Adenosine deaminases acting on RNA (ADARs) are enzymes that convert adenosine to inosine in duplex RNA, a modification that exhibits a multitude of effects on RNA structure and function. Recent studies have identified ADAR1 as a potential cancer therapeutic target. ADARs are also important in the development of directed RNA editing therapeutics. A comprehensive understanding of the molecular mechanism of the ADAR reaction will advance efforts to develop ADAR inhibitors and new tools for directed RNA editing. Here we report the X-ray crystal structure of a fragment of human ADAR2 comprising its deaminase domain and double stranded RNA binding domain 2 (dsRBD2) bound to an RNA duplex as an asymmetric homodimer. We identified a highly conserved ADAR dimerization interface and validated the importance of these sequence elements on dimer formation via gel mobility shift assays and size exclusion chromatography. We also show that mutation in the dimerization interface inhibits editing in an RNA substrate-dependent manner for both ADAR1 and ADAR2. RNA-editing involves altering a transcript's sequence by insertion, deletion, or modification of nucleotides resulting in a change in information content from that originally encoded in the genome . One of 2CR mRNA  Figure  27). We. We27). 2CR mRNA . The bas2CR mRNA . For insnclusion ,35. In cnclusion ,36. Thusnclusion . Additio2CR mRNA (Currently there are no structures of ADAR-RNA complexes that include both the deaminase domain and dsRBDs. An earlier study of the role of ADAR2′s dsRBDs described a highly active fragment bearing dsRBD2 and the deaminase domain (hADAR2 R2D) . In addi2CR mRNA . Thus, t32P] ATP was purchased from PerkinElmer Life Sciences. Avian myeloblastosis virus (AMV) reverse transcriptase, Thermus filiformis (Tfi) DNA polymerase, and dNTP mix were purchased from Promega. The QuikChange Xl II mutagenesis kit was purchased from Agilent Technologies. RNA oligonucleotides were synthesized at the University of Utah DNA/Peptide Core Facility or purchased from Dharmacon. DNA oligonucleotides were purchased from Integrated DNA Technologies. Storage phosphor imaging plates from Molecular Dynamics were imaged with a Molecular Dynamics 9400 Typhoon phosphor imager. Data were analyzed with Molecular Dynamics ImageQuant 5.2 software. Matrix Assisted Laser Desorption/Ionization (MALDI) mass spectrometry of oligonucleotide samples was performed at the Campus Mass Spectrometry Facilities, UC Davis. Oligonucleotide masses were determined with Mongo Oligo Mass Calculator 2.08.Unless otherwise stated, reagents were purchased from Fisher Scientific, Sigma-Aldrich, or Life Technologies. T4 polynucleotide kinase, molecular-biology-grade bovine serum albumin (BSA), and RNase inhibitor were purchased from New England BioLabs.  at the 5′ end with T4 PNK as described previously (32P labeled GLI1 91 5′ end (33 mer) RNA was phenol-chloroform extracted, ethanol precipitated, desalted with 70% ethanol, dried, resuspended in nuclease free water and hybridized 1:1:1:1 with 5′-phosphorylated hGLI1 3′ Extended top (28mer), 5′-phosphorylated hGli1 91 3′end (30mer) and 90 base complementary DNA splint (GLI1 DNA splint) in 1× T4 RNA ligase 2 buffer by heating to 95°C and slowly cooled to room temperature. T4 RNA ligase 2 (5 μl of 10 U/μl) was added along with 3 μl of 1.6 U/μl RNase inhibitor to a final reaction volume of 140 μl. The sample was incubated at room temperature overnight. Ligated product was phenol:chloroform extracted, ethanol precipitated and dried. Ligated product was resuspended in nuclease free water and DNA was digested with RQ1 RNase free DNase-I in 1× DNase buffer for one hour at 37°C. The splint ligation products were PAGE purified as described above. Labeled RNAs were hybridized with the complementary GLI1 bottom strand RNA in 10 mM Tris–HCl pH 7.5 and 100 mM NaCl. See Oligonucleotides were purified by denaturing PAGE and extracted by the crush and soak method similarly to RNA used for crystallography but were desalted only by ethanol precipitation and 70% ethanol wash. The hGLI1 91 5′end (80 pmol) was radiolabeled with  ATP (6000 Ci/mmol) and T4 polynucleotide kinase, purified by 19% denaturing polyacrylamide gel in 1× TBE, visualized by storage phosphor autoradiography, excised, and extracted by crush and soak. The labeled oligonucleotide was hybridized to its unlabeled complement by heating to 95°C for 5 min in 1× TE and 200 mM NaCl, followed by slow cooling to room temperature. Samples containing less than 1.1 nM RNA and different concentrations of hADAR2-R2D mutants  were equilibrated in 20 mM Tris–HCl, pH 7.0, 3.5% glycerol, 0.5% DTT, 60 mM KCl, 20 mM NaCl, 0.1 mM BME, 1.5 mM EDTA, 0.003% NP-40, 160 units/ml RNasin, 100 μg/ml BSA, and 1.0 μg/ml yeast tRNA for 30 min at 30°C. Samples were loaded onto a running 6% nondenaturing polyacrylamide gel (79:1 acrylamide:bisacrylamide) and electrophoresed in 1× TBE buffer at 4°C for 90 min. The gels were dried on a gel drier (Biorad) for 60 min at 80°C under vacuum followed by exposure to storage phosphor imaging plates (Kodak) for 3 h in the dark. After exposure, the dried polyacrylamide gels were removed, and the phosphor imaging plates were scanned by Typhoon Trio Variable Mode Imager . See Oligonucleotides were 5′-end labeled using t = α[1 = ekobst−] where [P]t is percent edited, α is the end point fitted to 95% and kobs is the observed rate constant. See Deamination reactions had a final volume of 10 μl with concentrations of 10 nM RNA and 100 nM ADAR2-R2D. The final reaction solution contained 17 mM Tris pH 7.4, 60 mM KCl, 16 mM NaCl, 2 mM EDTA, 0.003% Nonidet 2. At 70–90% confluency, 8 × 104 cells for ADAR2 experiments, or 6.4 × 103 cells for ADAR1 experiments were seeded into a 96-well plate. After 24 h, cells were transfected with 750 ng pcDNA3.1 plasmid containing ADAR1 p110 or full length hADAR2 WT or mutant with HA tag. Transfection was carried out using Lipofectamine 2000. After incubation of transfection reagent and plasmid in Opti-MEM, the solution was added to designated well and incubated at 37°C and 5% CO2 for 48 h. Total RNA was isolated using RNAqueous Total RNA Isolation Kit and DNase treated with RQ1 RNase-free DNase (Promega). Nested PCR was performed by first using Access RT-PCR Kit (Promega) for 20 cycles for ADAR1 or 24 cycles for ADAR2 followed by a second PCR using Phusion Hot Start DNA Polymerase for 30 cycles for ADAR1 or 19 cycles for ADAR2. The PCR products were purified by agarose gel and Qiagen Gel Extraction Kit. The purified product was subjected to Sanger sequencing and sequence traces were analyzed by 4Peaks (Nucleobytes) to measure percent editing. See HEK293T cells were cultured in Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum (FBS) and 1% anti–anti at 37°C and 5% CO32P. To enhance protein–RNA complex formation, we also used the hyperactive ADAR2 mutation E488Q, which exhibits a higher binding affinity to duplex RNA  has been well established . Here, wplex RNA . We thenplex RNA . Compariplex RNA . The ADAplex RNA  and suggElectrophoretic mobility gel shift assays (EMSA) are useful for the study of protein-RNA interactions in solution . In prevWe next sought to obtain a high-resolution crystal structure of the ADAR2 R2D + dsRNA complex. We were able to form protein/RNA crystals using a 32 bp 8-AN-containing duplex and ADAR2-R2D E488Q diffracting X-rays beyond 2.8 Å resolution Table  Figure . The sec2 for each monomer) with low temperature factors, suggesting the interface is quite stable. Interestingly, residues such as G452, R455, F457, S458, H460, R481, Q488, T490, R590 and G593 in monomer B, which comprises the catalytic pocket or RNA binding surface, instead participate in forming protein-protein contacts to an exposed α-helix from monomer A (consisting of residues 501–509), which we will refer to here as the dimerization helix  were not previously known to have a role in editing despite their high degree of conservation . For this purpose, each of the residues T501, W502 and D503 were individually mutated to alanine. Focus laid strictly upon residues within the dimerization helix since many of the amino acids in monomer B that comprise the protein-protein interface correspond to key residues of the deaminase catalytic pocket of monomer A. Each alanine mutation resulted in changes to the EMSA banding pattern observed with the 3′ extended 8-AN containing RNA compared to the protein with the wild type dimerization helix Figure –D. For eTo confirm the banding pattern observed by gel shift represents formation of 1:1 and 2:1 protein–RNA complexes, we performed analytical size-exclusion chromatography (SEC) of ADAR2-R2D and observed its change in retention upon incubation with RNA. Gel filtration of ADAR2-R2D or 8-AN-bearing duplex RNA individually, resulted in a major peak with a retention time consistent with monomeric free protein and a smaller peak corresponding to high molecular weight aggregates. However, combining the protein and RNA together resulted in a gel filtration trace with four major peaks with retention times most consistent with free RNA, 1:1 protein–RNA complex (monomer), 2:1 protein-RNA complex (dimer) and an aggregate peak Figure  Supplem. Importain vitro editing of the 5-HT2cR substrate . For comparison, we also measured rate constants for editing of a 147 nt RNA duplex resembling the GLI1 site, a substrate where dsRBD binding has been shown to be less important for editing efficiency . Fo. Foin visay used .in vitro editing experiments using the ADAR2 R2D mutants. Once again, D503A exhibited the greatest perturbation on editing followed by W502A and T501A . Th. Th62). A Figure . We alsoA Figure .Since there is high sequence conservation for residues in the dimerization helix for the different ADAR family members, we also considered the effect of mutation in this location for ADAR1 editing. For this purpose, we generated the human ADAR1 p110 D1023A mutant corresponding to the D503A mutation in human ADAR2 and expressed this protein in HEK293T cells. We measured editing for five known ADAR1 sites in these cells Figure  62,63).,63.62,63Drosophila ADAR is capable of dimerization in an RNA-dependent manner  5′ to the editing site. It is possible that this protection arises from RNA contact with the dsRBD from monomer A not resolved in our structure. However, additional crystallization or cryoEM studies with longer RNAs will be necessary to test this hypothesis. Finally, no high-resolution structural data has been reported for ADAR1 in complex with RNA. Such data are needed to fully understand differences in RNA substrate selectivity for the different members of the ADAR family.Atomic coordinates and structure factors have been deposited in the Protein Data bank under the accession code 6VFF.gkaa532_Supplemental_FileClick here for additional data file.


