functional TOST-1 . Small RNA sequencing from animals shifted to nonpermissive temperatures revealed no significant defects in 21U RNA populations .To further understand TOST-1 function, we generated loss-of-function alleles for pid-1 and tost-1 mutants is found in mutants for the other PETISCO subunits, strongly suggesting that PID-1 and TOST-1 define two distinct aspects of PETISCO function.In conclusion, both TOST-1 and PID-1 bind to the same PETISCO subunit, and loss of these two proteins results in different phenotypes. The combined phenotypes of both pid-1(xf35) mutants in triplicate. This revealed a significant reduction of 21U precursors in pid-1 mutants  experiments on IFE-3 and PID-3, including RppH treatment to remove 5′ caps before cloning, and checked the abundance of precursor molecules, as defined above. While PID-3 immunoprecipitations did not show enrichment, we detected significant enrichment of 21U RNA precursors in immunoprecipitations for IFE-3 only after decapping A, indicaSupplemental Fig. S9A,B). Such accumulation was less pronounced or absent for another small noncoding RNA that is produced from the same gene cluster as SL1: 5S rRNA  but point out that SL2 read counts are comparatively very low. U-snRNA-derived fragments were only mildly affected in these mutants .We noticed a striking accumulation of a 3′ fragment of SL1 RNA in a number of the mutants that we tested . In contrast, 5S rRNA and U-snRNAs are not enriched or are even depleted . The read coverage over SL1 and SL2 shows that two fragments are detected: one at the 5′ end and another at the 3′ end . The 5′ fragment is capped, as this fragment is absent from libraries from non-RppH-treated RNA . Finally, ERH-1 is more strongly conserved than ERH-2, with ERH-2 generally being absent in species in which PID-1 is not detected. Possibly, the ERH-2:PID-1 combination represents a specialization of PETISCO to 21U RNA biogenesis in the Elegans and Drosophilae supergroups.Many nematodes outside the st PRG-1 . We therst PRG-1 A, also iWe identified a protein complex, PETISCO, that is involved in at least two different pathways. PID-1:PETISCO acts in 21U RNA biogenesis, whereas TOST-1:PETISCO is essential for embryogenesis B. Here wWe found that PETISCO contains two proteins with predicted 5′ end interaction domains: a 5′ cap-binding activity in IFE-3 and a potential 5′P-binding domain, the MID domain , in PID-C. elegans  and an RNA processing machinery. Our data suggest that ERH-2 in C. elegans may have a similar function in that it connects PETISCO to a 21U and/or a SL1 precursor processing enzyme. The size of PETISCO, as determined by size exclusion chromatography (∼400 kDa) .ERH-2 is one of the two “enhancer of rudimentary” proteins of ws PID-1 A, may be complex . Hence, 400 kDa) E, sugges dimeric  and thatWe show that PETISCO has at least two functions: 21U RNA biogenesis and another essential function in embryogenesis. The latter may be related to SL1/2 snRNP homeostasis but may also relate to other yet unidentified RNA species. Independent of what this additional function may be, an image arises in which the 21U RNA pathway may have evolved out of an already existing small noncoding RNA network that became linked to an Argonaute-driven gene silencing program.Supplemental Fig. S1A), and also no PID-1 was detected in TOST-1 immunoprecipitations , suggesting that stable interactions within PID-1:PETISCO and TOST-1:PETISCO are very similar, if not identical.We show that the different functionalities of PETISCO can be separated through two different “adapter” proteins (PID-1 and TOST-1) that bind to PETISCO via ERH-2. The overall sequence similarity between PID-1 and TOST-1 is very low. Nevertheless, we identified a motif that is required for binding to ERH-2; hence, both proteins likely bind to PETISCO in a mutually exclusive way. Indeed, no TOST-1 was detected in PID-1 immunoprecipitations . Further details on these protocols, including details on bioinformatic analysis of next-generation sequencing data, are in the Supplemental Material.RNA was isolated by lysing worms with protK followed by TRIzol LS extraction. In RIP experiments, TRIzol LS was added directly to the immunoprecipitation beads after washing. Quantitative PCR was done in an Applied Biosystems ViiA7 real-time PCR system (Thermo Fisher Scientific) with cDNA made from 500 ng of total RNA using ProtoScript first strand cDNA synthesis kit  and Oligo d(T)Supplemental Material.Worm pellets were thawed on ice and mixed 1:1 with 2× lysis buffer  with 2× protease inhibitors . A Bioruptor Plus (Diagenode) sonicator was used to lyse worms (10 cycles of 30/30 sec at 4°C and high energy), and debris was removed by spinning. Lysate protein concentration was determined with Pierce BCA protein assay kit . Lysates were diluted in 1× lysis buffer plus 1× protease inhibitors to a final concentration of 1.5 mg of protein per milliliter, and a total of 0.75 mg of protein was used per immunoprecipitation. At this step, input samples were collected into 2× NuPAGE LDS sample buffer  plus 200 mM DTT and boiled for 10 min. Anti-mCherry immunoprecipitations were performed with RFP-Trap_MA beads , and anti-GFP immunoprecipitations were performed with GFP-Trap_MA beads ; in both cases, 25 µL of bead slurry was used, and samples were rotated for 2 h at 4°C. Subsequent washes were made with wash buffer  plus protease inhibitors in accordance with Chromotek protocols. When appropriate, RNase treatment was done with 20 µL of RNase A/T1 mix per 1 mL of sample. Samples were rotated for 20 min at 4°C followed by the described immunoprecipitation protocol. For RIP-seq experiments, lysates were diluted in 1× lysis buffer + 1× protease inhibitors + 1× SUPERaseIn RNase inhibitor to a final concentration of 1.5 mg/mL protein, and a total of 2.1 mg of protein was used per immunoprecipitation. Further details are in the Supplemental Material.Measurement was done on a Q Exactive Plus mass spectrometer (Thermo) operated with a Top10 data-dependent tandem mass spectrometry acquisition method per full scan . Measure


