C. elegans. Using forward genetic screens and candidate approaches, we find that a core set of nuclear RNAi and chromatin factors is required for multigenerational inheritance of environmental RNAi and piRNA silencing. These include a germline-specific nuclear Argonaute HRDE1/WAGO-9, a HP1 ortholog HPL-2, and two putative histone methyltransferases, SET-25 and SET-32. piRNAs can trigger highly stable long-term silencing lasting at least 20 generations. Once established, this long-term memory becomes independent of the piRNA trigger but remains dependent on the nuclear RNAi/chromatin pathway. Our data present a multigenerational epigenetic inheritance mechanism induced by piRNAs.Transgenerational effects have wide-ranging implications for human health, biological adaptation, and evolution; however, their mechanisms and biology remain poorly understood. Here, we demonstrate that a germline nuclear small RNA/chromatin pathway can maintain stable inheritance for many generations when triggered by a piRNA-dependent foreign RNA response in  ► Multigenerational inheritance and piRNAs converge on same nuclear silencing pathway ► HRDE1/WAGO-9 and chromatin factors required for inheritance of piRNA silencing ► piRNAs can induce multigenerational silencing for more than 20 generations. ► Long-term memory independent of piRNA triggers but remains dependent on nuclear pathway Multigenerational inheritance and piRNAs converge on same silencing pathway, in which both nuclear WAGOs and chromatin factors are required. The piRNA trigger can be lost, but the nuclear silencing pathway maintains the silencing for more than 20 generations. This newly silenced allele can now itself act in a paramutagenic fashion to silence other alleles. Paramutation has been described in multiple species, and it seems likely that small RNAs play a key role in the process, although the full mechanisms involved still remain unclear (Since August Weismann (1834–1914) formulated the distinction between innate and acquired characteristics at the end of the 19th century, the debate relating to the inheritance of acquired traits has raised many controversies in the scientific community . August  unclear .C. elegans has emerged as a key model for the analysis of several related pathways that regulate genes via small RNAs. C. elegans is well suited to the analysis of multigenerational effects, due to its short generation time (∼3 days) and the ease with which they can be maintained under tightly controlled experimental conditions. In eukaryotes, 20–30 nucleotide (nt) RNAs bound to Argonaute (AGO) protein cofactors are the effectors of a number of gene regulation pathways . Secondary siRNA pathways and RNA-dependent RNA polymerases (RdRPs) have not been found in vertebrates or Drosophila, but have been found in many other organisms, including nematodes, plants, fungi, and viruses. Secondary siRNA pathways in C. elegans are complex, can involve many different AGO proteins, and are only partly understood  (C. elegans piRNAs derive from two broad clusters on chromosome IV (trans to regulate endogenous targets in the germline (Piwi-interacting RNAs (piRNAs) are distinct from siRNAs and have an evolutionarily conserved role in transposon silencing in the germline in many animals, including nematodes . C. elegfunction . C. eleggermline . The piR1U-RNAs) . C. elegosome IV  and act germline .Here, we report how transgenerational inheritance of environmental RNAi and the piRNA pathway converge on the same germline nuclear RNAi/chromatin pathway. Both nuclear RNAi factors and chromatin regulators are essential for silencing. This pathway can elicit a long-term epigenetic memory for more than 24 generations. Once established, the initial silencing trigger is no longer required.C. elegans, we developed a heritable environmental RNAi paradigm. Taking advantage of the recent advance in technologies to generate single-copy intrachromosomal transgenes in C. elegans transcriptional gene regulation. Using quantitative RT-PCR, we tested whether Hrde sensor silencing in the F2 generation affected either. We were able to robustly detect both primary transcript (pre-mRNA) and Hrde transgene mRNA. However, mRNA levels were significantly repressed (p < 0.05) in silenced animals as compared to nonsilenced animals D. pre-mRC. elegans germline results in secondary siRNA-dependent silencing of a “piRNA sensor” and endogenous piRNA targets  orthologs, HPL-2  are dependent on the Piwi protein PRG-1 . Silencing of the piRNA sensor is established through an endogenous piRNA (21-UR-1) with perfect complementarity to a corresponding sequence in the piRNA sensor  described above remains unaffected in the soma , we generated F1 animals homozygous for the piRNA sensor but heterozygous for the recessive mutation in prg-1  , F20, and F24 (six strains) generations. We also observed that silencing can become PRG-1 independent using a second piRNA sensor construct integrated on a different chromosome  A and 5B.prg-1, mutation of nrde-1 (yp4 or yp5) or mutation of nrde-2 (gg95) triggered reactivation of outcrossed piRNA sensors   C. All nrrsd-6 or rsd-2 suggested that they could function to promote systemic spreading of RNAi from soma to germline . These results suggest that the original RNAi model of multicopy transgene silencing needs to be revised. Indeed, there appears to be no requirement for dsRNA intermediates in the silencing phenomena reported here either, as factors required for dsRNA-induced RNAi in the germline such as RSD-2 and RSD-6 , licensing of germline transcripts (self RNA recognition), and unpaired genomic DNA in meiosis. Based on this and related work, we propose that the piRNA pathway can detect transgenes as sources of foreign RNA (nonself) and initiates targeted silencing of these transgenes (Cell]). The piRNA pathway is perfectly suited for this task, as it provides a diverse and large