Drosophila AdarE374A mutant strain encoding a catalytically inactive Adar with CRISPR/Cas9. We demonstrate that Adar adenosine deamination activity is necessary for normal locomotion and prevents age-dependent neurodegeneration. The catalytically inactive protein, when expressed at a higher than physiological level, can rescue neurodegeneration in Adar mutants, suggesting also editing-independent effects. Furthermore, loss of Adar RNA editing activity leads to innate immune induction, indicating that Drosophila Adar, despite being the homolog of mammalian ADAR2, also has functions similar to mammalian ADAR1. The innate immune induction in fly Adar mutants is suppressed by silencing of Dicer-2, which has a RNA helicase domain similar to MDA5 that senses unedited dsRNAs in mammalian Adar1 mutants. Our work demonstrates that the single Adar enzyme in Drosophila unexpectedly has dual functions.ADAR RNA editing enzymes are high-affinity dsRNA-binding proteins that deaminate adenosines to inosines in pre-mRNA hairpins and also exert editing-independent effects. We generated a  Drosophila Adar has both innate immune and brain functions, despite being the homolog of mammalian ADAR2.Human RNA editing enzymes ADAR1 and ADAR2 are required for innate immune functions and neurological functions, respectively. Here, the authors show that  In A-to-I RNA editing, the ADAR (adenosine deaminase acting on RNA) enzyme binds to double-stranded RNA (dsRNA) and deaminates specific adenosines to inosines. Inosines are generally read by the cellular machinery as guanosines and RNA editing can change encoded protein sequences, as well as altering RNA structure and RNA splicing. ADAR RNA editing occurs throughout the transcriptome at about fifty specific coding sites in humans but site-specific editing is greatly expanded in some invertebrates such as Drosophila, where it occurs at over a thousand specific coding sites in transcripts. Additional promiscuous RNA editing occurs in long dsRNAs formed by inverted repetitive elements in both mammals and Drosophila; in humans this additional ADAR RNA editing occurs at millions of sites, mostly non-specifically and at low efficiency in Alu dsRNA hairpins embedded in protein-coding transcripts5. Unlike DNA mutations, RNA editing usually changes only some proportion of the transcripts of particular genes, and editing levels at individual sites can be different in different tissues and developmental stages8, due to different ADAR expression levels and other tissue-specific factors. Thus, RNA editing is a powerful tool that can over-write and enrich the genomically encoded information during the process of gene expression.Adenosine-to-inosine (A-to-I) RNA editing is an important co-transcriptional RNA modification . Mice lacking Adar1 die by embryonic day E12.5 due to the aberrant activation of cytoplasmic, antiviral, innate immune dsRNA sensors, which causes an upregulation of interferon expression and innate immune responses19. Elimination of MAVS, a mitochondria-associated adaptor protein required for innate immune activation by RIG-I-like receptors (RLRs), antiviral cytoplasmic dsRNA sensors, allows Adar1, Mavs double mutant mice to survive to birth, although they die soon after birth19. The immune regulatory role of ADAR1 is largely dependent on its RNA editing activity; Adar1E861A mice expressing catalytically inactive Adar1 E861A protein also show upregulation of interferon-stimulated genes (ISG) and embryonic lethality17. The Adar1E861A mutant mice die two days later than Adar1 null mutants, by E14.5. Elimination of MDA5 (encoded by Ifih1), one of three antiviral dsRNA sensor helicases, allows the Adar1E861A, Ifih1 double mutant to survive to a full life span with no obvious defects17. The differences between inactive Adar1E861A mutant and Adar1 null mutant mice suggest that editing-independent effects are also significant, although restoring Adar1E861A by transfection in Adar1 mutant cells prevents aberrant innate immune induction only very partially18.In vertebrates, the major defects in Adar2 die within 3 weeks of birth with increasingly severe seizures associated with effects of increased influx of calcium ions through glutamate receptors on synaptic plasticity across the brain21. These severe neurological defects are caused by loss of editing at a single glutamine (Q) to arginine (R) editing site in the Gria2 transcript encoding the key GluA2 subunit of AMPA class glutamate gated ion channels20.In contrast to the innate immune role of Adar1, vertebrate Adar2 is critical for neurological function and shows an even clearer dependence on RNA editing activity. Mice lacking Drosophila offers an opportunity to study Adar RNA editing-dependent and independent functions in more detail. Unlike mammals, Drosophila has only one Adar gene encoding a protein similar to mammalian ADAR223. Drosophila Adar null mutants such as Adar5G1 have severe locomotion defects, seldom fly, have aberrant neurotransmitter synaptic vesicle accumulation and sleep defects and develop age-dependent neurodegeneration25. All Adar5G1 null mutant phenotypes examined are rescued by expressing Drosophila Adar or human ADAR2 but not human ADAR122, showing the strong conservation of function between these ADAR2-type enzymes.Adar mutant phenotypes in Drosophila are fully dependent on RNA editing, we generated a fly strain (AdarE374A) that has a point mutation in the chromosomal Adar gene resulting in production of catalytically inactive Adar E374A enzyme. We find that loss of Adar RNA editing activity in Drosophila leads to severe locomotion defects and age-dependent neurodegeneration similar to Adar5G1 null mutant flies24. Furthermore, we show that the loss of RNA editing by Adar induces aberrant innate immune gene expression in Drosophila, through Dicer-2 acting as a dsRNA innate immune sensor. This is analogous to the findings in mice where loss of Adar1 editing leads to immune induction through the activation of MDA517; Drosophila Dicer-2 has a helicase domain of the same type as MDA526. These results show that Drosophila Adar has dual functions of both Adar1 and Adar2 proteins in vertebrates.To determine whether Drosophila deletion mutants with low to completely depleted levels of Adar have been used for characterizing Adar functions27. However, it is unclear whether Drosophila Adar functions are dependent on RNA editing activity. To uncouple the editing-dependent from the editing-independent functions of fly Adar, we used CRISPR/Cas9-mediated homologous recombination to generate a fly strain (AdarE374A) with a point mutation  chromosome and we confirmed that RNA editing was eliminated by sequencing of total, ribosome-depleted head RNA of male AdarE374A and wild type w1118 flies. Wild-type flies have site-specific editing events with a wide range of editing levels in hundreds of transcripts  which drives expression in the numerous brain cholinergic neurons that express the acetylcholine synthesizing enzyme choline acetyltransferase30. We note that Adar5G1 is close to being fully rescued by ChAT-Gal4, UAS-Adar 3/4S (i.e. ChAT > Adar 3/4S) expression while AdarE374A is partially rescued  in AdarE374A and Adar5G1 respectively  > = 0.6, Fig. AdarE374A are also affected in Adar5G1, suggesting that they are affected by loss of RNA editing. Gene expression changes in Adar5G1 only are likely a response to the loss of Adar protein, but not solely due to loss of RNA editing activity.Next, we hypothesized that, because there are such severe CNS defects in the AdarE374A mutant  > = 0.6). Although Adar mutants display CNS defects and RNA editing sites are enriched in neuronal genes in Drosophila8, we did not observe an enrichment of neurological terms in the genes with increased or decreased expression in the AdarE374A mutant  term analysis to examine the functions and categories of the most differentially expressed genes in the ant Fig. . HoweverAdarE374A was “Immune-induced protein”   Fig. . Similar5G1 Fig. . The 9 Dons Fig. , stronglAdarE374A head RNA-seq data  in RNA from heads versus whole bodies of Adar mutant flies. We observed that the aberrant induction is much stronger in heads   that kill bacteria. Different AMP transcripts are transcriptionally induced by different pathogens through different defined innate immune signaling pathways. We compared the induction of ads Fig. , indicatng) Fig. . The VagRNA Fig. . We concUAS-AdarEA ubiquitously under armadillo-GAL4 driver control suppresses aberrant expression of AMPs as effectively as UAS-Adar wild type expression , which is considered more of a stress-induced transcript is not well suppressed. We conclude that a partial inhibition of aberrant Dcr-2 innate immune signaling by AdarE374A protein when expressed at normal levels, becomes much stronger when the catalytically inactive protein is expressed ubiquitously from UAS constructs under arm-GAL4 driver control. This nearly total suppression of innate immune induction by increased inactive Adar  still has dsRNA sensor activity then the increased long dsRNA could cause the increased innate immune induction in the Adar5G1; Dcr-2R416X double mutant. Intriguingly this increased immune induction is much weaker in the AdarE374A; Dcr-2R416X double mutant, suggesting that it is prevented by catalytically inactive Adar protein. To test this idea, we knocked down Dcr-2 by RNAi in cholinergic neurons under ChAT-GAL4 driver control; The knockdown of Dicer-2 transcripts in head RNA is 40–60% complete  that is usually activated by the IMD-Rel pathway, and Drs (Drosomycin) that is usually activated through the Toll-Dif pathway  transcript inductions are clearly stronger in Adar5G1 than in AdarE374A, suggesting that the presence of catalytically inactive Adar protein may impede the immune induction. The Adar editing-independent effects are most clearly seen when the catalytically inactive AdarEA is overexpressed and is able to rescue some Adar mutant phenotypes. For example, neurodegeneration  protein still mediates innate immune induction but it retains the Hel1 and Hel2i subdomains of the helicase domain and their potential dsRNA contacts; and it may also have interactions with other proteins that help it recognize dsRNA. The Hel2i subdomain of helicase DICER proteins is a hotbed of interactions with dsRBD proteins in, for example, human DICER interacting with specialized dsRBDs of TRBP and PACT and with dsRBD3 of human ADAR1, which affects microRNA and siRNA processing14. When expressed in Drosophila, human ADAR1 strongly inhibits Dicer-2-mediated RNA silencing through siRNA production, even when catalytically inactive9 and it may also inhibit innate immune signaling by Dcr-2. We show here that Drosophila AdarEA also strongly blocks Dcr-2 innate immune signaling  flies are from31. The Cha-Gal4 driver expresses GAL4 in cholinergic neurons under the control of the choline acetyl transferase promoter (Cha is now named ChAT in Flybase http://flybase.org/reports/FBti0024050.html); this GAL4 driver line is Bloomington Stock number 6798, w1118; P48s19B/CyO, P{sevRas1.V12}FK1. The UAS-Adar 3/4 transgene expresses an editable cDNA encoding the adult ADAR 3/4 splice form30. The UAS-hADAR2 transgene expresses cDNA encoding human ADAR2. RNAi lines  are Adar (VDRC-7763), Dcr-2 (VDRC-25090), Rel (VDRC-105491), Dif (Bloomington-29514), Stat92E (Bloomington-35600). For the innate immune qPCR, flies were grown at 25 °C and collected at 2-3 days old. Total RNA was extracted from 7–15 whole flies or 20 heads using RNAdvance magnetic beads (Agencourt), and this was treated using TURBO DNase (Thermo Fisher Scientific).All stocks were grown on standard fly media. For the locomotion assays (negative geotaxis and open field locomotion), flies were kept in 12 h/12 h light/dark cycles at 25 °C. Replicate experiments were performed at the same time of day. Flies were not exposed to carbon dioxide for at least 24 h before the assays.AdarE374A fly line28 was generated with plasmid and nos-Cas9 fly line CRISPR reagents as described in54. sgRNA target sequence CCTGGAGACTATTTCAGCAT was inserted into the U6b-sgRNA-short plasmid and injected into the nos-Cas9 flies (attP2 insertion line) by BestGene. The sgRNA plasmid was injected at 75 ng/ul, and the donor oligo .The Three to four-day-old male flies were placed in two glass cylinders with a fly vial plug at the bottom. The following was repeated five times for each set of flies: The cylinders were banged onto a mouse pad and after 30 s, the number of flies above the 10.5 cm mark was counted. Then there was a 30 s rest period. The average of the percentage of flies above the 10.5 cm mark over the five trials was the “climbing success rate”. 