Eucheuma clade E, Kappaphycus clade A and B, Gracilaria salicornia, and Acanthophora spicifera) are established within Kāne‘ohe Bay , and reducing invasive macroalgae cover is a coral reef conservation and management priority. Invasive macroalgae control techniques, however, are limited and few successful large-scale applications exist. Therefore, a two-tiered invasive macroalgae control approach was designed, where first, divers manually remove invasive macroalgae (Eucheuma and Kappaphycus) aided by an underwater vacuum system (“The Super Sucker”). Second, hatchery-raised juvenile sea urchins (Tripneustes gratilla), were outplanted to graze and control invasive macroalgae regrowth. To test the effectiveness of this approach in a natural reef ecosystem, four discrete patch reefs with high invasive macroalgae cover (15–26%) were selected, and macroalgae removal plus urchin biocontrol , or no treatment , was applied at the patch reef-scale. In applying the invasive macroalgae treatment, the control effort manually removed ∼19,000 kg of invasive macroalgae and ∼99,000 juvenile sea urchins were outplanted across to two patch reefs, totaling ∼24,000 m2 of reef area. Changes in benthic cover were monitored over 2 years (five sampling periods) before-and-after the treatment was applied. Over the study period, removal and biocontrol reduced invasive macroalgae cover by 85% at treatment reefs. Our results show manual removal in combination with hatchery raised urchin biocontrol to be an effective management approach in controlling invasive macroalgae at reef-wide spatial scales and temporal scales of months to years.Invasive macroalgae pose a serious threat to coral reef biodiversity by monopolizing reef habitats, competing with native species, and directly overgrowing, and smothering reef corals. Several invasive macroalgae ( Caulerpa spp., Eucheuma spp., Gracilaria spp., and Kappaphycus spp.  . Macroalin situ desiccation  is a generalist herbivore native to Hawai‘i and will feed on at least five species of invasive macroalgae . Finally, T. gratilla achieves its maximum growth rate within the first two-years of life, and test size can reach 5.6–8.3 cm while grazing on invasive macroalgae species  F.S. Collins & Hervey, and Kappaphycus clade A and clade B (Doty) Doty ex P.C. Silva (Acanthophora spicifera (Vahl) Børgesen, and Gracilaria salicornia (C. Agardh) E.Y. Dawson. The introduction of these macroalgae to Hawai‘i in the mid-20th century occurred through a variety of pathways including ship biofouling, ballast water discharge, and mariculture experimentation and production  at the Hawai‘i Institute of Marine Biology (HIMB)  for experimentation in the 1970’s . Prior to this analysis, nomenclature for these species has been inconsistent; therefore, we will refer to this group collectively as E/K hereafter unless referring specifically to species. E/K was left unchecked in Kāne‘ohe Bay for over two decades, and by 1996, E/K had spread >5 km from Moku o Lo‘e Island and were found throughout Kāne‘ohe Bay  and increasing sedimentation surrounding reef corals , G. salicornia, A. spicifera) at a reef-wide scale over 2 years using a Before After Control Impact (BACI) experimental design. We hypothesized that our proposed invasive macroalgae removal and control methods would be effective at maintaining low invasive macroalgae abundance (percent cover) over time at treatment reefs relative to untreated-control reefs. While, a factorial design testing each treatment type separately  might be preferred, this, was not possible due to logistic and financial challenges associated with implementing and replicating three separate treatment types at the reef-wide scale. However, previous findings of E/K mats. In addition, applying urchins to a large standing crop of macroalgae would increase the amount of urchins, grazing time, and ultimate cost required to successfully treat a reef. Therefore, our goal was to use a single, most-effective treatment type  and test whether this treatment was effective at reducing invasive algae cover long term among replicate patch reefs.The overarching goal of the project was the rehabilitation and preservation of coral reef habitat and associated biodiversity with specific management objectives to: (i) reduce invasive macroalgae on Kāne‘ohe Bay patch reefs, and (ii) stop the spread of E/K and sea urchin biocontrol were applied, and two patch reefs (Reef 16 and 28) were designated as control reefs where no macroalgae manual removal or biocontrol were applied (2 (treatment Reef 26), 12,700 m2 (treatment Reef 27), 3,100 m2 (control Reef 16), and 14,500 m2 (control Reef 28). Each patch reef has a distinct reef slope composed primarily of live coral and a shallower reef flat consisting of a mix of live coral, dead coral, rubble, and sand. E/K occurred on reef slopes and reef flats and ranged in size from single low growing thalli to dense mats 1 m2 in area and ∼0.3 m thick  patch reefs located in central Kāne‘ohe Bay, on the windward side of O‘ahu, Hawai‘i , which is the largest embayment in the Hawaiian Islands and contains over 70 distinct patch reefs surrounded by a barrier reef and fringing reef system . The pat applied . Study r m thick –2B. G. s m thick –2D.E/K were manually removed from reefs by divers aided by an underwater vacuum system (“The Super Sucker”) that transported macroalgae from the reef to a support vessel , small clumps (<400 cm3) and holdfasts to maximize the yield to effort ratio and minimize disturbance to other benthic organisms and habitats. Invasive macroalgae species G. salicornia and A. spicifera were not directly targeted by divers for manual removal.Invasive macroalgae were controlled in two phases. First, t vessel  (Conklint vessel . To a let vessel . Divers T. gratilla were collected from the wild and spawned at an urchin hatchery. Urchin larvae were settled and reared in tanks on land until they reached approximately 2.5 cm diameter test size (∼4–6 months after spawning). A new cohort was produced every 30–60 days throughout the duration of the study. Following E/K manual removal, juvenile urchins were transported to the reef in trays and manually deployed on the treatment reefs where G. salicornia, A. spicifera, and E/K occurred  were outplanted from December 2011 to October 2012, with supplemental outplanting from July to December 2013 (19%) one additional outplanting in July of 2014   . On treactively) , primari Reef 27 .Control costs were calculated for field operations  and sea urchin hatchery operations. Cost estimates included salaries and operating expenses . The total invasive macroalgae control cost of the project was divided by the total reef area treated to estimate cost per square meter.Eucheuma, Kappaphycus clade B, G. salicornia, A. spicifera), native macroalgae, coral, crustose coralline algae (CCA), and the combined sand/rubble, bare space, turf  (SBT) at treatment and control reefs.Baseline benthic surveys were performed at all patch reefs from November 2011 to February 2012  prior to macroalgal removal and urchin outplanting, representing the “before” period of the analysis. Subsequently, benthic surveys were repeated during the treatment period at four additional times during summer and winter seasons from 2012–2014, representing the “after” periods of the analysis. Sampling periods were defined as: May–June 2012 , December 2012–February 2013 , May–June 2013 , and February 2014 . Using these five time points we analyzed changes in percent cover of invasive macroalgae , 18 (control Reef 28), 13 (treatment Reef 26), and 14 (treatment Reef 27). The number of transects were allocated in proportion to the total reef area first, then by primary reef habitats (aggregate and non-aggregate), then by non-aggregate sub-strata (mixed/unconsolidated reef and pavement/consolidated reef). Mean benthic cover was estimated using a point intercept transect method (n = 126 points transect−1). T. gratilla were surveyed at each transect location, counting all observed individuals within a 25 × 1 m belt. A correction factor of 90% detectability  was used to estimate the density of urchins from transect counts.Fixed transect locations were randomly selected initially