set  of small RNA triggers that are mismatch tolerant but do not depend on dsRNA generation  transcriptional pathways both in yeasts and plants . Many yepathways . DespiteC. elegans. Our study of transgenes targeted by an endogenous piRNA may recapitulate the fate of transposons that are transmitted in rare horizontal transfer events, in which a single transposon insertion could be subjected to dual layers of silencing, as the locus would likely remain heterozygous for a number of generations before potentially becoming fixed. It will be of great interest to identify the factor(s) that determines these distinct states of silencing.Transgenerational phenomena have been observed over one or multiple generations . In someC. elegans were grown under standard conditions at 20°C unless otherwise indicated. The food source used was E. coli strain HB101 . Bleaching followed by starvation-induced L1 arrest was used to generate synchronized cultures. The wild-type strain was var. Bristol N2  was used to simultaneously measure length (time of flight), absorbance (extinction), and fluorescence. Data handling and analysis were performed using FlowJo  and R.Extended Experimental Proceduresrol-6; dpy-17 or unc-13; rol-6 were first created to make prg-1; mjIs144 or mjIs144, nrde-2 or mjIs144; nrde-1 strains. To facilitate making strains with mjIs144, the unc-119(ed3) mutation was first removed from SX1316 by crossing rol-6 e187 / + males with single SX1316 hermaphrodites, selecting for F1 with Rol F2, singling many non-Rol siblings, selecting for F2 with Rol F3 but no Unc F3, and then selecting against rol-6 to obtain mjIs144 homozygotes. unc-13; mjIs144 and mjIs144; dpy-17 strains were then created by crossing rol-6; dpy-17 or unc-13; rol-6 with N2 wild-type males, crossing F1 rol-6; dpy-17 / +; + or unc-13; rol-6 / +; + males with single mjIs144 hermaphrodites, selecting for F1 that segregated both marker mutations, and then selecting for F2 that lacked rol-6 and then unc-13 or dpy-17 F3. Heterozygous prg-1 / unc-13 males stock were maintained by backcrossing heterozygous males with unc-13 homozygotes. Heterozygous nrde-1 / dpy-17 males stock were maintained by backcrossing heterozygous males with dpy-17 homozygotes. prg-1; rol-6 strains were crossed with prg-1 / unc-13 males, and F1 males were crossed with unc-13; mjIs144 hermaphrodites, and F1 with both marker mutations were transferred for several generations to obtain strains that lacked Unc or Rol mutations, which were prg-1; mjIs144 strains. rol-6; nrde-1 animals were crossed with nrde-1 / dpy-17 males, F1 males were crossed with mjIs144; dpy-17 hermaphrodites, and F1 with both marker mutations were transferred for several generations to obtain strains that lacked Rol or Dpy mutations, which were mjIs144; nrde-1. nrde-2 is tightly linked to the piRNA sensor (mjIs144) on chromosome II, so dpy-10, piRNA sensor, unc-4 strains were first created, and then F2 Dpy-non-Unc recombinants from dpy-10, mjIs144, unc-4 / +, +, nrde-2 F1 were singled, allowed to self, and genotyped for the presence of the piRNA sensor. Three of 6 F2 Dpy-non-Unc recombinants possessed the piRNA sensor, and F4 progeny of F3 homozygotes for piRNA sensor, nrde-2 displayed uniformly GFP. To construct piRNA sensor; rsd-2 strains, rsd-2; rol-6 and unc-24; mjIs144 strains were created using a rol-6; unc-24 strain. rol-6; rsd-2 hermaphrodites were then crossed with rsd-2 males. Male offspring were crossed with unc-24; mjIs144 hermaphrodites. F1 from this cross were singled. F2 with an unaffected phenotype but with both rol-6 and unc-24 siblings were singled. F2 that segregated neither unc-24, nor rol-6 offspring were selected, producing strains homozygous for both rsd-2 the piRNA sensor. To construct rsd-6; piRNA sensor strains, rsd-6; rol-6 and dpy-5 unc −55; mjIs144 strains were created using a dpy-5 unc-55; rol-6 strain. rsd-6; dpy-10 unc-4 hermaphrodites were then crossed with rsd-6 males. Male offspring were selected and crossed with dpy-5 unc-55; mjIs144 hermaphrodites. F1 were singled and those which produced dpy-5 unc-55 and dpy-10 unc-4 F2 were selected. Unaffected F2 were singled from these plates. Finally, F2 which segregated neither dpy-5 unc-55 nor dpy-10 unc-4 were selected. Independent strains that were homozygous for both rsd-6 and the piRNA sensor were tested for GFP fluorescence.Marker double mutants SNP mapping of mutations was performed as described previously . Primer prg-1; mjIs144 strains had both the prg-1 n4357 allele and the mjIs144 transgene, DNA was extracted from a recently starved plate of each prg-1; mjIs144 strain. PCR was performed using Bam-GFP-FW and Bam-GFP 2 Stop-RV primers to confirm the presence of the transgene. DNA from wild-type worms and mjIs144 worms were used as controls. The PCR products revealed an approximately 800 bp band indicating the presence of the GFP DNA sequence in the mjIs114 control as well as all in eight prg-1; mjIs144 strains, but not in the wild-type control. PCR using n4357 FW and n4357 RV primers was used to verify the presence of the prg-1 n4357 allele. DNA from wild-type worms and prg-1 n4357 worms were used as controls. The PCR products revealed a long band non mutant band for wild-type but only a shorter band for the n4357 deletion control as well as for all eight prg-1; mjIs144 strains, confirming that the prg-1; mjIs144 n4357 strains were homozygous for n4357.In order to verify that the rsd-6 was amplified from the fosmid WRM064bA10 (Source Bioscience) and subcloned into pDONR 221. Ubiquitously expressed dpy-30 promoter and the 3′UTR of rsd-6 were amplified from N2 wild-type genomic DNA and subcloned into pDONR P4-P1R and pDONR P2R-P3 vectors, respectively. Germline-specific pgl-3 promoter is a gift from Kyle Wang. Fragments for promoter, gene and 3′UTR were combined into pCFJ150 vector (Caenorhabditis briggsae unc-119(+) gene. All pDONR subclones