14–38 flies were examined for each genotype.Two-day-old individual male flies were placed in 35 mm petri dishes divided into eight sections by radii, with an additional center circle. The number of lines crossed in 2 min was recorded three times for each fly, and these values were averaged per fly. 15 flies were examined for each genotype.Flies were aged and then fixed in Carnoy’s fixation solution  for 3.5 h and flies were then placed in 95% ethanol for 30 min twice and then they were placed in 100% ethanol for 45 min. Flies were kept in methyl benzoate overnight. On the next day, flies were kept in solution of methyl benzoate and paraffin both in equal volume at 60 °C for 1 h, then transferred to fresh liquid paraffin for 1 h twice at 60 °C and then kept in liquid paraffin overnight at 60 °C. Flies were transferred to a plastic petri dish with fresh liquid paraffin at 60 °C and then allowed to cool down. Paraffin blocks were made from this. Sections of 5-μm thickness were taken using a LEICA RM 2235 rotary microtome and then dried overnight on the slide warmer at 37 °C and then processed for hematoxylin and eosin staining.28 (GEO GSE86056). DESeq255 were used to obtain gene expression levels. DEXSeq57 was used to examine differential exon usage. To determine the editing event, we required the editing level to be ≥2% in at least one of the datasets. 89% of the editing sites have significantly higher editing levels with this cut off than the typical A-to-G sequencing error rate. Editing levels were only used if there were at least 20 reads at the editing site.Reads and mapped reads are as inp-value was not NA) were used as the background. The GO terms used are from these lists (DAVID default): COG_ONTOLOGY, UP_KEYWORDS, UP_SEQ_FEATURE, GOTERM_BP_DIRECT, GOTERM_CC_DIRECT, GOTERM_MF_DIRECT, KEGG_PATHWAY, INTERPRO, PIR_SUPERFAMILY, SMART. P-values were corrected for multiple hypothesis testing using the Benjamini method, and only the top statistically significant GO terms (adjusted p < 0.01) are shown.DAVID was used for the GO term analysis. The lists of genes tested by DESeq2  or RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). qPCR reactions using the KAPA-SYBR FAST qPCR kit (Kapa Biosystems) or LightCycler® 480 SYBR Green I Master mix and the primers listed in Supplementary Table t-test.Heads of male flies (minimum 20 flies) of the desired genotype were collected and aged for 2 days and then homogenized in NB Buffer . Protein concentration was determined with Pierce BCA Protein Assay Kit. An equal amount of protein was loaded in each lane of a Tris-Glycine SDS Gel and transferred to a nitrocellulose membrane. The membrane was blocked with 5% Milk, incubated with primary antibody overnight. The next day it the membrane was incubated with secondary antibody and developed with Clarity™ Western Blotting Substrate from Bio-Rad. Anti-ADAR2 (HPA018277) (1:250) anti-Tubulin  (1:5000). Imaging was performed with ChemiDoc™ XRS + System and signal intensity was quantified with Image J software and Statistical analyses were done using the t test. A p-value of <0.05 was considered statistically significant. Locomotion and Climbing data p-values were calculated by a one-way ANOVA followed by Tukey’s test. The significance of differences between variables was described based on p-values *p-value < 0.05. **p-value < 0.005. ***p-value < 0.001 and n.s (not significant). Error bars: SEM .qPCR data and immunoblot data were analyzed by Student’s Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary


Below we highlight the latest additions.The European Centre for Disease Prevention and Control (ECDC) provides regularly updated information relevant to Europe through a dedicated document provides guidance on the application of non-pharmaceutical countermeasures to minimise the spread of the 2019 novel coronavirus (2019-nCoV) in the population. Some of the measures proposed refer specifically to certain phases of the epidemic (containment or mitigation phases), and can be adapted depending on the assessed severity/impact of the infection. Other measures are valid for all phases of an epidemic. The guidance is based on the current knowledge of the 2019-nCoV and evidence available on other viral respiratory pathogens, mainly the Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), the Middle East Respiratory Syndrome-related coronavirus (MERS-CoV) and seasonal or pandemic influenza viruses. ECDC will update this guidance as and when new relevant information becomes available or as required by the epidemiological situation.This document is to support public health preparedness planning with regard to personal protective equipment (PPE) needs in healthcare settings where patients suspected or confirmed to have been infected with the novel coronavirus 2019-nCoV are being treated.The aim of this This infographic provides basic information about the virus, symptoms, prevention and transmission.Eurosurveillance about our dedicated ‘Coronavirus disease 2019 (COVID-19) collection’ that we update regularly with new articles. The most recent addition was ‘First cases of coronavirus disease 2019 (COVID-19) in France: surveillance, investigations and control measures, January 2020’ while it already contained several articles about, for example, the development of a diagnostic methodology based on RT-PCR without the need for virus material, the severity among hospitalised cases, early-stage estimates of the importation risk to Europe, and estimated patterns of early human-to-human transmission.We would also like to remind the readers of


The measurement of RNA abundance derived from massively parallel sequencing experiments is an essential technique. Methods that reduce ribosomal RNA levels are usually required prior to sequencing library construction because ribosomal RNA typically comprises the vast majority of a total RNA sample. For some experiments, ribosomal RNA depletion is favored over poly(A) selection because it offers a more inclusive representation of the transcriptome. However, methods to deplete ribosomal RNA are generally proprietary, complex, inefficient, applicable to only specific species, or compatible with only a narrow range of RNA input levels. Here, we describe Ribo-Pop , a simple workflow and antisense oligo design strategy that we demonstrate works over a wide input range and can be easily adapted to any organism with a sequenced genome. We provide a computational pipeline for probe selection, a streamlined 20-min protocol, and ready-to-use oligo sequences for several organisms. We anticipate that our simple and generalizable “open source” design strategy would enable virtually any laboratory to pursue full transcriptome sequencing in their organism of interest with minimal time and resource investment. Researchers across the biological sciences use RNA sequencing to generate novel insights or test hypotheses. However, in most organisms, ribosomal RNA comprises the majority of the RNA sample, making it economically impractical to analyze sequences of interest such as protein-coding mRNAs and other nonribosomal transcripts at substantial coverage. Therefore, efficient sequencing requires either capture of the RNAs of interest or depletion of ribosomal RNA.The most widely used capture method is poly(A) selection, in which the polyadenylated RNAs are purified by hybridization to oligo(dT). Ribosomal RNA, which is not polyadenylated, is greatly reduced in poly(A)-selected sequencing libraries. However, in recent years, interest in the nonpolyadenylated transcriptome has grown considerably. Studies which seek to characterize preadenylation steps in RNA processing such as nascent transcription and splicing cannot use poly(A) selection . FurtherDrosophila. Because of the proprietary nature of these kits, researchers working in other organisms cannot benefit from any knowledge used to produce these probe sets in order to design probe sets for their organism of interest. Secondly, even commercial kits are unsuitable for certain types of experiments in their target organism(s). Some of them have narrow RNA input ranges or are not made available as a separate component outside of a sequencing library construction kit, making them difficult to use for novel or exploratory experiments that may deviate from a traditional workflow. Finally, the cost of commercial rRNA depletion reagents is prohibitive for some research groups and prevents their wider adoption.To address these shortcomings, removal of ribosomal RNA, rather than selection of a poly(A) tail, has been introduced in many experimental workflows. Unfortunately, removal of ribosomal RNA is more complicated than poly(A) selection because there is no universal reagent equivalent to oligo(dT) available. Although many protocols and commercial products have been developed to deplete ribosomal RNA or its cDNA derivatives, most of these solutions suffer from various pitfalls that prevent their widespread use. First, each reagent is only applicable to a specific organism or family because hybridization approaches require a high degree of sequence complementarity. For example, as of writing, several commercial kits that deplete rRNA from mammalian samples are available, but none are available for Drosophila and S. cerevisiae, and we created an automated probe design pipeline to enable selection of new probe sets. We named our method Ribo-Pop  because it democratizes rRNA depletion, enabling researchers working with any organism or workflow to deplete ribosomal RNA in an efficient and cost-effective manner.In summary, we argue that the current lack of versatile and cost-effective rRNA subtraction reagents severely restricts exploration in many areas of RNA biology. Here, we address this problem by developing an “open source” rRNA depletion solution that is simple to use, cost-effective, and can be easily adapted to any organism of interest. We validated the use of our rRNA subtraction method using RNA purified from Supplemental Fig. S1; Drosophila total RNA at an estimated fivefold molar excess in a single-probe depletion assay . These results show that many different probes are effective, despite the highly structured nature of the ribosomal RNA molecule.In an effort to develop high-efficiency ribosomal RNA removal, we decided to test short, biotinylated oligonucleotides for their ability to deplete ribosomal RNA by hybridization and subsequent removal of the rRNA/probe hybrids via pulldown with streptavidin-coupled beads. First, we tested how much rRNA depletion is achieved from a single probe/target interaction, in order to determine the minimum number of high affinity probes required to purify unfragmented rRNA. We designed and biotinylated 20 short ∼30mer probes (26–32 nt) targeting the small 18S rRNA transcript using parameters developed for microarray probe design and used in smFISH experiments . We hypothesize that the additional nucleotides present in the longer probes make the propensity for self-structure greater and offset the theoretical gain in target affinity. Therefore, we decided to continue using small probes and turned our focus to optimization of probe selection within the 30 nt size range.We chose to use short ∼30mer probes for several practical and theoretical reasons. Short probes are inexpensive to manufacture at high purity, and they have lower potential for hairpin formation, unwanted dimerization, and off-target binding . HoweverSupplemental Fig. S2B,C). In contrast, the predicted Tm of each probe bound to its target was convincingly correlated with target depletion, with higher Tm probes achieving greater depletion on average  .We examined our depletion data in order to discover probe properties favorable for ribosomal RNA depletion. Although all probes were able to achieve some degree of depletion, we observed large differences between the best- and worst-performing probes B. To detTms that are relatively high (>72°C) compared to the surrounding sequence area would perform better, we designed probes corresponding to Tm peaks in the first 60% of the 18S target region and tested them for their ability to deplete 18S rRNA. We observed that higher Tm probes did perform better than lower Tm probes on average (3.3-fold vs. 2.7-fold median depletion), although the comparison was not statistically significant (P = 0.12) . Nevertheless, when we include all our tested ∼30mer probes in the analysis, Tm and probe performance are significantly correlated . Given this striking correlation, we decided to set high Tm as one of our probe selection criteria.To test whether probes with Tms higher than the denaturation temperature (70°C), we reasoned that annealing would likely begin during the denaturation step, and that the annealing step of the protocol could be eliminated without affecting performance. We tested three different probes with and without the annealing step . Removing the annealing step completely by adding the denatured RNA directly to beads that had been preheated to 50°C increased the remaining 18S by an average of 11% across three probes tested, which ranged in Tm from 65°C to 80°C (P = 0.04). Given the small size of the effect, we decided to eliminate the 10 min annealing step from our further experiments and moved quickly from the denaturation step to streptavidin binding, allowing the hybridization reactions to rest only briefly at room temperature (∼1 min) before proceeding to bead capture. Thus, simply by choosing high Tm probes, we were able to create a rapid and robust rRNA depletion strategy.Because we planned to select probes with Supplemental Fig. S4; Tm probe sets based on their proximity to the ES3/ES6 helix, then the difference between high and low Tm performance