using ArcGIS random point tool  within tt method . Surveyolme4 package  and time (before treatment applied vs. periods after treatment applied) were included as fixed effects. To account for spatial structure of the benthos habitat types within patch reefs  habitat was designated as a random effect nested within individual reefs. Reef transects were included as a repeated-measure random effect. Considering that surveys conducted over the two-year study period spanning different months and seasons, we first tested ‘season’  separately as a fixed effect in a linear model; no effects were observed (p ≥ 0.408) and season was not included in the final analysis. Normality of residuals and homogeneity of variance was verified using graphical inspection of standardized residuals, and transformations were applied where assumptions of ANOVA were not met. An arcsine transformation was used for invasive algae and abiotic cover and a square root transformation was used for CCA and native macroalgae. Analysis of variance tables were generated using type-II sum of squares with Satterthwaite approximations of degrees of freedom using the package lmerTest (lsmeans (10.5281/zenodo.1285551).Response variables  were monitored over time, with baseline surveys at each patch reef (Winter 2011) designated as the “before” period and four subsequent surveys  designated as “after” periods. Treatment application  was partial in Summer 2012 and complete by Winter 2012 . Changes package  in R veron 3.3.0 . TreatmelmerTest . Where s at treatment and control reefs at the start of the study (Dictyosphaeria versusii (74 %) and Dictyosphaeria cavernosa (19%). Invasive macroalgae on control reefs was predominantly G. salicornia (11%), and Eucheuma (7%), whereas invasive macroalgae cover at treatment reefs had similar cover of G. salicornia, A. spicifera, and Eucheuma (5–7%)  on all study reefs  (he study –4E. In Wa (5–7%) –5B. Kappdy reefs –5B. T. g−2) of E/K from different areas of treatment Reef 26 and 7,095 kg wet weight (0.622 ± 0.05 kg m−2) from treatment Reef 27  removal effort was greater for Reef 26 (2.36 ± 0.27 m2 min−1) compared to treatment Reef 27 (1.23 ± 0.10 m2 min−1) as well as the E/K biomass removed 0.81 ± 0.14 kg m−2(Reef 26) versus 0.62 ± 0.05 kg m−2 (Reef 27). While stocking density of juvenile urchins was designed to be ∼4 urchins m−2 (−2 (treatment Reef 26) and 0.74 urchins m−2 (treatment Reef 27). No presence of T. gratilla was reported in benthic surveys on control Reefs 16 and 28 in post treatment surveys.Divers removed a total of 11,963 kg wet weight , approximately 6 months after the treatment had been applied (posthoc: p = 0.029). By Winter 2012 total invasive macroalgae cover had declined by 29% relative to Winter 2011 levels. The mean invasive macroalgae cover at control reefs fluctuated between 14–25% over the entire study period (Winter 2011 to Winter 2013) (posthoc: p ≥ 0.080) except Winter 2013 where invasive algae declined relative to start of the study (posthoc: p = 0.005). G. salicornia and Eucheuma consistently dominated the invasive macroalgae community at control reefs  , 6 and col reefs , represep < 0.001) but not in response to treatments (p = 0.906) (p < 0.001) and CCA cover (p = 0.037), and both coral and CCA increased over the study period (p < 0.001). However, mean coral and CCA cover did not differ among control and treatment reefs at each discrete time point (posthoc: p ≥ 0.286). SBT (sand/bare/turf) was not affected by time, treatment, or their interaction (p ≥ 0.255)   Fig. 4Bp < 0.001≥ 0.255) , but ten≥ 0.255) .2 ($33 m−2) of affected reef.Field components of macroalgae removal and control operations cost an estimated $255,000 and roughly 3,000 human hours of work. Hatchery operations cost $562,000, and accounted for approximately 19,000 human hours to run the hatchery facility, which required daily oversight. The total project cost $817,000 to treat 24,600 kmT. gratilla) for biocontrol, can be an effective approach for reducing the benthic cover of invasive macroalgae at a reef-wide scale. Invasive macroalgae was reduced by 85%, two-years after macroalgae removal and sea urchin biocontrol was applied—a result consistent with a small-scale experiment that employed a similar control technique over a shorter time period  and was also an efficient means of moving thousands of kilograms of macroalgae from the reef to the support vessel at a mean removal rate of 1.48 ± 0.14 m2 min−1. In addition, the vacuum system captured loose macroalgae fragments created by dislodging the macroalgae, reducing the risk of unintentional dispersal. Following manual removal, invasive macroalgae continued to decline by 61% from Winter 2012 to Winter 2013  offer greater replication, but results may not necessarily be extrapolated to larger scales. In regards to this study, treatments were not fully crossed and replication was low, however, our method are promising, especially in demonstrating the potential for a native, mobile invertebrate as an effective biontrol agent. Despite shortcomings, our results show a clear and lasting results of reduced invasive macroalgae on treatment reefs, indicating our approach was successful and effective in controlling invasive macroalgae over 24,600 kmT. gratilla, are well suited for mariculture and outplanting for the biocontrol of invasive macroalgae. T. gratilla are able to be propagated in a hatchery using wild stock, producing large numbers of juvenile urchins   without impacting wild T. gratilla populations. The small size (∼2.5 cm test diameter) of outplanted T. gratilla may also be an important factor in treating invasive macroalgae. T. gratilla (∼4 cm test diameter) within in situ enclosures. Thus, small test-size urchins appear more capable of grazing holdfasts within the small interstitial spaces of the reef. As juvenile urchins mature, they continue to contribute to invasive macroalgae biocontrol  also declined over the study period  invasive macroalgae. In feeding trials, T. gratilla consumed all four species of invasive macroalgae found in this study, but given the choice, urchins preferred A. spicifera, especially among smaller test-size urchins . However, since urchin movement was naturally confined by 10–15 m deep sandy habitats surrounding patch reefs in Kāne‘ohe Bay, this raises the question as to whether T. gratilla would be as effective in treating larger continuous reefs. T. gratilla are present in high densities (>4 m−2) at Lord Howe Island. T. gratilla movement to be <1 m d−1 and suggested that this low vagility may explain its generalist diet of a wide range of macroalgae species including non-natives. The low movement rates have allowed Hawai‘i managers to utilize T. gratilla in spot-treating areas with high invasive macroalgae biomass and apply a manipulated urchin density in problematic locales. Therfore, T. gratilla shows promise as a macroalgae biocontrol agent, but assessing its function in different reef systems should be a priority for future research.This study demonstrates that T. gratilla population growth. However, we believe this risk is unlikely due to a number of factors, including T. gratilla