were sequenced to confirm lack of mutations, and att recombination sites were sequenced in the final promoter:gene:3′UTR constructs prior to microinjection. Primers were following; attB1-RSD6FW, ggggacaagtttgtacaaaaaagcaggctatgaatgaaaaagagctggcggatt; attB2-RSD6RV, ggggaccactttgtacaagaaagctgggttcagataaagacgtctttgatattc; attB4-dpy30p-FW, ggggacaactttgtatagaaaagttggtctattctcacacctctcc; attB1-dpy30p-RV, ggggactgcttttttgtacaaacttgcttggtttttgctcgatttct; attB2-rsd6-3U-FW, ggggacagctttcttgtacaaagtggacttcaaatcatgtttctatctaaa; attB3-rsd6-3U-RV, ggggacaactttgtataataaagttgtctcatgtatattgtttgatgtgaa.Rescue constructs were prepared using MultiSite Gateway Three-Fragment Vector Construction Kit (Invitrogen). A PCR product corresponding to the coding region of 0 vector  includinrsd-6(pk3300) I, ttTi5605 II (Mos1); unc-119(ed3)III and the integrants were obtained using the ‘Mos1 excision-induced transgene-instructed gene conversion’ method  and fluorescence imaging was performed using standard methods using an AxioImager A1 upright microscope . Images were captured using an ORCA-ER digital camera  and processed using OpenLabs 4.0 software . For RNAi inheritance assay was performed as described above. Silenced animals were sorted using the biosorter and used to generate the F2 generation. Silenced F2 worms were sorted and collected into Trizol ®. Control worms were grown alongside under identical conditions. RNA extraction was performed using standard protocols. cDNA was synthesized from 5 μg total RNA using Superscript III reverse transcriptase (Invitrogen) with random hexamers. qRT-PCR was performed using a ABI7300 Real Time PCR system (Applied Biosystems). Primers sequences are as follows: nascent transcript F: TCTGTCAGTGGAGAGGGTGA; nascent transcript R: TTTAAACTTACCCATGGAACAGG; mRNA F: CTACCTGTTCCATGGCCAAC; mRNA R: GGCATGGCACTCTTGAAAAA.RNAi bacteria was inoculated into LB Broth containing Ampicillin (50 μg/ml) for 6 hr at 37°C with shaking. Bacterial cultures were then seeded on to NGM plates containing IPTG (1 mM) and Carbenicillin (25 μg/ml) and grown overnight at room temperature. Adult animals were placed on the bacteria and the cultures grown at 20°C for 4 days. GFP fluorescence in the germline and oocytes of adult offspring was assayed using either a large particle biosorter (Union Biometrica) or a fluorescent microscope (Kramer scientific). Ten (biosorter) or one (microscope) silenced adults were selected and placed on HB101 growth plates to produce the next generation. This procedure was repeated for up to five generations.After EMS treatment following standard protocols, synchronized P2 larvae (L1) were placed onto GFP RNAi plates for 3 days at 20°C. Adult RNAi P0 animals (EMS P2s) were individually picked onto standard HB101 NGM plates and grown at 20°C for 4 days. The resultant F1 (EMS P3) animals were screened for GFP phenotype and any plates with a substantial number of GFP expressing animals kept for secondary screening. Secondary screening was performed as described above for the RNAi inheritance assay. Screening was performed in 8 batches with 600 P2s individualized each time.F2 or F3 offspring of mutagenized worms were sorted using a Copas Biosort Large-Particle Sorter as described in . Worms sorted as ‘GFP ON’ were re-selected for sensor de-silencing using an FBS10 Fluorescence Microscope System (Kramer Scientific).We used a pellet of approximately 60-100 μl of packed mixed stage worms and froze at −80°C. For extraction of genomic DNA we followed the Quiagen DNeasy Blood and Tissue kit  including an RNase treatment step. We eluted DNA in 2x 200 μl buffer and quantified using the QuBit dsDNA BR assay kit (Invitrogen). Prior to library preparation we cleaned 150 ng of genomic DNA using the DNA Clean& Concentrator-5 Kit (Zymo Research Corp.). We eluted in 15 μl pre-heated water and re-measured DNA concentration using the QuBit dsDNA HS DNA assay kit.We prepared libraries from genomic DNA using the EPICENTRE Biotechnologies Nextera DNA Sample Prep Kit. Tagmentation was performed in HMW Buffer and PCR-amplified libraries were purified either using the DNA Clean & Concentrator-5 Kit (Zymo Research Corp.) or Agencourt AMPure XP beads (Beckman Coulter). Library concentration was measured using the QuBit dsDNA HS DNA assay kit, then fragment length and distribution were determined with the Agilent High Sensitivity DNA assay (Agilent Technologies Inc.). Whole-genome sequencing of simplex libraries was performed on either the Genome Analyzer IIx or the High-Seq 2000 Illumina platform. Sequencing data were analyzed as described previously .Total RNA was isolated using the mirVana miRNA isolation kit (Ambion). The total RNA samples were separated on denaturing 15% polyacrylamide gels and 18-30 nt length species selected. Following poly-A tailing, small RNAs were treated with tobacco acid pyrophosphatase (Epicenter). Adapters were then ligated to the 5′ phosphate of the RNA. First-strand cDNA synthesis was performed using an oligo(dT)-adaptor primer and the M-MLV reverse transcriptase. The resulting cDNAs were PCR-amplified to about 20-30 ng/μl in 18-20 PCR cycles using a high fidelity DNA polymerase. cDNA were purified using the Macherey & Nagel NucleoSpin Extract II kit. Libraries were sequenced at CRI using an Illumina GA2x instrument.Total RNA was isolated using TRIsure reagent (Bioline). Total RNA was treated with RNA 5′ polyphosphatase (Epicenter). cDNA libraries were then prepared following the TruSeq Small RNA protocol (Illumina). Libraries were sequenced on a MiSeq machine (Illumina).C. elegans genome (assembly WS190/ce6) and refGene annotation were downloaded from the UCSC Genome Browser website (http://genome.ucsc.edu/) . Reads were trimmed by removing 5′ barcodes, 3′ adapters or 3′ As depending on the protocol used for library generation. Inserts with length 18-30 nucleotides were collapsed to unique sequences, retaining