becomes statistically significant , spaced evenly along the transcripts to yield a probe density of approximately 1 probe/400 nt. We did not include probes for the smaller transcripts (5S and 5.8S) because we observed that they are not well captured by most RNA-seq protocols and do not contribute substantially to the total rRNA contamination. For the 18S probes, we selected five probes from those tested in Tm for potential probes. We also screened probes for potential matches against processed and unprocessed transcripts, using an alignment score cutoff that excludes probes matching more than 15 consecutive base pairs to nontargets , we successfully used an estimated fivefold molar excess of probe mix to rRNA (40 pmol probe mix per µg of input RNA) to deplete targets to <1% of their input levels  were prepared in parallel. After sequencing, we mapped the reads to all annotated transcripts and counted the sum of reads counted per transcript class . The lack of substantial complementarity between the observed decreased genes and both the probes and the rRNA transcripts themselves suggests that Ribo-Pop does not cause codepletion of these RNAs by off-target hybridization. The RNA pools before and after depletion differ greatly in concentration and composition. It is possible that these differences cause over or under recovery of some specific RNAs during library preparation that we are detecting here. Overall, depletion does not substantially bias gene quantification.To test the specificity of our probe pool, we compared poly(A)-primed sequencing from depleted samples to poly(A)-primed sequencing of input samples. We chose to use poly(A)-primed data for this analysis because the low depth of sequencing in nonpoly(A)-primed, nondepleted input samples precludes quantification of most genes. We found that Ribo-Pop depletion had little effect on our gene quantification. The correlation between input and depleted RNA levels for mRNAs and ncRNAs was high . For example, the pipeline designed a probe set targeting both S. cerevisiae and S. pombe yeast rRNA by finding regions of identity between each target and then choosing probes corresponding to our selection criteria  of interest. To this end we wrote a fully automated pipeline for probe selection, which includes selection of evenly spaced, high checking A. The pichecking , which mcriteria B. The yecriteria C, similaTm, and structural effects. We argue that these insights and the computational probe design pipeline that we have developed will be helpful to any researchers wishing to design their own probes and may even have relevance beyond rRNA removal for other types of RNA pulldown experiments, such as viral RNA purification.We have described a simple, effective, and affordable strategy for ribosomal RNA removal that can be extended to any organism with a sequenced genome. Using only 15 short ∼30mer probes, we were able to reduce rRNA levels greatly and increase the percent of non-rRNA reads from 3% to 73%. Furthermore, by testing individual probes, we elicited information about the importance (or lack thereof) of probe length, Ribo-Pop has low start-up costs compared to many other noncommerical rRNA subtraction methods. By testing individual probes, we were able to identify features of successful probes and assess how many probes would be needed to obtain the desired results, without resorting to an unnecessarily costly excess of probes and streptavidin matrix. The oligos can be ordered biotinylated for around $1000–$2000, but thereafter used at a relatively low cost per reaction. As described here, oligos can also be ordered unmodified and biotinylated in-house for a start-up cost of around $50 per probe set and the additional cost of the TdT enzyme and ddUTP for a total start-up cost of around $150. The low costs and ease of adding or subtracting probes from the mix will make it simple for researchers to test and optimize custom mixes if needed.A researcher wishing to deplete as much rRNA as possible may wish to add more probes to target the other contaminants or to tile across the 18S and 28S more densely. However, one should consider whether such a strategy is optimal from a cost-benefit perspective. To do so would more than double the size of the probe set and the cost of the experiment. If the expanded probe set were to decrease rRNA-mapping reads to 5%, then the space available for non-rRNA reads would increase from 73% to 95%, an increase of only 30%. In other words, greatly increasing the target space of the probe set and the cost per experiment is expected to increase the effective sequencing depth only modestly. For this reason, we chose to remain focused on the major contaminants, the processed rRNA transcripts of the small and large cytosolic ribosome. However, our probe design pipeline can accept user parameters to design more densely packed probes or to include more targets if desired.One potential pitfall of our 15 oligo probe set is that it would not be expected to perform as well on fragmented rRNA. Use of a larger probe set entails a higher cost per reaction but would be predicted to perform better on fragmented samples. Another option for highly fragmented RNA may be an enzymatic depletion method where rRNA is bound by tiling DNA probes and then subjected to RNase H digestion. The RNase H treatment is sometimes preceded by a reverse transcriptase step to extend the DNA/rRNA hybrids and is always followed by a DNAse treatment to remove the DNA probes . These sDuring preparation of this study, a handful of similar protocols using short probes were published using biotinylated oligos of approximately 50, 40, or 30 nt, followed by streptavidin pulldown . Two of Tm than the Ribo-Pop pipeline. They achieved similar depletion levels to Ribo-Pop, although a direct comparison is difficult because bacteria lack 5.8S and mitochondrial rRNAs. Impressively, they were able to deplete rRNA from highly distant species. The Ribo-Pop probe design pipeline does allow the user to design probe sets targeting multiple species. However, it does not currently support probe design at alignment sites containing mismatches between the target species. Given the prevalence of short stretches of nucleotide identity suitable for probe design and the relatively low cost of creating additional Ribo-Pop probe sets, we do not feel that this feature is required at this time. Users that need to deplete rRNA from a pool of distantly related microbial communities may find the DIY design better suited to their needs, although another option could be to use Ribo-Pop to select high Tm probes from subsets of more closely related species and then pool them to create a probe set against a very diverse set of species. It is difficult to know whether inclusion of more high Tm probes or fewer low Tm probes would lead to better rRNA depletion in this type of experiment. In any case, either of these tools should provide an effective open source option to design rRNA removal probes that target multiple species.The most recently published protocol, the DIY method, which used 21 30-nt probes is most similar to ours in probe length, number, and depletion protocol . In thatDrosophila) and other organisms of ecological interest effectively prevent or severely limit the application of many RNA sequencing protocols in these systems. This discrepancy further widens the gap between often better-funded mammalian work and frequently underfunded work on invertebrate models. We argue that rRNA depletion is the most appropriate approach to informative RNA enrichment for many biological questions, and that “open source” reagents as cost effective and readily available as oligo-dT for poly(A) selection should be made available. We believe that the Ribo-Pop method will help bridge this resource gap.Ribosomal RNA removal reagents should be available to the entire scientific community, not only those that happen to work with the organisms best served by the biotechnology industry. The cost of RNA sequencing has been decreasing rapidly over the last 10 years . While pDrosophila 18S from the three annotated nonpseudogene variants  using MAFFT . For the longer 46–52mer probes, we changed the desired probe length to these ranges and set the ΔGmin = −56 and ΔGmax = −48. We then screened the returned probes to remove probes with five or more consecutive identical nucleotides, four or more consecutive As, or four or more consecutive Cs in the first half of the probe sequence, as previously recommended (Tm probes corresponding to Tm peaks on the left side of the 18S. The high Tm probes have otherwise the same length and thermodynamic property ranges (predicted ΔG for homodimer and heterodimer formation) and meet the same sequence composition criteria as the first set of 20 short probes. The predicted Tm of each probe/target interaction was calculated using Biopython . The long 46–52 nt probes were subjected to in-house PAGE purification before use. Probes were biotinylated with terminal deoxynucleotidyl transferase  and dideoxy-UTP-biotin  as previously described . To biotDrosophila melanogaster wild type strain Oregon-R flies were raised on standard cornmeal-agar medium at 25°C. Three samples of 40 wandering third instar larvae each were collected and RNA was extracted with TRIzol . Yeast were diluted to low OD600 and grown overnight at 30°C with rapid agitation, then harvested in mid-log phase at OD 0.5. Each sample was derived from an independent colony. RNA was extracted from cell pellets using the hot acid phenol method. Pellets from 10 mL of culture were resuspended in 200 µL TES  and 200 µl acidic phenol (Sigma Aldrich P4682) and heated with agitation at 65°C for 10 min in a thermomixer (Eppendorf ThermoMixer C) at 1000 rpm. After 5 min of cooling on ice, then 5 min at room temperature, the sample was mixed vigorously with chloroform and centrifuged 5 min at 16,000g to separate the phases. The aqueous phase was then subjected to extraction with 1 volume phenol:chloroform:iaa  and a final extraction with 1 volume chloroform. The aqueous phase was brought to 300 mM NaOAc with 3M NaOAc, pH 5.2, and 1 volume of isopropanol was added to precipitate the RNA. Prior to rRNA depletion, 50 µg aliquots of RNA were treated with 10U of TURBO DNAse (Thermo Fisher) for 30 min at 37°C, then purified with the RNA Clean & Concentrator kit (Zymo Research) and eluted with water.h TRIzol . SaccharSupplemental Table S3. Standard curves with cDNA dilutions were routinely performed to ensure the amplification efficiency was close to 100% and no RT controls were also assessed to verify the success of DNA removal from the RNA samples. The amplification Ct values were called automatically by Bio-Rad CFX Manager. Fold changes are derived from the ΔΔCt method. Each target Ct value was first normalized by Act5c (ΔCt), followed by normalization by a control sample  to produce ΔΔCt.cDNA synthesis was performed with UltraScript Reverse Transcriptase (PCR Biosystems) in 10 µL reactions containing a mix of RNA, random hexamer (5 µM), anchored oligo-dT primer , buffer, and 100U UltraScript enzyme. Reactions were denatured 5 min at 70°C with RNA and primers alone, then placed on ice for 5 min. The remaining components were then added and the reactions were incubated for 10 min at 25°C, 30 min at 42˚C, followed by 10 min at 85°C. cDNA was amplified with qPCRBIO SyGreen Mix Lo-Rox (PCR Biosystems) according the manufacturer's instructions in 10 µL reactions with a Bio-Rad CFX96 instrument. The qPCR primer sequences, some of which were taken from a previous study , are lisSupplemental Fig. S3), the same protocol was followed except that the no annealing samples were added directly to beads that had been preheated to 50°C for 5 min.Biotinylated probes were brought to 0.2 µM in probe resuspension buffer  and used to deplete rRNA from 200 ng of larval total RNA. To minimize the effect of pipetting error on probe efficacy measurements, each replicate uses a different sample of larval RNA and an independent dilution of biotinylated probe. Each probe was hybridized in a 10 µL reaction containing 200 ng total RNA and 0.5 pmol of probe in Ribohyb buffer . Reactions were heated to 70°C for 5 min in 1.7 mL tubes in a thermomixer and then placed at room temperature for 10 min before capture with Dynabeads MyOne Streptavidin C1 (Thermo Fisher). To prepare beads for capture, 2 µL beads per sample were washed 3× in MyOne B & W buffer + 0.01% Tween, 2× in Dynabeads buffer A + 0.01% Tween, 2× in Dynabeads buffer B + 0.01% Tween, 3× in Ribohyb buffer and resuspended in a final volume of 10 µL Ribohyb buffer in a nonstick tube (Eppendorf Protein LoBind). The 10 µL hybridization reactions were added to 10 µL of resuspended beads for a binding reaction volume of 20 µL. Samples were then incubated in a thermomixer at 50°C for 10 min at 1000 rpm, then moved to a magnetic rack. The supernatant (rRNA depleted) was taken, the beads were washed with 20 µL of Ribowash buffer , and the second supernatant combined with the first supernatant. The supernatant was diluted 1:8 in water and 4 µL of the dilution was used for cDNA synthesis. A no probe control sample, which consists of RNA subjected to hybridization conditions and bead binding in the absence of an added probe, was included in every experiment. In the test of the requirement of the annealing step , hybridization was performed in 10 µL reactions containing 16 pmol of probe mix and 4U RNasin Plus (Promega). For the 1 µg input samples, hybridization was performed in 10 µL with 40 pmol of probe mix and 4U RNasin Plus. The 5 µg input samples (used for RNA-seq) were hybridized in 40 µL reactions containing 200 pmol of probe mix and 40U RNasin Plus. Reactions were denatured at 70°C for 5 min and then added to prewashed MyOne C1 streptavidin beads with the supernatant removed such that the volume of the binding reaction remained the same as the hybridization reaction . Beads were prepared as for the single-probe assay, except Tween was withheld from Dynabeads buffer A. (We observed that Tween forms precipitates in buffer A after storage for >1 wk and thus decided to omit it for further experiments.) After binding, the supernatant was collected, and the beads were washed with 1 volume of Ribowash buffer and the supernatant collected again. The Ribo-Pop depleted supernatant was cleaned up with 1.8X volume of Mag-Bind TotalPure NGS (Omega Bio-tek) beads. After cleanup, Ribo-Pop depleted RNA was subjected to cDNA synthesis and qPCR analysis, where it was compared to a nondepleted input RNA sample from the same source material. 