stocking densities were similar to those observed in natural populations on Hawaiian reefs .It is reasonable to acknowledge the potential risk of urchin stocking in Kāne‘ohe Bay to facilitate rapid an reefs . In addiG. salicornia was the second most prevalent macroalgae species in gut contents of herbivorous reef fishes in Kāne‘ohe Bay. While E/K and A. spicifera were also identified in fishes gut contents, these species were far less prevalent  was lower than −2, it still may be advisable to reduce urchin densities once urchins have grazed invasive macroalgae to <2% cover in order to limit potential negative effects on native macroalgae colonization and growth. grazers , however grazers , newly s grazers . CCA are grazers , in addi grazers . HoweverE/K was carefully hand removed from the reef and fed into the vacuum system, the process does cause low levels of disturbance to the reef including abrasion to coral tissue, dislodgment of benthic organisms and habitat, and the potential for bycatch of small or cryptic organisms associated with macroalgae. It is therefore advised that macroalgae manual removal be performed once or infrequently to reduce the potential for environmental disturbance.The observed decrease in invasive macroalgae on control reefs over the course of the study did not result in a significant increase in any single benthic cover type as a result of the treatment . HoweverManual removal of invasive macroalgae in combination with sea urchin outplanting proved to be a successful approach for invasive macroalgae mitigation in Hawai‘i. However, the substantial costs and labor requirements, as well as the necessity of a native herbivore amenable to culturing and outplanting, may limit the broad application of this approach in other reefs or at broader scales than those tested here. Therefore, the effectiveness of this approach on other reef systems required appropriate testing at small experimental scales before reef-wide treatments are applied , in addi2 ($33 m−2) of affected reef. This figure not only demonstrates the need to invest in invasive species prevention through strict vector management and importation rules, but also indicates the importance of Hawai‘i’s reefs in order to justify such a large expense. 2 of treated reef can be reduced by further advances in sea urchin aquaculture.The control of invasive macroalgae for this study was a substantial investment by managers at a cost of $817,000 to treat 24,600 kmOur findings show that manual removal and sea urchin biocontrol applied at a reef-wide scale is an effective approach for controlling invasive macroalgae, but should not be viewed as a replacement for managing some of the other drivers of macroalgae phase shifts, including increased nutrients , and red10.7717/peerj.5332/supp-1Data S1Click here for additional data file.10.7717/peerj.5332/supp-2Supplemental Information 1Click here for additional data file.10.7717/peerj.5332/supp-3Supplemental Information 2Click here for additional data file.


It is widely believed that tertiary nucleotide-nucleotide interactions are essential in determining RNA structure and function. Currently, direct coupling analysis (DCA) infers nucleotide contacts in a sequence from its homologous sequence alignment across different species. DCA and similar approaches that use sequence information alone typically yield a low accuracy, especially when the available homologous sequences are limited. Therefore, new methods for RNA structural contact inference are desirable because even a single correctly predicted tertiary contact can potentially make the difference between a correct and incorrectly predicted structure. Here we present a new method DIRECT (Direct Information REweighted by Contact Templates) that incorporates a Restricted Boltzmann Machine (RBM) to augment the information on sequence co-variations with structural features in contact inference.Benchmark tests demonstrate that DIRECT achieves better overall performance than DCA approaches. Compared to mfDCA and plmDCA, DIRECT produces a substantial increase of 41 and 18%, respectively, in accuracy on average for contact prediction. DIRECT improves predictions for long-range contacts and captures more tertiary structural features.We developed a hybrid approach that incorporates a Restricted Boltzmann Machine (RBM) to augment the information on sequence co-variations with structural templates in contact inference. Our results demonstrate that DIRECT is able to improve the RNA contact prediction. RNA molecules play critical roles in various biological processes –8. ThereOne can exploit what is known about nucleotide-nucleotide interactions from experimental studies to heuristically provide data about the distances involved in such interactions. One of the most successful methods for contact prediction, based on this approach, is direct coupling analysis (DCA). DCA infers the interacting nucleotides in a sequence from the sequence coevolution across different species –33. A reAn alternative to contact prediction from sequence co-variations is to incorporate structural information as well. Skwark et al. applied a pattern-recognition approach to the contact prediction of a residue pair by examining the expected pattern of nearby contacts surrounding the pair . SpecifiHowever, it is more important and difficult to pinpoint the RNA interactions in loop-loop and junction regions than to identify its secondary structure of base-pair interactions. Existing methods on proteins only consider local structural patterns modeled as statistical potential. This approach ignores global structural features that might be useful in improving the RNA prediction accuracy. Therefore, we introduce a new method that first learns a lookup table of contact weights by a Restricted Boltzmann Machine (RBM) from non-redundant and known RNA structures. Then, this lookup table is used to improve RNA contact prediction obtained from sequence co-evolution by DCA. We call our method Direct Information REweighted by Contact Templates (DIRECT). In a benchmark testing on riboswitch, DIRECT outperforms the state-of-the-art DCA predictions for long-range contacts and loop-loop contacts. Moreover, DIRECT maintains better predictions when the number of available sequences is limited. Here, we examined the accuracy of contact prediction for the 5 RNAs using only 50 randomly chosen homologous sequences that represent about 11 to 43% of all available sequences for the 5 RNAs.Traditional direct coupling analysis (DCA) for RNA contact prediction has some drawbacks. For one, DCA requires a sufficient number of homologous sequences for accurate sequence co-evolution analysis, which may not be readily available. Moreover, a co-evolving pair of nucleotides can interact within the same molecule (intra-molecule) or across the homodimer interface (inter-molecule) of the RNA. In addition, several unknown factors, other than intra- or inter-molecular interactions, can result in co-evolving pairs and make it difficult to detect the true contacts among the evolving pairs without additional information. One way to overcome this difficulty is to augment the