the number of reads for each sequence. The sc.edu/) . Perfectsc.edu/) .The anti-HRDE-1/WAGO-9 polyclonal antisera were generated using Genomic Antibody Technology (GAT) by SDIX . In brief, DNA immunization was done using cDNA encoding amino acids 12 to 161 of HRDE-1/WAGO-9. Polyclonal sera were subsequently affinity-purified and tested for specificity in Western blotting (data not shown) and immunostaining. Immunostainings were performed on dissected gonads. Animals were dissected in 10 mM Tetramisol on microscopy slides. After freeze-cracking, the tissue was fixed in ice-cold Methanol for 20 min and rehydrated by three washes in 1xPBS/0.2% Tween 20 .Primary antibodies were added over night at 4°C in 1xPBS/0.2% Tween 20/1% BSA. Slides were washed three times in 1x PBS/0.2% Tween 20 . Secondary antibodies were added for one hour at 37°C. The secondary antibody mix contained 4′,6-diamidino-2-phenylindole (DAPI) at 100 ng/ml final concentration. Slides were washed three times in 1xPBS/0.2% Tween 20  and mounted using Vectashield. We carried out differential interference contrast (DIC) and confocal fluorescence imaging using standard methods and using an Olympus FluoView FV1000 upright microscope using 40x and 63x objective magnification. Images were processed using the FluoView image software (Olympus) and ImageJ (version 1.43u). Antibodies and concentrations used are: rabbit anti-HRDE-1/WAGO-9 1:4000, mouse OIC1D4  1:100, mouse anti-α-Tubulin  1:10,000, Alexa Fluor 488 anti-rabbit IgG, 1:1000, Alexa Fluor 568 anti-mouse IgG, 1:1000 (both Life Technologies).


T-705 (Favipiravir) is a broad-spectrum antiviral molecule currently in late stage clinical development for the treatment of influenza virus infection. Although it is believed that T-705 potency is mediated by its ribofuranosyl triphosphate (T-705 RTP) metabolite that could be mutagenic, the exact molecular interaction with the polymerase of influenza A virus (IAVpol) has not been elucidated. Here, we developed a biochemical assay to measure the kinetics of nucleotide incorporation by IAVpol in the elongation mode. In this assay, T-705 RTP was recognized by IAVpol as an efficient substrate for incorporation to the RNA both as a guanosine and an adenosine analog. Compared to natural GTP and ATP, the discrimination of T-705 RTP was about 19- and 30-fold, respectively. Although the single incorporation of the ribonucleotide monophosphate form of T-705 did not efficiently block RNA synthesis, two consecutive incorporation events prevented further primer extension. In comparison, 3′-deoxy GTP caused immediate chain termination but was incorporated less efficiently by the enzyme, with a discrimination of 4,900-fold relative to natural GTP. Collectively, these results provide the first detailed biochemical characterization to evaluate the substrate efficiency and the inhibition potency of nucleotide analogs against influenza virus polymerase. The combination of ambiguous base-pairing with low discrimination of T-705 RTP provides a mechanistic basis for the in vitro mutagenic effect of T-705 towards influenza virus. Orthomyxoviridae family and is the causative agent of recurring seasonal and pandemic influenza outbreaks. There are currently only two classes of FDA-approved drugs for the treatment of influenza virus infection, the M2 ion channel blockers and the neuraminidase inhibitors. The M2 ion channel blockers amantadine and rimantadine are of limited use mainly due to rapid emergence of resistant viruses, to the point that currently circulating seasonal H1N1 and H3N2 influenza viruses harbor mutations in the M2 protein and are amantadine resistant The influenza virus belongs to the In vitro, T-705 is efficiently converted to its ribofuranosyl 5′-triphosphate (T-705 RTP) form by cellular enzymes The antiviral 6-fluoro-3-hydroxy-2-pyrazinecarboxamide  is a nucleoside precursor entering phase III clinical trials in the United States for the treatment of influenza virus infections Here we report the detailed mechanism of inhibition of influenza A virus polymerase by T-705 RTP. We designed specific short RNA templates used in enzyme kinetic assays to probe the selectivity of recombinant IAVpol against different nucleotide analogs through single- and multiple-nucleotide incorporation experiments. We show that, (1) T-705 RTP is efficiently recognized by IAVpol both as a guanosine and as an adenosine analog, (2) single incorporation of T-705 RMP into RNA delays but does not block primer extension, and (3) two consecutive events of incorporation of T-705 RMP completely block subsequent RNA synthesis. Collectively, this study provides the first biochemical characterization of IAVpol interaction with T-705 RTP, further supporting the observation that T-705 could induce lethal mutagenesis during virus replication. In addition, our results offer new insights into the enzymatic fidelity of influenza virus polymerase, as they suggest that the overall error rate during RNA synthesis could be higher than previously measured.2, EDTA, NaCl solutions and Tris-Cl buffers were purchased from Life Technologies. Radiolabeled nucleoside triphosphates were purchased from Perkin Elmer. RNA oligonucleotides . Rabbit globin mRNA and heparin sodium salt were from Sigma-Aldrich. MgClCloning, expression and purification of the influenza A virus PA/PB1/PB2 polymerase complex were performed as previously described 7 IU/ml virus was incubated for 10 min in a reaction buffer containing 50 mM Tris-HCl, pH 8, 50 mM KOAc, 5 mM MgCl2, 1 mM DTT and 0.5% Triton X- 100. Viral genomic RNA transcription was started by mixing the lysed viruses with 12.5 ng/µl rabbit globin mRNA, 50 µM ATP, 50 µM UTP, 50 µM CTP, 2 µM GTP, and 0.17 µM [α-33P]GTP in the same reaction buffer at 37°C for 1 h. The reactions were quenched with a solution containing 90% formamide, 50 mM EDTA, 0.1% bromophenol blue and 0.1% xylene cyanol. The samples were denatured at 95°C for 5 min. The samples underwent electrophoresis on a 6% denaturing PAGE gel (Invitrogen) at 190 volts for 50 minutes.Influenza virus (A/WSN/33 (H1N1)) was purchased from Virapur . 