100 ng of Ribo-Pop rRNA removal was applied to 5 µg of larval RNA as described in the previous section. A synthetic RNA spike-in mix, Lexogen E0  was added to the sample before rRNA removal at a ratio of 0.09 ng per 100 ng total RNA for quality control. After rRNA removal, fractions of both the input RNA and the rRNA-depleted RNA were used to construct total RNA libraries or poly(A)-primed libraries. We used the QuantSeq 3′ mRNA-Seq Library Prep Kit FWD for Illumina  to prepare poly(A)-primed libraries. For the input samples, 500 ng of total RNA was used for library construction. For rRNA-depleted samples, the equivalent amount RNA corresponding to 500 ng of total RNA predepletion was used (11 ng). Libraries were amplified with 12 cycles of PCR. We used the NEBNext Ultra II Directional RNA Library Prep Kit (E7765S) to construct the total RNA libraries. We used the equivalent amount of RNA corresponding to 1 µg of total RNA predepletion (22 ng) or a matched amount of nondepleted total RNA. Libraries were amplified for eight PCR cycles. Libraries were loaded onto an Illumina NextSeq 500/550 High Output v2 cartridge and subjected to single-end sequencing on a NextSeq 500 instrument for 85 cycles.https://sourceforge.net/projects/bbmap/) with the parameters “k = 13 ktrim = r useshortkmers = t mink=5 qtrim = r trimq = 10 minlength = 20”, as recommended by Lexogen. Drosophila melanogaster genomic and transcript sequences were downloaded from Ensembl . A STAR . Snakemake (https://github.com/marykthompson/ribopop_rnaseq. The specific versions of software used are listed in Supplemental Table S3.Gene-level quantification presented in nakemake  and Condnakemake  were usenakemake . IllustrRNA sequencing data, including processed files summarizing the gene-level quantification and differential expression analysis, have been deposited in the Gene Expression Omnibus with accession number GSE150332.Supplemental material is available for this article.


P = 0.010); however, its effect was insignificant when applied over the left S1 (P = 0.74). Moreover, a comparison of the change in 50% TPD threshold among the regions revealed that α-tACS applied over the left PPC significantly reduced TPD threshold compared with that applied over the left S1 (P = 0.003). Although we did not reveal the actual changes in cortical activity induced by α-tACS, this is the first empirical evidence that α-tACS applied over the left PPC and left S1 exerts region-specific effects on determining TPD threshold assessed in the contralateral index finger pad by stimulation.Changes in α-band cortical oscillatory activity (8–13 Hz) affect perception; however, how these changes in the left posterior parietal cortex (PPC) and primary somatosensory cortex (S1), which play different roles in determining the two-point discrimination (TPD) threshold, affect TPD threshold remains unelucidated. Therefore, to determine TPD threshold, we aimed to investigate the function of the left PPC and S1 by applying α-band transcranial alternating current stimulation . TPD threshold was examined at the pad of the right index finger, contralateral to the stimulation site, in 17 healthy adults using a custom-made, computer-controlled, two-point tactile stimulation device, with random application of either active or sham α-tACS over the left PPC (Experiment 1) and left S1 (Experiment 2). Then, 50% TPD threshold was obtained in the active and sham conditions via logistic regression analysis. Afterward, we compared the difference between the active and sham conditions at 50% TPD threshold in each region and found that α-tACS reduced TPD threshold when applied over the left PPC ( Haptic information from the outside world is input to areas 3b and 1 of the primary somatosensory cortex (S1) by ascending afferent fibers, such as the slowly adapting fibers and rapidly adapting fibers, and is then sent to the secondary somatosensory cortex (S2) and higher-order areas, such as the posterior parietal cortex (PPC) . Two-poiFurthermore, alpha (α)-band oscillatory activity (8–13 Hz) in the cortex reportedly affects the process of sensory perception in the brain. Perceptual performance negatively correlates with the power of α-band activity in the visual  and somaConversely, increased α-band activity is reportedly associated with improved performance requiring sustained attention, such as in a working-memory task, where higher-order functions are involved . For exaHowever, recent studies have suggested that modulation of α-band activity affects decision confidence and awareness of sensory experiences rather than sensitivity during discrimination tasks in the visual domain . In addiTranscranial alternating current stimulation (tACS) has attracted considerable attention as a non-invasive method to modulate cortical oscillatory activity . This tyConsidering these findings, although the change in the actual oscillatory brain activity caused by α-tACS remains unknown in the present study, we hypothesized that α-tACS may decrease TPD threshold when applied over the left PPC because increased α-band activities are reported to facilitate sustained attention . ConversOverall, 21 healthy college students  with no history of neurological, orthopedic, or psychiatric disorders and who were not receiving any medication during the experimental period were included in this study. All participants underwent the experiments via the application of α-tACS to the left PPC (Experiment 1) and left S1 (Experiment 2). In the TPD task, four participants who answered two points to a one-point stimulus were excluded from the analysis to maintain the consistency of the task; as a result, final analysis included 17 participants. This experiment was conducted in accordance with the Declaration of Helsinki and was approved by the Ethics Committee of Niigata University of Health and Welfare (18264-19092). Full written informed consent was obtained from all participants.2). One of the electrodes was placed over the left PPC  or left S1 , whereas the other electrode was placed on the lateral side of the contralateral shoulder  through a pair of saline-soaked surface sponge electrodes . In the sham condition, stimulation was initiated 1 min before the onset of the TPD task but was only applied for 20 s (10 s of fade in and 10 s of fade out) to avoid stimulation of the cortex during the TPD task. The order of the blocks was randomly assigned for each participant and a 5-min break was introduced between blocks to avoid any carry-over effects from the previous block .X-axis and Y-axis, respectively, following which we fitted a binomial logistic regression model [“glmfit” function on MATLAB (MathWorks Inc.) with “binomial” and “logit” settings] to the data to draw a psychometric function. Thresholds at the correct rates of 25, 50, and 75% were calculated for each stimulus condition (tACS or sham) based on the psychophysical curve via logistic regression analysis. Moreover, 50% threshold was defined as TPD threshold and 75% threshold minus 25% threshold was defined as just the noticeable difference (JND), indicating discrimination sensitivity, as previously described . Moreover, we analyzed discrimination sensitivity using statistical software  in both Experiments 1 and 2. Furthermore, the differences in the 50% threshold recorded in the left PPC and left S1 were compared using the two-tailed paired t-test. To directly compare the possible regions and to demonstrate the reliability of the TPD measures used in our experiments, we used the two-tailed paired t-test to compare the means of the 50% threshold data obtained from the sham conditions in Experiments 1 and 2. The significance level was set at 5% for all statistical analyses. Data are expressed as mean ± standard error (SE).For statistical analysis, we performed the Shapiro–Wilk test to verify the normal distribution of each stimulus region for 25, 50, and 75% thresholds, followed by the two-tailed paired t-test between the two groups revealed a significant decrease in the tACS condition compared with the sham condition  ( = 0.54] . JND wast-test  ( = 0.08] . JND wast(16) = 3.507, p = 0.003, r = 0.66, two-tailed] (t(16) = 0.014, p = 0.93, r = 0.00, two-tailed]  in the left PPC and left S1. The differences in α-tACS-induced threshold in the left PPC and S1 were 0.2 ± 0.28 and –0.02 ± 0.27 mm, respectively, with the changes being significantly larger in the left PPC than in the left S1 [-tailed] . However-tailed] . TherefoThe present study aimed to clarify the effects of non-invasive brain stimulation over the left PPC (Experiment 1) and left S1 (Experiment 2), which were suggested to be related to TPD, on TPD threshold in the right index finger pad using 10-Hz α-tACS. The results of Experiment 1 illustrated that 10-Hz α-tACS of the left PPC, which is the area that plays a vital role in the TPD process and is responsible for attentional control, decreased TPD threshold. In contrast, in Experiment 2, similar stimulation with α-tACS over the left S1, which is suggested to be involved in tactile signal transfer to the higher-order regions of TPD, yielded no changes in TPD threshold. Furthermore, a comparison of the effects of α-tACS on the left PPC and left S1 based on the differences in 50% TPD threshold revealed that α-tACS applied over the left PPC significantly decreases TPD threshold compared with that applied over the left S1.A possible explanation for the improvement of TPD following 10-Hz α-tACS over the left PPC is the increased α-band activity caused by α-tACS. Previous studies have shown that α-tACS enhances α-band activity at the stimulated region ; therefoUnlike Experiment 1, α-tACS of the left S1 in Experiment 2 did not yield any change in TPD. Similar to visual attention, previous studies have shown that when an electrical stimulus is administered to the index finger, the α-band activity in the S1 on the opposite side of the stimulus decreases . This isFurthermore, on comparing the differences in TPD threshold because of α-tACS (50% threshold in the sham condition – 50% threshold in the tACS condition) applied over the left PPC and left S1 in Experiments 1 and 2, respectively, the amount of change in the left PPC was significantly larger than that in the left S1. The reliability of the TPD measurement method was a major issue in the present study, as previous studies questioned the reliability of TPD as a measure of perceptual function . Slowly This study had some limitations in its experimental method. Previous studies have reported changes in cortical oscillatory activity induced by α-tACS ; therefoThe present study reveals that the use of 10-Hz α-tACS over the left PPC effectively reduces TPD threshold; however, a similar stimulation over the left S1 does not yield any significant changes. The effect of 10-Hz α-tACS on the determination of TPD threshold was region-specific in each cortical region involved in TPD. However, our results are speculative because actual cortical activity was not measured; therefore, additional studies are warranted to confirm the causal relationship between α-tACS in the left PPC and sustained attention or α-tACS in the S1, and information processing during the determination of TPD threshold using a more precise experimental paradigm, such as the concurrent measurement of cortical activity, during 10-Hz α-tACS.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by the Niigata University of Health and Welfare. The patients/participants provided their written informed consent to participate in this study.HY and HO conceived the study and designed the experiments. KS and SK performed the experiments. NO interpreted the data. SM, YI, and KN helped draft the manuscript. All authors approved the final version of the submitted manuscript.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.