contact detection of a target RNA sequence with additional information on the structural contact template expected of the RNA class to which the target RNA belongs. To this end, we employ a Restricted Boltzmann Machine to learn the contact template of RNAs by using the structures and then improve the contact predictions  and medium-range (13~24 nt) contacts, but less accurately for long-range (24 nt+) contacts. DIRECT, however, utilizes the structural contact template to re-rank DCA predictions and is able to improve the long-range contact prediction  on a given riboswitch to build a number of tertiary structures and evaluated the percentage of top contacts by DIRECT that were actually retained as the structure deviates from the native one. The results of riboswitch 1Y26 are shown in Fig. We then used another three non-redundant RNA training sets to evaluate the prediction accuracy of DIRECT. The non-redundant RNA training set 2 contains all the representative high-quality structures of 147 classes with length from 50 nt to 120 nt. The homology sequence or structure between training and testing sets were removed to ensure that RNAs in the training and testing sets have no sequence and structural overlap. As shown in Additional file Another two non-redundant RNA training sets are as follows: (1) non-redundant RNAs with length from 50 nt to 120 nt without any riboswitch structure (training set 3 in Methods Section), and (2) all non-redundant RNAs without any riboswitch structure (training set 4 in Method Section). The results showed an average accuracy increase of 15 and 4% compared to mfDCA and plmDCA using training set 3, and 7 and 11% using the training set 4. The predictions clearly indicate that there are indeed generic and useful RNA structural features discerned by DIRECT that can improve contact prediction for a specific class of RNA.The hypothesis of direct coupling analysis stipulates that co-evolving nucleotides in an RNA molecule may form intra-molecular contacts to support its structure and function. DCA thus aims to disentangle the direct pairwise couplings from indirect correlations of the sequence variations. While highly conserved contacts are critical for RNA structural stability and function, their detection by DCA may be difficult due to insufficient information on variations. To examine if DIRECT can improve the prediction in this case, we divided nucleotides into different types based on their conservation scores calculated by the ConSurf program . The conPrevious research suggests the number of sequences should be more than three times the length of the molecule for reliable contact prediction . HoweverTo investigate the predictive accuracy on different structural templates, we incorporated a Restricted Boltzmann Machine (RBM) to augment the information on sequence co-variations with four different training sets in contact inference. The contacts learned by Restricted Boltzmann Machine fall mainly into two categories  to augment the information on sequence co-variations with structural templates in contact inference. Our results demonstrated a 41 and 18% precision increase for RNA contact prediction in comparison to the mfDCA and plmDCA when structural templates are utilized. In fact, our approach establishes a straightforward framework that can incorporate any additional information, such as NMR spectroscopy data, by training a corresponding Restrictive Boltzmann Machine to further improve the prediction on RNA contacts.DIRECT (Direct Information REweighted by Contact Templates) improves the prediction of tertiary contacts by using both sequence and structure information. Figure The Restricted Boltzmann Machine (RBM) is a graphical model for unsupervised learning that can extract features from the input data . RBM hasT above is a set of samples from the empirical data. By minimizing the loss function, we can update the parameters W, b, c according to the equations below:Two nucleotides are considered in contact if they contain a pair of heavy atoms, one from each nucleotide, less than a pre-defined cutoff –51. PrevRiboswitch is a regulatory portion of a messenger RNA. When binding with a small ligand, this regulatory segment will regulate the translation of the entire mRNA. In this study, we constructed four different training sets ranging from containing no homologous riboswitch with similar RNA size to strictly no riboswitch with all RNA sizes. This is to ensure that there is no data leakage between the training set and testing set so that the features captured by DIRECT are generic and useful RNA template patterns. Details of the datasets are as follows.http://www.genome.jp/tools-bin/clustalw) and structural similarity via RMSD value  between each pair of RNAs in the training set  RNA non-redundant training set 1. To generate a representative set of riboswitch families for our study, we systematically selected riboswitch families from the Rfam database. The ten representative riboswitches in the training set are shown in Additional file  RNA non-(2) RNA non-redundant training set 2. We collected all the representative high-quality structures with resolution ≤3.0 Å of 147 classes of RNA 3D Hub non-redundant RNA set (version 3.21) with length from 50 nt to 120 nt . RNA 3D (3) RNA non-redundant training set 3. We collected all the representative high-quality structures in RNA non-redundant training set 2, then removed all riboswitch structures in this training set. These steps ensure no riboswitch structure in the training set.(4) RNA non-redundant training set 4. Unlike RNA non-redundant training set 3 that collected similar size RNAs as a testing set (from 50 nt to 120 nt), we collected all the representative high-quality tertiary structures with resolution ≤3.0 Å of 1023 classes of RNA 3D Hub non-redundant RNA set (version 3.21). Then, we also removed all the riboswitch structures in this training set. These steps ensure no riboswitch structure in the training set. The lists of RNA non-redundant training set 3 and 4 can be downloaded from our website.For the testing set, we used the published testing dataset including six riboswitches  is used to extract the contact knowledge from riboswitch structures in the training set Fig. .Fig. 5FStep 1: Prepare the training set Fig. a and b. Step 2: Learning the shared weights Fig. c. TrainiStep 3: Gibbs sampling Fig. d. After Step 4: Contact weight learned by RBM Fig. e. We couThe direct coupling analysis (DCA) is performed to infer the interacting nucleotides from sequence coevolution across different species , 35, 56.http://dca.rice.edu/portal/dca/download. The plmDCA was downloaded from the Marks lab at Harvard Medical School (http://evfold.org/evfold-web/code.do).coupling , 57. We The final contact prediction is DI scores reweighted by structural information learned by RBM with better contact prediction accuracy.We predicted RNA tertiary structures using 3dRNA, RNAcomposer, simRNA and Vfold3D , 17, 58.Regularization is a strategy that aims to reduce the generalization errors . Most maAdditional file 1. Supplementary material, including all supplementary figures and supplementary tables.