5×102, 0.5 mM EDTA, 2 mM DTT, 5% glycerol), 0.15 µM polymerase complex, 0.4 mM 5′-ApG primer, 1.5 µM 50-nt 3′vRNA template, 1.6 µM 15-nt 5′vRNA, 0.20 U/µl RNaseIn, 500 µM UTP, ATP, CTP, and 1 µM GTP mixed with 2.5 µCi [α-33P]GTP. The competition assays were performed under similar conditions, except that each competing nucleotide was tested at low (3 µM) or high (300 µM) concentration, while keeping the other NTPs at 300 µM. For the GTP and ATP competition assays, we replaced the isotope tracer to [α-33P]CTP. The reactions were stopped by the quench solution containing formamide with 50 mM EDTA. The samples were denatured at 95°C for 5 min. The samples were run on a 15% denaturing PAGE gel (Invitrogen) at 190 volts for 50 minutes.The activity of the purified polymerase complex was measured using the 5′-ApG replication assay as described previously 2, and 2 mM DTT), 0.15 µM polymerase complex, 1 µM 15-nt RNA promoter, 2.5 µM 14-nt RNA template, 0.4 mM 5′-pApG primer, 0.2 U/µl RNaseIn, 25 µM UTP, 25 µM CTP, 0.033 µM [α-33P]CTP, and other NTPs at various concentrations as indicated in figure legends. The replication reactions were stopped after 35 min by mixing with 2x volume of the formamide quench solution. The quenched reactions were denatured at 95°C for 3 min and then were loaded onto a 22.5% denaturing polyacrylamide gel with 7 M urea . The electrophoresis was performed at 80 watts using a Sequi-Gen GT system from Bio-Rad .The recombinant polymerase complex catalyzed the primer extension reaction using 5′-pApG as primer, a 14-nt synthetic RNA oligonucleotide as template and a 15-nt RNA as promoter. The 14-nt RNA template and 15-nt RNA promoter form a panhandle structure that was required for RNA replication activity of the influenza polymerase complex Denaturing PAGE gels were dried at 80°C for one hour with a Model-583 gel drier . Dried gels were exposed to storage phosphor screens and visualized by a Typhoon scanner . The fraction of RNA products were calculated based on the intensities of bands on the gel that were quantified using the ImageQuant software .50- The compound concentration at which the enzyme-catalyzed rate was reduced by 50% (IC50) was calculated by fitting the data to the equationIC50 value was derived from the mean of a minimum of two independent experiments.Where Y corresponds to the percent relative enzyme activity, % Min is the residual relative activity at saturating compound concentration, % Max is the relative maximum enzyme activity, and X corresponds to the compound concentration. The ICKapp - The phosphor images of the reactions were analyzed by ImageQuant software. For each lane, the intensity for each band starting from 9-mer up to 14-mer was quantified. The percentage of the bands intensity (10-mer to 14-mer) in the total intensity (9-mer to 14-mer) represented the percentage of the products converted from 9-mer. The data of concentration dependent 9-mer conversion to product were fit to a hyperbolic equation to obtain the Kapp concentration.Y is the percentage of all observed products, %Min is the percent product from mis-incorporation when the nucleotide in the study was not added, %Max is the maximal product percentage, S is the concentration of NTP in the study, and Kapp is the concentration of NTP when half of the 9-mer was converted to products. All Kapp values were derived from the mean of a minimum of two independent experiments.Where correct) and an analog or incorrect nucleotide (NTP) can be calculated asDiscrimination defines the relative incorporation efficiency of a nucleotide analog or an incorrect nucleotide to the incorporation efficiency of the natural correct nucleotide. According to our equation derivation  for IAVpol can be calculated asWhere We initially evaluated two nucleotide analogs against the RdRp activity of viral ribonucleoprotein (RNP) complex prepared from purified influenza virus particles. We used 3′dGTP as a positive control for polymerase inhibition because it is an obligate chain terminator 50 of 43±12 and 2.9±0.14 µM, respectively . This new template sequence was designed to maintain partial base-pairing with the modified 5′vRNA complementary strand (t14-1 promoter), while allowing for single incorporation of GMP or GMP analogs at position +10 . When weKapp . We found that T-705 RTP was discriminated only by 19-fold, compared to 330,000- and 4,900-fold for ATP (A:C mismatch) and 3′dGTP, respectively , instead of guanosine opposite C . We suppposition . Using t003 µM−1 . We calcKapp of AMP with this new sequence was 0.038 µM, a value that is similar to the result obtained with the t14-2 RNA template  containing a series of 3 pyrimidines (UCC) at positions +10, +11, and +12 . When juT-705 is currently the most advanced small molecule clinical candidate for the treatment of influenza A and B virus et al. Kapp  result showing the decrease in radiolabeled viral RNA product from the enzymatic reaction in the presence of increasing concentrations of a nucleozin analog used as nucleoprotein inhibitor. Concentrations of inhibitor are as follows: lane 1 (0), lane 2 (0.015), lane 3 (0.046), lane 4 (0.14), lane 5 (0.41), lane 6 (1.2), lane 7 (3.7), lane 8 (11.1), lane 9 (33.3), and lane 10 (100 µM). Figure S2. Determination of ATP substrate efficiency using t14-3 RNA Template. (A) Natural ATP was added to the reaction at increasing concentrations up to 10 µM and the quantitative analysis of AMP incorporation based on the extension of the 9-mer RNA product to the +10 position. (B) The percentage of the extended products from 9-mer was plotted against ATP concentration and the data was fitted to a hyperbolic equation Click here for additional data file.