APC, ATM, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, MLH1, MSH2, MSH6, MUTYH, NF1, PALB2, PMS2, PTEN, RAD51C, RAD51D, and TP53) in 345 whole-blood samples from healthy donors. We subsequently demonstrated that this approach can detect mis-splicing by comparing splicing profiles from the control dataset to profiles generated from whole blood of individuals previously identified with pathogenic germline splicing variants in these genes. To assess the utility of our TSG splicing profile to prospectively identify pathogenic splicing variants, we performed concurrent capture DNA and RNA-seq in a cohort of 1000 patients with suspected hereditary cancer syndromes. This approach improved the diagnostic yield in this cohort, resulting in a 9.1% relative increase in the detection of pathogenic variants, demonstrating the utility of performing simultaneous DNA and RNA genetic testing in a clinical context.Germline variants in tumor suppressor genes (TSGs) can result in RNA mis-splicing and predisposition to cancer. However, identification of variants that impact splicing remains a challenge, contributing to a substantial proportion of patients with suspected hereditary cancer syndromes remaining without a molecular diagnosis. To address this, we used capture RNA-sequencing (RNA-seq) to generate a splicing profile of 18 TSGs ( Aberrant splicing data associated with a DNA variant can be used as evidence of pathogenicity, whereas normal splicing data can be used as evidence of neutrality.3 RNA-sequencing (RNA-seq) has shown significant potential for improving the diagnostic yield and resolution of DNA genetic testing, primarily because of the functional splicing data generated by this analysis.4 Importantly, RNA-seq also addresses a technical limitation of most clinically available DNA genetic tests, which typically capture only exons and short stretches of the flanking introns. Pathogenic variants (PVs) outside the captured sequence will be missed with a DNA-only approach; however, the addition of RNA-seq provides an opportunity to uncover mis-splicing caused by intronic events, leading to the identification of PVs in the non-coding region of genes.2Splicing is the removal of non-coding sequences (introns) from an RNA molecule followed by the ligation of exons, the protein coding regions of genes.8 The addition of WTS has been shown to increase diagnostic yield by 7–36%, depending on the disease studied. Across all studies, pathogenic splicing variants were identified in regions typically captured by current DNA testing methods as well as deep-intronic regions, highlighting the utility of RNA-seq in both identification and interpretation of disease-causing splicing variants.The first attempts to incorporate RNA-seq into clinical diagnostics have involved whole-transcriptome sequencing (WTS) for patients with rare Mendelian disorders who have remained without a molecular diagnosis despite receiving whole-exome or whole-genome sequencing.9 Thus, a substantial proportion of patients currently receiving DNA testing are likely to benefit from the addition of RNA genetic testing. Several studies have also identified pathogenic deep-intronic variants across a range of hereditary cancer conditions, including hereditary breast and ovarian cancer (HBOC),11 Lynch syndrome,13 familial adenomatous polyposis,14 neurofibromatosis,15 and Li-Fraumeni syndrome.16 However, the prevalence of cancer-predisposing deep-intronic variants has not been fully explored due to the limited scalability of previous RNA testing methods.Studies have also shown the benefits of RNA-seq for hereditary cancer predisposition genes; however, this approach has been traditionally performed as a follow-up to inconclusive DNA testing. In a recent study, RNA genetic test results facilitated classification of 88% of the cancer gene splicing variants selected for analysis as either pathogenic or benign, and was predicted to impact 1 in 43 individuals if performed simultaneously with DNA testing.17 By obtaining RNA splicing data in parallel to DNA, we identified PVs that would have been either missed or classified as inconclusive (variant of unknown significance) with DNA testing alone. To determine whether a given splicing event was aberrant, we built a reference control dataset from 345 healthy blood donors using the same RNA-seq capture approach. This allowed us to generate a normal splicing profile for these TSG against which we compared patient’s splicing profiles. We then confirmed the ability of this approach to detect abnormal splicing by testing 25 positive controls that have been previously identified with a PV known to result in mis-splicing. Finally, we demonstrated that this approach can increase the positive yield of genetic testing through the identification of PVs in these cancer predisposition genes.In this study, we test the clinical utility of performing simultaneous capture RNA-seq and DNA multi-gene panel testing (MGPT) analysis on whole blood of 1000 patients receiving genetic testing for hereditary cancer syndromes. The 18 tumor suppressor genes (TSGs) tested were selected because loss of function in these genes have been previously associated with increased cancer risk.18 was performed in 345 healthy individuals, 25 positive controls, and 1000 patients referred to a clinical diagnostic laboratory for suspicion of hereditary cancer  (see Methods). While a minority of alternative splicing events were highly expressed, most exhibited low PSI . In order to determine if the observed data might be explained by the number of exons, we calculated the Pearson’s correlation coefficient between median splice event count and exon number for all 18 genes. Overall, we observed that there is a correlation between number of exons and median splice event count . While 26 recurrent splicing events were identified in BRCA1  . Different genes appeared to tolerate different degrees of alternative splicing. CA1 Fig. , only niCA2 Fig. . In ATM,PSI Fig. . Some alels Fig. . In MUTY20% Fig. . Interes13) Fig. . Four pat test, p < 1 × 10−55) , or hereditary diffuse gastric cancer. For all tested variants, the PSI for the variant-associated splicing event was greater than the mean PSI among controls  after performing consecutive DNA-sequencing and RNA-seq, versus 77 individuals who would have received positive results if MGPT was performed alone, resulting in a 9.1% relative increase in the diagnostic rate. All abnormal splicing events were confirmed by Sanger sequencing of cloned RT-PCR products , which was not detected in controls , which is predicted in silico32 to weaken the native splice acceptor and create a cryptic acceptor, as confirmed by the RNA data. The resulting aberrant transcript is predicted to cause a glutamic acid to leucine substitution at position 29 in the amino acid chain and result in a frameshift causing a premature termination codon (p.E29Lfs*5). In the second case, the proband is a female Caucasian of Ashkenazi Jewish descent diagnosed with breast cancer at the age of 49 years. Skipping of exon 17 (r.5075_5152del) was observed in the proband but not in controls , which is predicted to abolish the splice donor. This in turn is predicted to result in the deletion of amino acids at positions 1692 through 1718 and the insertion of a glycine (p.D1692_W1718delinsG). Saturation mutagenesis studies in BRCA130 support that such a change would result in a non-functional protein.Two cases exemplify the utility of splicing profile by capture RNA-seq in identifying PVs in the HBOC predisposition gene ols Fig. . Analysiols Fig. . A BRCA1BRCA2, full skipping of exon 5 (r. 426_475del) was observed at approximately twice the highest PSI observed in controls (57.7%)  Fig.  in a CauATM, two splicing variants were reclassified from inconclusive to positive due to RNA evidence. The first variant was detected in a Hispanic female proband diagnosed with breast cancer at the age of 64 years. The ATM variant is a rare guanine to adenine substitution five nucleotides downstream from exon 56 (c.8418 + 5G > A). The splicing in silico tools predicted a weakened donor splice site. The aberrant transcript identified by capture RNA-seq , which was absent from controls . Predictions in silico indicated that this cytosine to guanine substitution results in the creation of a novel donor site that is 13 base pairs upstream from the native splice donor site, and this was confirmed by the RNA data. The aberrant transcript is predicted to result in an alanine to valine substitution as position 4 with a frameshift and premature termination codon (p.A4VFS*26).Finally, we detected in a Caucasian female diagnosed with breast cancer at the age of 65 years partial skipping of ols Fig. . This sp38 In this cohort, the 9.1% relative increase in the diagnostic yield for high-risk cancer genes is comparable if not superior to the relative increase in yield gained by the introduction of DNA copy number variation analysis.17 We anticipate that RNA-seq will have an even higher impact in the diagnostic yield among specific genes and phenotypic sub-groups, which will be evaluated as the testing cohort continues to expand.Results from our study demonstrate the feasibility and utility of capture RNA-seq in identifying patients with clinically actionable variants that would have been either missed with DNA testing alone or classified as inconclusive. This whole-blood assay also offers a clinical alternative to other less scalable methods for analysis of splicing variants, such as minigene assays.BRCA1 and BRCA2 have implications not only for early detection and risk reduction of several associated cancer types but also have the potential to inform eligibility for PARP inhibitor therapy.39 Similarly, early detection and prevention options are recommended for individuals with PMS2 PVs.40 For individuals with pathogenic ATM variants, increased breast cancer surveillance is recommended.39The additional increase in diagnostic yield offered by RNA-seq improves a clinician’s ability to accurately apply personalized management strategies for early detection, cancer risk reduction, and treatment. In six of the seven cases with RNA-seq-related positive test results, substantial changes to medical management would be recommended based on current guidelines—not only for the probands but also for family members who test positive as well. PVs in Our capture RNA-seq approach required the generation of a unique control dataset to characterize the splicing landscape of TSGs in healthy individuals. Evaluating whether alternative splicing events are part of normal biological variation is an essential quality component of a clinical grade RNA-seq assay. In the absence of control data, laboratories risk over-interpreting splicing events as pathogenic. Considering the frequency of alternative splicing events in the healthy control population reported this study, failure to consider the natural splicing landscape in cancer genes could result in misclassifications.BRCA1 and BRCA2). Along with further development of standards for the interpretation of RNA findings and the generation of publicly available databases for alternative splicing events, this approach should also be considered to accompany DNA testing panels for other hereditary diseases.Previous studies have leveraged WTS approaches to assess for pathogenic splicing variants across the genome. While our capture RNA-seq approach is more limited in the number of genes analyzed, it has a distinct advantage over WTS as it offers increased depth of coverage for genes that are not highly expressed in blood yet have a known clinical impact . All participants provided written informed consent to take part in the study.n = 1000). Harvested