The interaction between non-coding RNAs (ncRNAs) and proteins is crucial for the stability, localization and function of the different classes of ncRNAs. Although ncRNAs, when embedded in various ribonucleoprotein (RNP) complexes, control the fundamental processes of gene expression, their biological functions and mechanisms of action are still largely unexplored. Mass Spectrometry (MS)-based proteomics has emerged as powerful tool to study the ncRNA world: on the one hand, by identifying the proteins interacting with distinct ncRNAs; on the other hand, by measuring the impact of ncRNAs on global protein levels. Here, we will first provide a concise overview on the basic principles of MS-based proteomics for systematic protein identification and quantification; then, we will recapitulate the main approaches that have been implemented for the screening of ncRNA interactors and the dissection of ncRNA-protein complex composition. Finally, we will describe examples of various proteomics strategies developed to characterize the effect of ncRNAs on gene expression, with a focus on the systematic identification of microRNA (miRNA) targets. Non-coding RNAs (ncRNAs) are generally defined as transcribed, but not translated RNAs. With the exception of ribosomal RNAs (rRNAs) and transfer RNAs (tRNAs)-whose role and function have long been known- the majority of ncRNAs were considered as mere transcriptional noise until their role as key-modulators of gene expression began to be unraveled. At present, it is generally accepted that ncRNAs are central players in many biological processes, such as cell proliferation, apoptosis, differentiation and development  the identification and characterization of ncRNA interactomes; (ii) the dissection of protein complexes involved in ncRNA biogenesis and function; and (iii) the measurement of the impact of ncRNAs on global gene expression Figure .2), coupled to high performance liquid-chromatography (LC). Shotgun MS-proteomics typically is carried out using a “bottom-up” MS-approach, whereby a complex protein mixture, e.g., a whole cell extract, is first digested into peptides by a specific protease. The most frequently used enzyme is Trypsin, thanks to its selectivity in cleaving the C-terminal peptide bond of lysines and arginines and its efficiency both in-solution and in-gel conditions ; the most common are: SEQUEST  and stable isotope-labeling approaches, summarized in Table LFQ strategies consist in the quantification of proteins using either intensity-based or spectral counting approaches. Intensity-based approaches use the extracted ion chromatogram (XIC) corresponding to the m/z ratios of each peptide. Under the assumption that the XIC value linearly correlates with the peptide abundance, this value can be employed to compare the quantity of the same peptide in different samples . Nevertheless, these approaches require very high technical and experimental reproducibility and numerous biological replicates to reach confident protein quantification. LFQ approaches are reviewed in Bantscheff et al. , Lai et In isotope-labeling strategies, proteins or peptides are labeled with stable (heavy) isotopes of various elements that make them chemo-physical identical to their natural (light) counterparts, except for a specific difference in their nominal mass. Therefore, when heavy and light proteomes are mixed, each peptide is detected in the mass spectrum as a peptide-pair, whereby the two peaks are virtually identical- except for a specific mass difference (delta mass) distinguishing them- and the ratio of the intensities of the heavy and light peptides is directly proportional to the respective abundances in the samples of origin. Hence, a relative quantification is extrapolated within the same spectrum, circumventing run-to-run variability.in vitro, using a tag added covalently to the reactive side chains of amino acids, through a chemical reaction, either before or after the proteolytic cleavage. This is suitable for profiling biological samples not amenable to in vivo labeling, or when multiple (>3) samples must be compared. Most common methods for chemical labeling are: Isotope-Coded Affinity Tagging (ICAT)     or light  versions of lysine and arginine for a number of passages that enable their full incorporation into the proteomes  are used as baits to characterize the respective RNA interactors by RNA-sequencing analysis. Given the focus of this review on proteomic methods in ncRNA research, we will focus only on RNA-centric strategies.In the last two decades, various strategies have been developed to comprehensively analyze ribonucleoprotein (RNP) complexes. They can be grouped based on whether the RNAs or the proteins are used as baits for RNP complex enrichment and dissection: in the in vitro RNA pull-down coupled to MS-based proteomics has been the most successful approach to identify RBPs regulating various RNAs. Improvements of this method led to the development of in vivo RNA immuno-purification assays, which enabled the purification of native RNPs from living cells. In the last few years, more global approaches to study RBPs have shifted the focus from the analysis of single RNAs to the comprehensive investigation of global cellular RNA-interactomes  is the most common tag used in RNA pull-down experiments. It can either be incorporated during in vivo and in vitro experiments. Some aptamers are naturally-occurring RNA stem-loop sequences , while others are derived from library screenings . The MS2 and PP7 aptamers are based on the bacteriophage system: they bind with high specificity to the Escherichia coli bacteriophage coat protein MS2 (MS2cp) and Pseudomonas aeruginosa bacteriophage coat protein PP7, respectively , was developed by Hogg and colleagues for the characterization of proteins associated to the 7SK RNA  method that employs a MS2-based fluorescent reporter (MS2-CP-GFP) fused with the streptavidin-binding protein (SBP) tag, thus allowing both the visualization of the mRNAs bearing the MS2 aptamer and the purification of the protein interactors of the MS2-tagged RNAs by streptavidin beads , whereby the MS2-tagged RNAs and the chimeric MS2-glutathione-S-transferase (MS2-GST) protein are co-expressed in mammalian cells, thus permitting the purification of native RNP complexes formed on the MS2-tagged RNA with a glutathione-sepharose resin , to enable the large-scale identification of lncRNA-bound proteins in vivo. The workflow of both methods is the same: cells are first cross-linked in vivo, chromatin is extracted and sheared by sonication, and then biotinylated oligonucleotides complementary to the complete lncRNA sequence are added and let hybridize with the lncRNA. The hybrids, which comprise the target lncRNA, the associated cross-linked proteins and the chromatin, are purified using streptavidin beads. While in ChIRP-Seq the aim is to capture and sequence the genomic DNA binding to the lncRNA of interest, in ChIRP-MS the biotin-elution is followed by de-cross-linking, protein extraction and MS-analysis. This method was successfully applied to comprehensively identifying the Xist- interactome , a derivate of CHART-Seq  method -Assisted Identification of RBPs), which was successfully applied to study proteins associated to ank, a pan-neuronal dendritic mRNA  the expression level and folding of the RNA used as bait; (2) the cellular localization of the RNA; (3) the type of cell-lysis protocol employed; (4) the RNA-to-RBP stoichiometry; and (5) the stability of the RNP complex under investigation. These experimental issues are reviewed in more detail in Oeffinger .In vitro RNA affinity-purification approaches are generally the first experimental choice because they are relatively fast and manageable and require overall small amount of starting material. However, in vitro purifications usually suffer from a large excess of bait-RNA that affects the physiological RNA-to-RBP stoichiometry. In addition, in vitro synthesized RNA might not fold properly and the protein extract used for the purification is not restricted to the cellular compartment where the endogenous RNA resides. All together, these aspects might favor the formation of artificial and unspecific protein-RNA interactions  the possibility of rescuing the cleavage activity of Csy4 H29A by imidazole. The peculiar conditional activity of Csy4 H29A, together with the high selectivity and affinity of the enzyme for its substrate, permits a very selective affinity-purification of RNP complexes. Typically, pre-miRNAs are tagged with 16-nt hairpin sequences at their 5′-end. Upon incubation with the cellular extract, the complex comprising the Csy4 hairpin tagged-pre-miRNA and the co-associated proteins is captured by biotinylated-Csy4 H29A, immobilized on avidin resin; upon stringent washing steps, the RNPs are eluted by imidazole. The selective transcript isolation with imidazole ensures very low background contamination which allows omitting a SDS-page purification step prior to MS, thus favoring the detection of low-abundant RBPs.Various RNA-centric methods have been adopted for the characterization of RBPs involved in miRNA biogenesis. For instance, a method exploiting the endoribonuclease Csy4, a part of the CRISPR system in in vitro synthetized pre-mRNA bearing the same 5′ extension, complementary to biotinylated 2′-O-methyloligonucleotides, to uncover 180 proteins that show preferential or selective binding to single, or small sets of precursor-miRNAs . These auxiliary proteins are required to modulate the ribonuclease activity of the core dimer  was characterized with a similar strategy, in the same period  of the transcript. One mRNA may have different variants bearing 3′ UTRs regions of different length. Any variation in the 3′ UTR length of a transcript can affect its regulation and stability as a consequence of the changes in the repertoire of regulatory elements present within the region, including regulatory RBPs and binding sites for miRNAs  strategy, which was developed by M. Selbach in 2008 and represents a technological milestone for experimental miRNA target identification by MS  method, coupled with MS-proteomics analysis  from Salmonella Typhimurium  has gained much attention. It has been suggested that various PTMs, such as phosphorylation, methylation, glycosylation, acetylation, NEDDylation and ubiquitination, modulate both protein-protein and protein-RNA interactions within RNPs, thus influencing both the processing, stability, turnover and translation of mRNAs (Will and Luhrmann, MS-based modification-proteomics will play a pivotal role in the future to dissect the extent and regulatory impact of arginine methylation, as well as other PTMs, in defining the biogenesis, stability, location and function of RBPs.RG and MM wrote the draft. TB proposed the topic and revised the text.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.