Eukaryotes use RNA interference and RNA-based epigenetic regulation to control transposon activity. In the standard pathways of RNA-based transcriptional and post-transcriptional silencing the protein complex RNA-dependent RNA polymerase (RdRP) plays a crucial role. However, alternative pathways that bypass RdRP have recently been described. Hence two important questions are: is RdRP truly a necessary component for transposon control, and are the alternative RNA-based strategies also capable of controlling transposable elements?We have studied the interplay between host RNAi pathways and transposons using mathematical models. We show that the canonical RdRP-based model controls transposons tightly, mainly via the feedback of cytoplasmic small RNA amplification. Next, we consider two variants lacking RdRP and instead employing antisense transcription of transposons. We show that transposon activity is also controlled by the alternative pathways, although cytoplasmic small RNA amplification is absent. Instead, control occurs in the nucleus, through a feedback in the epigenetic regulation.Concluding, our models show that the control of transposon activity can be achieved by alternative pathways that lack RdRP and act through different feedback mechanisms. Thus, although RdRP activity is ubiquitous in eukaryotes, it need not be a general requirement for transposon control. Transposons are found in virtually all eukaryotes. They are DNA sequences that have the ability to create copies of themselves in the genome. This copying activity has been linked to a range of deleterious mutations, for instance chromosomal aberrations, and faulty expression of genes . On the The large diversity of TEs, their different sequences and copy strategies have resulted in a variety of host responses . In thisPTGS takes place in the cytoplasm. A double-stranded RNA molecule (dsRNA) is cleaved into small interfering RNAs (siRNAs), 21-25 nucleotides long, by a protein of the Dicer family. Next, single siRNA are loaded onto Argonaute proteins, which are part of a RNA Induced Silencing Complex (RISC). RISC identifies complementary RNA transcripts and subsequently degrades them by cleavage. In this manner TE mRNAs cannot be translated and thus TE activity is inhibited. In many eukaryotes the silencing response is enhanced and sustained by amplifying the number of siRNAs by means of creating secondary siRNAs . Complemcis [trans-effects as well [In the nucleus TEs are silenced by TGS. Both DNA methylation and histone modification patterns lead to the inhibition of expression of the underlying DNA sequence. Here we focus on (di)methylation of histone 3 at lysine 9 (H3K9me) as the signal resulting in heterochromatization. Such modifications are initiated and maintained by small RNAs . Schematcis , we assu as well .D. melanogaster) and mammals (mouse and human), canonical RdRP has not been found [In both above described silencing processes the protein complex RdRP appears to be a crucial component: it is required for the formation of dsRNA and for sustaining the cytoplasmic silencing response. In lower eukaryotes this role is fulfilled by the 'canonical' RdRP complex. In higher eukaryotes, such as fly  to two antisense-based models (the antisense and hairpin model). Specifically, we address this question in terms of active and silenced transposons, and abundance of TE products in the cytoplasm. Furthermore, we study the robustness of model behavior against parameter changes and how each of the models accomplishes transposon control by means of feedback loops.We studied mathematical models describing the dynamics of TEs, euchromatin and heterochromatin formation and cytoplasmic RNAi-based TE silencing. We report on three models composed of ordinary differential equations, which we introduce below , chromatin-silenced TEs (silT) and the number of virus-like particles in the cytoplasm (VLP) over a simulation time of 2 years and the final numbers of each of these observables we use in our comparison. In order to quantify how well a host controls its transposons, we take a single, rather strict condition that if VLP <1.0, we regard the host to be in control.We compared three different TE control mechanisms by studying the scenario in which a single transposon invades a new genome: act T+ sil ≤ T2.0). Clearly, in these simulations the transposable element has hardly been able to copy itself within the two year time span. There are two underlying causes. the TE is not capable of increasing its copy number due to a very strict silencing regime of the host or the TE has an intrinsic low copy rate, i.e. it is a rare event that simply occurred only once. We refer to this group of simulations as cluster 1, or the "no invasion" cluster.The first observation we make, is that the RdRP model and the two antisense variants produce results in similar orders of magnitude, both with respect to active against silenced TEs, and active TEs against VLPs Figure . If we f>2 to ~104. Thus TEs invaded the host genome, but if we look at the level of VLPs, we also find most runs satisfy the condition of VLP <1.0. We find that in the RdRP model the host silences a fraction of 0.90, while in the antisense model a fraction of 0.98 is under control and in the hairpin model a fraction of 0.91  of VLPs . In other words, due to an insufficiently strong feedback in siRNA production, the RdRP-based silencing response is inadequate for the control of transposable elements.Secondly, the RdRP and antisense model have an additional third cluster in which a high number of silenced TEs has accumulated relatively to the number of active TEs. However, there is a distinct difference between the two models with respect to the levels of VLP in the cytoplasm. In the RdRP model, this third cluster corresponds to high levels (sil T≈ 104) and thus a relatively large number of asRNA is available for silencing TEs.In contrast to the high levels of VLPs in the RdRP model, the antisense model shows the opposite behavior Figure . Along tThis behavior does not occur in the hairpin variant. The main difference between the antisense and hairpin model is that in the antisense model the asRNA is also available in the cytoplasm, thus resulting in the observed low VLP levels.Given the above observations, we come to the interesting conclusion that in all three models the large majority of parameter settings result in invading TEs being silenced (controlled) by the host. At least within our modeling framework the two pathways based on antisense transcription appear to be viable, robust alternatives to the standard RdRP-based pathway. In other words, our results suggest RdRP does not need to be as essential for silencing transposons as has been implicitly