blood was collected and stored in PAXgene™ Blood RNA tubes . For the retrospective cohort, RNA was extracted using either a manual method  or an automated one . RNA yield and RNA integrity number were quantified using a TapeStation with RNA ScreenTape (Agilent).For the healthy controls and retrospective cohort, peripheral blood was drawn from 345 healthy donors and 83 patients participating in the Ambry Genetics Family Studies program, respectively. For the prospective cohort, patients were referred for paired DNA and RNA MGPT to Ambry Genetics by their physician or healthcare provider from 17 medical centers across the United States as part of routine clinical genetic cancer risk assessment  with D1000 ScreenTape. Libraries passing the quality assessment were pooled and subjected to bait capture as described in Mercer et al.18 The post-capture libraries were subjected to a second round of index PCR amplification, SPRI cleaned, and then assessed with the Tapestation. Twelve library pools (96 samples) were sequenced at 2 × 150 bp on a NextSeq 500 (Illumina). CloneSeq and Sanger sequencing of individual colonies was performed as previously described.27 DNA MGPT was performed as previously described.42For capture RNA-seq, complementary DNA (cDNA) libraries were generated using a RiboErase + RNA Hyper Prep Kit (KAPA Biosystems) as previously described.43 De-multiplexing was done with the RTA software 1.17.21.3  and bcl2fastq Conversion software v1.8 . RNA samples that passed sequencing quality control (QC) criteria  were used for downstream analysis. Paired-end RNA-seq reads (2 × 150 bp) were first aligned to the hg19 human reference genome using the STAR aligner v2.6.0c. Average coverage for each exon from the 18 genes .Bar plots, box plots, and violin plots were generated using ggplot2 package (v3.5.3) from R v3.5.1 with default settings. Hierarchical clustering was generated using pheatmap v3.5.2. Sashimi plots were generated using rmats2sashimiplot .Classification of germline variants identified by MGPT was performed following the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines.n = 77). This calculation excluded moderate risk mutations . Physicians/healthcare providers could select up to 18 of the TSGs on the RNA panel for testing . The numerator for diagnostic rate was the difference between the number of patients with a PV after incorporating RNA data (n = 84) and the number of patients with a PV prior to RNA data (n = 77). This yielded a relative percent increase of 9.1%.Among the 1000 patients referred to Ambry for suspicion of hereditary cancer, patients with a PV within any of the 18 TSGs prior to the incorporation of RNA data were considered the denominator for diagnostic rate (Further information on research design is available in the Supplementary MaterialReporting Summary


O-methylation of ribosomal (r)RNA, guiding pre-rRNA processing, and acting as molecular chaperones. Here, we reviewed their activity in promoting the assembly of ribosomes in eukaryotes with regards to chemical modification and pre-rRNA processing.Ribosomes are perhaps the most critical macromolecular machine as they are tasked with carrying out protein synthesis in cells. They are incredibly complex structures composed of protein components and heavily chemically modified RNAs. The task of assembling mature ribosomes from their component parts consumes a massive amount of energy and requires greater than 200 assembly factors. Among the most critical of these are small nucleolar ribonucleoproteins (snoRNPs). These are small RNAs complexed with diverse sets of proteins. As suggested by their name, they localize to the nucleolus, the site of ribosome biogenesis. There, they facilitate multiple roles in ribosomes biogenesis, such as pseudouridylation and 2′- Chemical modifications of RNA are found throughout all domains of life. The MODOMICS database currently lists 172 experimentally confirmed RNA modifications ,4. RibosO-methylation (2′Ome) whose deposition is most commonly facilitated by small nucleolar ribonucleoproteins (snoRNPs). In addition to guiding the deposition of these modifications, certain snoRNPs have critical roles in the regulation the processing and maturation of rRNAs. Further integrating their role in ribosome biogenesis, many snoRNAs, the RNA component of snoRNPs, are found within the introns of ribosomal proteins or ribosome biogenesis factors. In this review, we will discuss the biogenesis of snoRNPs and their role in directing ribosome assembly.Within rRNAs, the vast majority of modifications are either pseudouridylation (Ψ) or 2′-Generally, snoRNPs come in two major classes: Box C/D and Box H/ACA. Although, there are non-canonical snoRNPs, such as RMRP, that have critical roles in ribosome biogenesis, or TERC, which aids in maintenance of telomeres. Regardless of the class, each snoRNP is composed of a snoRNA and an assemblage of proteins. The nomenclature denoting each class of snoRNP is derived from conserved sequence elements within each snoRNA.Xenopus laevis, Xenopus borealis, Rattus rattus, Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Dictyostelium revealed the conservation of Box C and Box D [Box C/D snoRNPs catalyze the methylation of the 2′ hydroxyl group of ribose in RNA A 5]. Th. Th5]. Tnd Box D ,7,8. Butnd Box D . Box C aIn addition to Box C/D snoRNAs, K-turns are found in multiple RNA species, including mRNAs, riboswitches, and small nuclear (sn)RNAs but were first discovered and described in ribosomal RNAs . A canonPhysarum [Box C/D snoRNAs associate with four evolutionarily conserved proteins: Fibrillarin (FBL)/Nop1p, SNU1315.5K)/Snu13p, NOP58/Nop58p, NOP56/Nop56p C. The caPhysarum . Immunop.5K/Snu13Physarum . CrystalPhysarum ,18. BindPhysarum .O-methylation through antisense guide base-pairing. Early work showed that specific snoRNA species could hybridize with mature rRNA species through canonical Watson–Crick base-pairing [The assembled snoRNP mediates site-specific 2′--pairing , which w-pairing ,21,22,23-pairing ,24,25. T-pairing ,27.O methylation using RNA-RNA base-pairing to direct target sites. FBL is the catalytic component of the Box C/D snoRNP.Take home message: Box C/D snoRNPs catalyze the deposition of 2′-6-N3 axis to generate a carbon-carbon glycosidic bond as compared to the carbon-nitrogen glycosidic bond in uridine /Cbf5 C. The fiTake home message: Box H/ACA snoRNPs catalyze the conversion of uridine to pseudouridine using RNA-RNA base-pairing to direct target sites. DCK1/Cbf1 is the catalytic component of the H/ACA snoRNP.SnoRNAs are found in different genomic contexts that broadly fall into two sub-dividable categories: Independent transcription units and intronically encoded snoRNAs . In yeasCaenorhabditis elegans, as many as 59 snoRNA genes are transcribed by RNA polymerase III [Drosophila [While RNA polymerase II (Pol II) transcribes the majority of independently transcribed snoRNA genes, there are some important exceptions. In yeast, all snoRNAs are transcribed by RNA Pol II except snR52, which is transcribed by RNA Pol III ,46,47. Irase III  and at losophila . Of partosophila ,51,52. Yosophila . In humaosophila , but in osophila .7G cap. However, this is either removed by Rnt1 [2,2,7G trimethylguanosine cap by TGS1 [Following transcription, pre-snoRNAs must be processed to generate a mature snoRNA A. This w by Rnt1 , a membe by TGS1 . If the  by TGS1 . Cleavag by TGS1 .Xenopus injection system [Higher eukaryotes have largely embraced a more nuanced mechanism of snoRNA transcription B. Rathern system . Later wn system . The disn system . NOP56 in system . When NOgas5, which hosts 10 snoRNAs (nine in mouse) of the Box C/D family [Another important set of host genes are those that appear to have no coding potential and also do not appear to function as long noncoding (lnc)RNAs. Among the most noteworthy is the U22 host gene (UHG), which, in addition to U22 (SNORD22), hosts seven additional snoRNAs (U25–U31) . All snoD family . SimilarD family . SubsequD family , U19HG [D family , and U50D family , have beD family . What isAgain the molecular intricacies of snoRNA excision from introns has been covered in depth recently , so we wAssembly of both Box C/D and Box H/ACA snoRNPs require multiple assembly factors that mediate RNP formation and trafficking from the site of transcription, through Cajal bodies and to the nucleolus. This complex process has recently been reviewed in . HoweverTake home message: Genomically, snoRNAs are found as independent transcription units or hosted within introns of mRNAs. Both require post-transcriptional processing and assembly with proteinaceous components.O-methylation increases hydrophobicity and pseudouridylation allows for additional hydrogen bonding. Both of these properties can stabilize interactions or provide rigidity. In yeast, global loss of either 2′Ome or Ψ generated through the expression of catalytically dead mutants of Nop1 (FBL) and Cbf5p (DKC1) results in severe phenotypes [2923 to Ψ2923 in the 25S rRNA. Deletion of this snoRNA renders cells more susceptible to osmotic and cold stresses [2918 in the 25S rRNA reduced growth and exhibited translational defects [Escherichia. coli is required for optimal fitness [In many cases, the roles of rRNA modifications remain enigmatic. The conservation and clustering of modification at functional centers, such as the peptidyl transferase center or the subunit interface, point to their importance. With regards to ribosome structure and stability, 2′-enotypes ,83. Inacenotypes . Howeverenotypes . Howeverstresses . Howeverstresses . Yet, thstresses . Again,  defects . The imp fitness . However fitness . An inte fitness .Take home message: Multiple chemical modifications of rRNA have a cumulative effect on ribosome activity.While most snoRNPs function in rRNA modifications, a subset directs rRNA processing. While loss of a single snoRNA that guides modification is rarely detrimental, the loss of a snoRNA that directs processing is often lethal. The rRNA maturation is a baroque process, requiring multiple endonucleolytic cleavage and exonucleolytic trimming reactions . RibosomO-methylation of ribose sugars  cap [The U3 snoRNA was first identified by fractionation of RNA from the rat liver . Further,7G) cap . Cloning,7G) cap ,102.The fact that the U3 snoRNA will interact with rRNA in deproteinized cell extracts suggested that it interacted with these RNAs through base-pairing . Using pSeveral studies proposed that U3 was involved in rRNA processing ,109,110;While the snoRNA itself and base-pairing of the snoRNA to the 5′ETS of the rRNA precursor is required for activity, it is not sufficient for cleavage. Assembly of the snoRNA into a snoRNP is necessary for activity. Autoantibodies against FBL coprecipitated the U3 snoRNA. In yeast, depletion of FBL was shown to have the same effect on rRNA processing as depletion of the U3 snoRNA, suggesting that the interaction between the two is required for activity . The U3 Chaetomium thermophilum [The identification of Mpp10 proved particularly fruitful in the next major discovery regarding the U3 snoRNP. The U3 snoRNP is a central component of a large processing complex called the small subunit (SSU) processome . Using tmophilum .Take home messages: The U3 snoRNP is required for small subunit assembly.RMRP is a non-canonical snoRNA that belongs to neither the Box C/D nor Box H/ACA classes. That RMRP has a role in human pre-rRNA processing has long been established, but the precise role was not understood until recently. Only with the generation of CRISPR/Cas9-mediated deletions of the RMRP gene was it definitively shown that it directs the cleavage at site 2 in ITS1 of human pre-rRNA . RMRP isSaccharomyces