The distribution of amyloid beta peptide 42 (Aβ42) between model exosomal membranes and a buffer solution was measured. The model membranes contained liquid-ordered regions or phosphatidylserine. Results demonstrated that up to ca. 20% of amyloid peptide, generated in the plasma (or intracellular) membrane as a result of proteolytic cleavage of amyloid precursor proteins by β- and γ-secretases, can stay within the membrane milieu. The selection of RNA aptamers that bind to Aβ42 incorporated into phosphatidylserine-containing liposomal membranes was performed using the selection-amplification (SELEX) method. After eight selection cycles, the pool of RNA aptamers was isolated and its binding to Aβ42-containing membranes was demonstrated using the gel filtration method. Since membranes can act as a catalytic surface for Aβ42 aggregation, these RNA aptamers may inhibit the formation of toxic amyloid aggregates that can permeabilize cellular membranes or disrupt membrane receptors. Strategies are proposed for using functional exosomes, loaded with RNA aptamers specific to membrane Aβ42, to reduce the oxidative stress in Alzheimer’s disease and Down’s syndrome. Oxidative stress occurs early in the course of Alzheimer’s disease (AD), which would support its key role in AD pathogenesis, in relation with the presence of amyloid peptide (Aβ) . Elevate2+ leakage, and a disruption of ionic homeostasis. It is also possible that a fraction of amyloid peptides do not fully penetrate the membrane, but rather associate with membrane surfaces, where they induce membrane thinning and leakage [The toxicity of amyloid peptides is correlated with their interactions with cell membranes , and witMolecules blocking Aβ channel formation and other forms of membrane disruption have the potential to alleviate Aβ-induced cytotoxicity . CurrentThe new concept, based on our studies of membrane RNAs ,10,11,122) and ca. 1250 Aβ42 molecules. After liposome preparation, a pool of the Aβ42 molecules dissociates from the liposomal membrane and equilibrium was reached in the distribution of Aβ42 between liposomes and the buffer. In order to observe the distribution of Aβ42 and RNA between liposomes and the buffer, liposomes were modified with a blue fluorescent dye (Pacific Blue-PE), incorporated into the liposomal membrane through the phospholipid PE (phosphatidylethanolamine). Amyloid peptide Aβ42 was modified with a fluorescein group attached to the N-terminus. In cells, the N-terminus is located outside the membrane during the cleavage of the amyloid precursor proteins by two proteolytic enzymes (β- and γ-secretases), while the C-terminus is at the end of hydrophobic residues located inside the membrane [32P-GTP.We prepared several different pools of liposomes (as model membranes), made of various lipid compositions, in order to evaluate the distribution of amyloid peptide Aβ42 and RNA between liposomal membranes and buffer solution. Amyloid-liposomes were prepared from a mixture of amyloid peptide and lipids in organic solvents dried under a vacuum. The mole ration of Aβ42 to lipid was 2:100. This means that initially, each 100-nm liposome consisted of ca. 63,000 lipid molecules (assuming the mean surface area of lipid molecule ca. 1 nm32P-GTP within the RNA chain).There is no binding of RNA 50N to the liposomal membranes, since no RNA peak associated with liposomes was observed . HoweverWe performed the selection-amplification procedure in order to select a pool of RNA aptamers specific to amyloid peptide embedded in lipid bilayers .Before cycle 1, there was no binding of the pool of RNA 50N to the liposomal membranes containing Aβ42 A. After 32P-radioactivity measurements for RNA, and fluorescence intensities for both liposomes and Aβ42  between liposomal membranes and buffer. We quantified all the components using and Aβ42 . To bettWe employed gel filtration that separates voided liposomes from included Aβ42. In our study, we found that the affinity of Aβ42 is higher to PS-containing liposomes in comparison with rafted liposomes. In contrast, the random pool of RNA sequences (RNA 50N) had very low affinity both to rafted and PS-containing liposomes. The obtained data demonstrate that up to ca. 20% (depending on the percentage of anionic lipids in the membrane) of amyloid peptide, generated in the plasma (or intracellular) membrane by proteolytic cleavage of amyloid precursor proteins (APP) by β- and γ-secretases, can stay within the membrane, while over 80% of the peptide is released into the extracellular solution (or into cytoplasm). These results are consistent with the finding of Bokvist et al.  that aciBesides the membrane Aβ peptide generated by proteolytic cleavage, soluble Aβ oligomers in the brain specifically bind neuron plasma membrane, evoking neurotoxicity and synapse deterioration . The proAbove, we have measured that up to 18% of Aβ42 peptide can stay in the lipid bilayer after liposome formation. Assuming there are ca. 30,000 lipid molecules on the liposomal surface (per one 100-nm-liposome), there is initially 2 mol% = ca 600 Aβ42 molecules within one liposomal membrane, and ca. 300 Aβ42 in the external leaflet per liposome. We used DOPS:DOPC liposomes, mole ratio 3:10, in the selection-amplification procedure, and we found that ca. 16.5% of Aβ42 stay within the liposomal membrane. Therefore, we can estimate that during the selection, there are ca. 50 Aβ42 molecules within the outer layer of one liposome as a target for RNAs aptamers.In order to show that a pool of RNA 50N binds membrane Aβ42, we used the selection-amplification (SELEX) method and gel filtration technique on a column that voids liposomes but includes RNAs of this size. RNA aptamers are single-stranded oligonucleotides usually composed of ca. 20 to 100 nucleotides . Their uD values remain questionable, and they were not tested for their cross-reactivity with fibrillary assemblies of Aβ or of other amyloidogenic proteins [RNA (or DNA) aptamers were previously selected only against a non-membrane Aβ40 peptide (neither Aβ42 nor membrane Aβ). These aptamers were selected against monomeric/oligomeric forms of amyloid peptide, and they showed very low affinity to amyloid peptide in these forms; however, they exhibited a strong non-specific affinity to amyloid high-molecular weight fibrils ,18. Therproteins .Below we demonstrate the potential applications of membrane RNA aptamers to amyloid beta peptide for exosome-based antioxidant strategies, using exosomes. Exosomes  are nanovesicles that originate from the endosome and are released from cells ,22. The 2+ homeostasis. The formation of Ca2+ channels in lipid bilayers was directly observed [In the diseased brain of AD patients, the level of soluble amyloids is significantly elevated. This could result in an accumulation of amyloid in the cerebrospinal fluid, insertion of amyloid peptide into membranes, and the formation of calcium-permeable amyloid channels in the cell plasma membrane. The membrane amyloid peptide can also be generated by proteolytic cleavage of APP by β- and γ-secretases. It is well accepted that neuronal death in AD is related to disturbances in Caobserved ,25.RNA aptamers specific for lipid rafts have been identified , and bifIn order to deliver RNA aptamers into the cytoplasm, the RNA molecules have to be encapsulated inside exosomes. Through endocytosis of exosomes by neural cells and the subsequent fusion of exosomal membrane with the endosomal membrane, RNA aptamers can be released into the cytoplasm and target membrane amyloid peptides located at the cytoplasmic leaflet of intracellular membranes (The intracellular amyloid peptide (iAβ) can be generated inside neural cells, or can appear in the cytoplasm as a result of a reuptake of extracellular Aβ (eAβ) , cholesterol (CHOL); N-stearoyl-d-erythro-sphingosylphosphorylcholine . H2DCF-DA  and fluorescent membrane probe Pacific Blue-PE were obtained from Invitrogen Molecular Probes . The amyloid peptides Aβ42 and Aβ42, modified with a fluorescein group attached to the N-terminus, were purchased from Biopeptide . T7 RNA polymerase (used for in vitro transcription) was obtained from Epicentre .Chemicals were analytical-reagent grade whenever available. Sephacryl S-1000 was obtained from Pharmacia. The following were purchased from Avanti Polar Lipids : 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC); 1,2-dioleoyl-sn-glycero-3-phospho-2, and 2 mM CaCl2, 3 mM KCl, 294 mOs/L), and multilamellar liposomes containing Aβ42 were formed by gentle vortex. The suspension was subjected to seven freeze–thaw cycles by repeated immersion in liquid nitrogen followed by warming in 60 °C water. LUV containing Aβ42 were prepared by extrusion using the Avanti MiniExtruder  with a filter pore diameter of 100 nm. The composition/pH/osmolarity of AM buffer was based on the ion composition/pH/osmolarity of the cerebrospinal fluid. In a procedure reported earlier [The appropriate lipids were dissolved in chloroform or chloroform/methanol (2/1), mixed with amyloid peptide solution in DMSO. Following this, solvents were evaporated under a stream of nitrogen gas, the pellet was desiccated under a vacuum for at least 2 h and resuspended in AM buffer  with LUV  was performed in AM buffer at room temperature for 5 min, followed by gel filtration on a Sephacryl S-1000 1-mL column. The eluted fractions were analyzed for 32P RNA content by scintillation counting, and for liposomes and amyloid by Pacific Blue fluorescence intensity and fluorescein fluorescence intensity, respectively, using NanoDrop spectrofluorometer .LUV containing fluorescent probe Pacific Blue-PE and amyloid peptide Aβ42, modified with a fluorescein group attached to the N-terminus, were prepared. Incubation of internally 32P-labeled RNA  with liposomes  was performed in AM buffer at room temperature for 5 min, followed by gel filtration on a Sephacryl S-1000 1-mL column. The initial RNA pool with 50-mer random region (50N) was generated by T7 polymerase by in vitro transcription of a DNA template strand of the sequence: 5′-TGG TCA TGT GAT CGG CGT ATG—50N—TAT CGT GTC ATC GTC GTC CCT ATA GTG AGT CGT ATT A-3′ (the underline sequence is the T7-promoter region). Approximately 1015 molecules of 91-nt RNA transcribed from independently synthesized DNA templates were heated in water at 65 °C for 5 min, 10xAM buffer was added, and solution was cooled to room temperature over 10 min to allow for RNA folding. The transcribed RNA 50N had the following sequence: 5′-TGG TCA TGT GAT CGG CGT ATG—50N—TAT CGT GTC ATC GTC GTC CC-5′.The selection was performed according to Vlassov et al. . Briefly15 sequences. In step B, ssRNA is first incubated (counterselection) alternatively with liposomes without Aβ42  or with buffer only (to remove non-specific RNA aggregates), then ssRNA is incubated with amyloid-liposomes, and next the amyloid/liposome/RNA complexes are separated from unbound species by gel chromatography using Sephacryl S-1000. The eluted fractions were analyzed for 32P RNA content by scintillation counting and for liposomes by A320 turbidity [AβRNA are RNA sequences that can bind to membrane amyloid peptides. In step C, AβRNA sequences are extracted from the liposome suspension by precipitation. In step D, the ssAβRNA sequences are reverse transcribed to give a cDNA copy of the winning sequences . For step E, PCR amplification completes the selection cycle and provides a dsDNA template enriched in the winning sequences for the next cycle.The selection-amplification (SELEX) procedure  starts wO-Me pyrimidines which are nuclease resistant [In order to improve the stability of RNA aptamers, both in cytoplasm and other fluids containing nucleases, it is possible in future applications to synthesize the aptamers using 2′-esistant  and inex2 and 95% air humidified atmosphere at 37 °C [2DCF-DA) [2DCF-DA. H2DCF-DA is incubated with cells and it enters the cells, then is cleaved by cytosolic esterases to 2′,7′-dichlorodihydrofluorescein, H2DCF (this prevents the back-diffusion of the dye to the extracellular space), and next is oxidized to the fluorescent DCF. Observed fluorescence is proportional to the concentration of intracellular ROS. Cells were cultured in 96-well plates, treated with RNA- and Aβ-bound liposomes , a Culture Collection of Public Health England  through Sigma-Aldrich , and were cultured in Eagle’s Minimum Essential Medium (Sigma) supplemented with 1% Non-Essential Amino Acids (NEAA), 2 mM glutamine, 0.5 mg/mL of streptomycin sulfate, 100 units/mL of penicillin G, and 10% fetal bovine serum (FBS) in a 5% COat 37 °C . Changes2DCF-DA) . The detIn this study, we searched for RNA aptamers specific for membrane amyloid peptides. The results demonstrate that up to ca. 20% of amyloid peptide, generated in the plasma (or intracellular) membranes as a result of proteolytic cleavage of amyloid precursor proteins by β- and γ-secretases, can stay within the membrane. Functional exosomes containing the selected pool of aptamers can inhibit the formation of toxic Aβ aggregates at the site of their nucleation, or can inhibit amyloid channel activity, and reduce reactive oxygen species production by mitochondria. RNA aptamers specific for membrane Aβ could potentially serve both in AD treatment and as dementia-preventive therapy for children and young adults with Down’s syndrome.