cerevisiae, the RMRP ortholog NME1  showed an essential role in cell viability, indicating a critical nuclear role for RNase MRP [3 cleavage site in yeast pre-rRNA, which is thought to be the functional equivalent of site 2 in humans [RMP1, for RNase MRP protein [In Nase MRP . MRP clen humans . Furthern humans ,142,143.n humans . Salinas protein . Charact protein .nme1 mutants [CLB2 (B-type cyclin) mRNA levels leading to increased Clb2p (B-cyclin) levels and a resulting late anaphase delay. One reason for the cell cycle delay in these mutants might be the increased level of CLB2 mRNA. Normally, the RNase MRP complex cleaves the 5′ UTR of CLB2 mRNA. That, in turn, causes a rapid degradation of CLB2 mRNA and efficient cell cycle progression [Another role has been assigned to this RMRP by observing a delay in the progression of the cell cycle at the end of mitosis in some  mutants . This isgression .Take home message: RMRP is a non-canonical snoRNA that is involved in processing of ITS1.Xenopus results in failure to mature large subunit rRNAs. U8 is also necessary for large subunit processing in mice and was recently shown to be necessary in humans as well [The U3 and RMRP are the two-best characterized snoRNPs that are necessary for rRNA maturation, but they are not unique. The U8 and U13 snoRNAs are also independently transcribed snoRNAs genes and are necessary for rRNA processing . Both of as well ,151. The as well . Upregul4N-acetylation of C1773 (ac4C1773) in yeast. Ultimately, they showed that this was dependent upon yeast acetyltransferase KRE33/RRA1. The human homolog of this protein, NAT10, performs the same function at C1842 of the human 18S rRNA [1337 of the 18S rRNA in humans and C1297 of the 18S rRNA in yeast [1773, whereas snR4 directs acetylation of C1280. As U13 has not been shown to direct any 2′-O-methylations, it is intriguing to believe that this snoRNA has evolved to direct acetylation that is required for processing.The U13 snoRNA was among the earliest discovered snoRNAs . Early w18S rRNA . Furtherin yeast . But theO-methylation. Like U3, U8, and U13, the U14 snoRNA is a member of the box C/D family, but unlike those, this snoRNA is intronically encoded, rather than being independently transcribed [O-methylation is required for processing or if this is a separate function of the snoRNP.U14 has undergone intense study over the last 30 years and is also necessary for pre-rRNA processing although this may be linked to its ability to direct 2′-nscribed . In yeasnscribed . Like otnscribed ,157. Thinscribed . Howevernscribed ,159. Unlnscribed . It is uIn addition, several other snoRNPs have been shown to be necessary for efficient pre-rRNA processing; however, the precise mechanism by which they perform these functions is unknown. These include the U22 (SNORD22) , U17 . Indeed, even those that guide RNA modification have been shown to have a role in rRNA processing . However, showing that a particular snoRNA is required for processing does not distinguish between two possibilities: (1) That snoRNP:rRNA interaction is required for processing or (2) that chemical modification of particular nucleotides is necessary for processing. That is, it is possible that certain snoRNPs act as chaperones, changing the secondary structure of the pre-rRNA, which promotes efficient cleavage. Therefore, the snoRNP:rRNA interaction could serve to chemically modify the rRNA and alter the rRNA secondary structure.O-methylation of ribosomal RNA was essential to pre-rRNA processing, but not for rRNA transcription [O-methylation delayed or reduced the efficiency of processing, but did not completely abolish it [O-methylation is evolutionarily conserved, the need for 2′-O-methylation in rRNA processing may not be. In E. coli, treatment with ethionine appears to have no effect on rRNA processing [E. coli grown in ethionine were defective, but this appears to be due to lack of methylation of ribosomal proteins. The fact that rRNA methylation is not required for assembly of E. coli ribosomes may not be surprising since E. coli ribosomes can be assembled in vitro with submethylated rRNA [O-methylation in E. coli is not meditated by snoRNAs. More surprising is that in yeast, some temperature-sensitive mutants in Nop1p/FBL that affect methylation do not impair rRNA biogenesis, but do prevent 2′-O-methylation [O-methylation for maturation of rRNA.Early work by the Darnell lab presented data demonstrating that 2′-cription . Later wcription . Using colish it . Surprisocessing . Howeverted rRNA . Furtherhylation . TherefoO-methylation in which Ψ is required for processing in higher eukaryotes, but appears to be more dispensable in yeast. However, as mentioned above, while unmodified rRNA can be matured, the ribosomes they comprise are defective.It has been more difficult to determine the role of Ψ in rRNA processing due to the lack of inhibitors of pseudouridylation, particularly in vertebrates; however, some progress has been made. In yeast, Cbf5p is essential for viability . However1191 to Ψ1191 which is further modified to 1-methyl-3-γ-(α-amino-α-carboxypropyl)-pseudouridine (m1acp3Ψ) [1191 to Ψ1191 prevents deposition of m1acp3. As a result, the 20S pre-rRNA never matures to the 18S rRNA [There are instances, such as the above-mentioned snR10, in which snoRNAs that direct modifications are necessary for processing ,87. Yet m1acp3Ψ) . This mo18S rRNA .Take home message: There are specific instances in which chemical modification of rRNA aids in pre-rRNA processing, but most are dispensable. The trend is that these modifications are more required in higher eukaryotes than in yeast or bacteria.Loss of specific snoRNAs or reduction in the activity of catalytic components of snoRNPs have been tied to multiple disease states including congenital disorders and cancer. Here, we only focused on those involved in the regulation of ribosome biogenesis or activity, but it is worth mentioning that there are certain instances, such as deletion of SNORD116, which causes Prader–Willi syndrome, but appears unconnected to ribosome function. Recent work also shows that certain snoRNAs can direct modification of mRNAs and tRNAs that likely have a role in maintaining cellular homeostasis ,174.Cartilage-hair hypoplasia (CHH) is a form of dwarfism, first identified by Victor McKusik in Amish communities . PatientO-methylation of rRNA.Another prominent disease related to snoRNP function is X-linked dyskeratosis congenita (X-DC). Classical X-DC is characterized by hematopoietic defects, such as bone marrow loss, and cutaneous abnormalities, such as abnormal pigmentation . MutatioIn recent years, it has also become clear that dysregulation of snoRNPs and rRNA modification is tied to oncogenesis. Much work on miRNA expression used snoRNAs as reference genes for qRT-PCR studies, until it was discovered that snoRNAs are misregulated in tumors . The p532848 and possibly G2863 in the 28S rRNA. However, it is also possible that U50 has a role in directly regulating the expression of KRAS [609 and U863 to Ψ609 and Ψ863, respectively. Particularly important here is Ψ609, which is found in the decoding center of the ribosome. Loss of these modifications had little effect on global translation but were defective in tRNA selection and ribosome translocation. Further investigations have identified dozens of additional snoRNAs whose expression is significantly altered in tumors, either positively or negatively . The meDespite the fact that snoRNPs have been under intense study for 40 years, there is still much to be learned. Recent cryo-EM data have suggested the existence of several unknown rRNA modification . There rWithin the last decade, it was discovered that mature tRNAs serve as precursor molecules for smaller RNA species that have been designated by various names, such as tiRNAs, tRFs, and tRNA halves ,205,206.An even less appreciated class of small RNAs are those derived from rRNAs, which were also discovered at the same time as tRNA-derived RNAs and sdRNAs . Recent Hox genes depends upon ribosomes with a precise set of associated proteins has accelerated research in this area [32P-orthophosphate and 14C-methyl-methionine and conducting RNA fingerprinting [That differential modification of rRNAs may play a role in the generation of rRNA-derived RNAs leads to the most provocative current topic with regards to snoRNPs: Their potential role in the generation of “specialized ribosomes”. While the possibility of such ribosomes has often been speculated, the discovery that mRNAs from his area . As a bahis area . Initialprinting ,217. Pseprinting . More siprinting ,220. CMCprinting ,222,223.printing ,225,226.O-me in RNA and have been particularly useful in mapping this modification in rRNA [O-Me-Seq [Several high-throughput techniques have emerged to specifically analyze both Ψ and 2′Ome. Multiple techniques aim at identifying 2′ in rRNA . These i in rRNA , 2′O-Me-O-Me-Seq , RiboOxiO-Me-Seq , and Nm-O-Me-Seq . RiboMetO-Me-Seq ,232,233.O-methylatiom at particular sites [O-methylation, there are specific sites of pseudouridylation that are differentially modified [Using these approaches, modification heterogeneity has been demonstrated by several groups and revealed with regards to 2′-O-methylation and pseudouridylation. Using RiboMethSeq in isogenic HCT116 cells that were either p53-positive or p53-negative, the Lafontaine and Motorin labs showed that loss of p53 affected the occupation of 2′-ar sites . Sites oar sites . The chaar sites . In thisRNAribonucleic acidbpbase pairntnucleotiderRNAribosomal RNAmRNAmessenger RNAsnoRNPsmall nucleolar ribonucleoproteinsnoRNAsmall nucleolar RNAsnRNAsmall nuclear RNAGguanosineCcytosineAadenosineTthymidineUuridineΨpseudouridine2′OmeO-methyl2′-RMRPRNA component of mitochondrial RNA processing endonucleaseTERCtelomerase, RNA componentSNU13small nuclear ribonucleoprotein 13NOP58nucleolar protein 58NOP56nucleolar protein 56FBLfibrillarinCRISPRclustered regularly interspaced short palindromic repeatsNHP2non-histone Protein 2NOP10nucleolar Protein 10Gar1glycine/arginine rich-domain containing protein 1DKC1dyskerinCbf5centromere binding factor 5TBF1TTAGGG repeat-binding factor 1Rap1repressor/activator site binding protein 1NME1nuclear mitochondrial processing endoribonucleaseRNaseribonucleaseTGS1trimethylguanosine synthetase 1RPS3ribosomal protein small subunit 3RPL7Aribosomal protein large subunit 7ARPL13Aribosomal protein large subunit 13AGAS5growth arrest specific 5mTORmammalian target of rapamycinHSP90heat shock protein 90R2TPRvb1, Rvb2, Tah1, Pih1 complexRvb1RuvB-like protein 1Rvb2RuvB-like protein 2Tah1TPR repeat-containing protein associated with Hsp90Pih1protein interacting with Hsp90 1RUVBL1RuvB-like AAA ATPase 1RUVBL2RuvB-like AAA ATPase 2RPAP3RNA polymerase II associated protein 3RIH1D1PIH1 domain containing 1snR10snoRNA 10snR30snoRNA 30Sbp1suppressor of PaB mutant 1SSUsmall subunitDNasedeoxyribonucleasehU3-55Khuman U3 component, 55 kDaRrp9ribosomal RNA processing enzyme 9cDNAcomplementary DNACRACcrosslinking and high-throughput analysis of cDNAsSof1suppressor of fibrillarin 1Mpp10M-phase phosphoprotein 10MDamegadaltonrDNAribosomal DNAU3uridine-rich RNA 3ETSexternal transcribed spacerITSinternal transcribed spacerSSvedberg unitsMRPmitochondrial RNA processing enzymeCas9CRISPR-associated protein 9UTRuntranslated regionKRE33/RRA1killer toxin resistant 33/ribosomal RNA cytosine acetyltransferase 1NAT10N-Acetyltransferase 10MEFmouse embryonic fibroblastKRASkirsten rat sarcoma viral oncogene homologRASrat sarcomaEMelectron microscopytiRNAtRNA-derived stress-induced RNAtRFtRNA fragmentYB-1Y-box protein 1CMCTN-Cyclohexyl-N′-(2′morholinoethyl)carbodiimide metho-p-toluenesulfonateHCT116human colon cancer 116