assumed up to now.In order to appreciate how the two antisense-based variants allow for equally robust TE control as the RdRP model, we need to understand the underlying mechanism of this silencing. How are TEs exactly silenced in the three models?First of all, all three models share an amplification loop of TE growth. If left unchecked, exponential growth is realized via transcription of transposons, several stages in the cytoplasm and the subsequent integration of a new transposon in the host genome.Secondly, what sets the RdRP model apart from the antisense variants is that there is an amplification of siRNAs in the cytoplasm. The production of siRNA promotes the recruitment of mRNA into dsRNA formation, hence leading to more small RNAs Figure . In thisIn contrast to the cytoplasmic control of the RdRP model, the two alternatives utilize a feedback loop in the nucleus via heterochromatization. As mentioned in the Introduction, heterochromatin is not equivalent to complete transcriptional silencing. Instead from genomic regions where TEs reside, RNA transcripts originate that are processed into small RNAs, which in turn guide the formation and expansion of heterochromatin into neighboring regions and other genomic locations with matching base pairs between the small RNA and the genomic DNA. That is to say, the small RNAs spread a silencing signal over the host genome. As more TEs become silenced in heterochromatin, the signal becomes stronger until an equilibrium is reached where active and silent transposons keep each other in check. In combination with sense-antisense duplex formation or hairpin formation in the cytoplasm, this nuclear-based control is sufficient for a robust silencing of TEs Figure  and 3. Tpn(x), and cytoplasm, pc(x) and cxxp. In agreement with the above observations, we find a clear distinction between the RdRP model and both antisense-based variants. In the RdRP model, cytoplasmic silencing dominates over nuclear silencing  via small RNA guided heterochromatin formation and post transcriptional silencing (PTGS) via small RNA mediated mRNA degradation. These processes have been subdivided into a set of pseudo-reactions, which are given in Table Below we highlight a few characteristics of our modeling approach. Firstly, transposon copying has been modeled according to the life cycle of retrotransposons, with a stage in the cytoplasm including a virus-like particle (VLP). This may seem rather specific, however the mathematical equations describing the TE life cycle allow for a more general interpretation: the equations capture a general reproductive cycle with intermediate steps (protein products etc) in the cytoplasm. Thus the model can be understood as describing a generalized TE with an intermediate phase in the cytoplasm.Secondly, we only take into account fully functional transposable elements, and we do not model defective or non-autonomous TEs. This modeling decision does not affect the generality of the models as the omission of these passive transposable elements provides us with a worst case scenario: every TE is capable of reproduction and thus every new copy of a transposon "puts host control under more pressure".Finally, from the reactions as given in Table http://www.r-project.org), Python (http://www.python.org) and Gnuplot (http://www.gnuplot.info).The ODE equations were numerically integrated using MATLAB, and subsequently analyzed using R (Stochastic versions of the models were simulated using in-house developed software, which is available upon request.Abbreviation DescriptionasRNA: antisense RNA; dsRNA: double-stranded RNA; mRNA: messenger RNA; PTGS: post-transcriptional gene silencing; RdRP: RNA-dependent RNA polymerase; RISC: RNA induced silencing complex; RITS: RNA induced transcription silencing; RNAi: RNA interference; TE: transposable element; TGS: transcriptional gene silencing; VLP: virus-like particleAC conceived of the study, analyzed the data and drafted the manuscript. PH participated in the design of the study, analyzed the data and helped to draft the manuscript. All authors read and approved the final manuscript.Model Description. Model description in terms of mathematical equations.Click here for fileStochastic Simulations. A short description of the stochastic simulations of each of the models.Click here for fileParameter Choice. Additional information to elaborate on the choice of parameter value ranges.Click here for file


C9orf72 gene is a common cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis. Non-coding repeat expansions may cause disease by reducing the expression level of the gene they reside in, by producing toxic aggregates of repeat RNA termed RNA foci, or by producing toxic proteins generated by repeat-associated non-ATG translation. We present the first definitive report of C9orf72 repeat sense and antisense RNA foci using a series of C9FTLD cases, and neurodegenerative disease and normal controls. A sensitive and specific fluorescence in situ hybridisation protocol was combined with protein immunostaining to show that both sense and antisense foci were frequent, specific to C9FTLD, and present in neurons of the frontal cortex, hippocampus and cerebellum. High-resolution imaging also allowed accurate analyses of foci number and subcellular localisation. RNA foci were most abundant in the frontal cortex, where 51 % of neurons contained foci. RNA foci also occurred in astrocytes, microglia and oligodendrocytes but to a lesser degree than in neurons. RNA foci were observed in both TDP-43- and p62-inclusion bearing neurons, but not at a greater frequency than expected by chance. RNA foci abundance in the frontal cortex showed a significant inverse correlation with age at onset of disease. These data establish that sense and antisense C9orf72 repeat RNA foci are a consistent and specific feature of C9FTLD, providing new insight into the pathogenesis of C9FTLD.An expanded GGGGCC repeat in a non-coding region of the The online version of this article (doi:10.1007/s00401-013-1200-z) contains supplementary material, which is available to authorized users. MAPT [GRN [C9orf72 [C9orf72 mutation is the most common known cause of ALS [C9orf72 cases fall into two of the four FTLD-TDP subtypes: FTLD-TDP type A, being characterised by many TDP-43-positive neuronal cytoplasmic inclusions and short dystrophic neurites, the majority of which are found in layer 2 of the cortex; or FLTD-TDP type B, characterised by moderate numbers of neuronal cytoplasmic inclusions and dystrophic neurites in all cortical layers [Frontotemporal lobar degeneration (FTLD) is a common cause of young-onset dementia , 24, chaMAPT , 23, 27,APT [GRN , and C9o[C9orf72 , 13, 25.e of ALS . FTLD hae of ALS . FTLD-TDe of ALS . Patholol layers , 20. In C9orf72 is an expanded GGGGCC repeat, located upstream of the translation start codon [C9orf72 GGGGCC repeat expansion causes RAN translation, leading to the formation of neuronal inclusions containing RAN protein products generated from sense transcripts [C9orf72 cases in one study [C9orf72 antisense repeat transcripts have been reported [C9orf72 FTLD (C9FTLD) are unknown. We report here, in a series of C9FTLD cases and controls, that sense and antisense RNA foci are a characteristic and specific feature of C9FTLD.The FTD- and ALS-causing mutation in rt codon , 13, 25.rt codon . These drt codon . Intrigurt codon . The C9onscripts . Nuclearne study . C9orf72reported , but wheC9orf72 expansion repeat confirmed by repeat primed PCR (rp-PCR) analysis and Southern blot as previously described [C9orf72 expansion carriers. Additional cases used as controls were three FTLD-TDP type B cases and four FTLD-TDP type A cases without a known genetic mutation ; three Alzheimer’s disease cases all pathologically diagnosed with Braak and Braak stage VI pathology and Thal phase 5 [Brain specimens  sequences were (GGCCCC)4, (GGGGCC)4 or (CAG)7, 5′ labelled with either Cy3 or Alexa488. Primary antibodies were for NeuN , GFAP , Iba1 , CAII , p62  and phospho TDP-43 .Supplier and catalogue numbers for FISH reagents are detailed in Supplementary Table 1. 7 μm-thick paraffin sections were dewaxed, followed by antigen retrieval in citrate buffer  for 20 min in a microwave. Sections were then dehydrated in a graded series of alcohols, air dried and rehydrated in phosphate-buffered saline (PBS), briefly washed in 2×SSC and incubated for 30 min in pre-hybridisation solution (50 % formamide/2×SSC) at 80 °C. Hybridisation solution  was heated at 80 °C for 5 min prior to incubation with sections for 2 h at 80 °C. Sections were firstly washed three times for 30 min each with a high-stringency wash solution (50 % formamide/0.5×SSC) at 80 °C, and then three times for 10 min at room temperature in 0.5×SSC. After a brief wash in PBS sections were blocked in 10 % foetal bovine serum/PBS for 30 min, and then incubated with primary antibody (diluted in PBS) overnight at 4 °C, washed with PBS, and then incubated with appropriate secondary antibodies  for 1 h at room temperature. Autofluorescent background was reduced using Sudan black B . After further washes in PBS, sections were mounted with ProlongGold containing DAPI (Life Technologies). Images were obtained using a LSM710 META confocal microscope (Zeiss). For FISH with both probes, the sense probe was applied first as above, and after the three 30 min 80 °C washes in 50 % formamide/0.5×SSC, the antisense probe was added for 2 h, followed by the protocol above. For DNase and RNase treatments the standard protocol above was followed with additional steps: after rehydration in PBS, sections were incubated in 0.5 % Triton/PBS for 10 min, washed once in PBS, and treated with either RNase A (1 mg/ml in PBS/0.05 % Tween-20) or Turbo DNase  for 2 h at 37 °C. After the treatment sections were washed three times in PBS, once in 2×SSC and then placed in pre-hybridisation solution and the standard FISH protocol resumed. 2′- z-stack images  were taken at high resolution (2048 × 2048 pixels) using a 1.4 NA objective, in the anterior frontal cortex, two in the dentate fascia of the hippocampus and two in the granule cell layer of the cerebellum, to ensure a minimum of 100 neurons were quantified in each brain region . Quantification was performed blinded. RNA foci were counted in maximum intensity projections of z-stack images using the touch count tool in Volocity image analysis software (Perkin Elmer), and then scored for co-occurrence with staining of the nucleus and cell type markers. RNA foci were determined to be on the edge of the nucleus if they were touching the edge of DAPI staining, and in the cytoplasm if they were outside of the nucleus but within the staining of the cell type markers. Cytoplasmic localisation was not determined for astrocytes or microglia, or neurons of the cerebellum as cytoplasm could not be clearly identified. Cytoplamsic p62 and phospho TDP-43 inclusions were identified by Volocity image analysis software using the same threshold for all images. Inclusions positive for both p62 and phospho TDP-43 were only included in the TDP-43 group, to enable specific quantification of p62-positive, TDP-43-negative inclusions. All data is presented as the mean ± standard error of the mean. The homozygous C9FTLD case is shown on all graphs, but is excluded from means and statistical analysis. Statistical testing was performed with GraphPad Prism software using either a paired t test or one-way ANOVA and post hoc Bonferroni test.Three to six 40×C9orf72 mutation, 3 Alzheimer’s disease cases and 10 age-matched controls (Table 4 2-O-methyl RNA probe. In order to improve specificity we utilised the very high melting temperature of the (GGCCCC)4 RNA probe (predicted Tm > 80 °C), using a hybridisation temperature of 80 °C and high-stringency washes. We used high-resolution confocal imaging to improve signal to noise and generated z-stacks through the entire volume of the tissue to ensure RNA foci were not missed. We also included an antigen retrieval step, allowing us to combine RNA FISH with protein immunostaining. RNA FISH and immunostaining for the neuronal marker NeuN was performed in the frontal cortex, dentate fascia of the hippocampus and granule cell layer of the cerebellum  or TDP-43-positive cytoplasmic inclusions Fig. a. QuantiTo gain further insights into pathomechanisms of C9FTLD, sense and antisense neuronal RNA foci burden (percentage of neurons containing foci) were correlated with age at onset and age at death. Sense foci burden in the frontal cortex was found to be significantly and inversely correlated with age at onset of disease Fig. . A consin sequence forms an i-motif and whether this has biological significance. The identification of cytoplasmic antisense RNA foci raises the possibility that antisense RAN translation products will be produced, as well as the sense RAN translation products already reported [We provide the first definitive evidence for the presence of both sense and antisense RNA foci in C9FTLD. This has important implications for disease mechanism. Firstly, it suggests that either or both sense and antisense RNA foci could play a pathogenic role in C9FTLD; presumably through sequestration of RNA-binding proteins, as has been observed in other non-coding repeat expansion diseases . The idereported , 22, furO-methyl RNA probes), and stringent washes, which might explain the greater specificity. We also note that the RNA foci were not visible using a standard fluorescence microscope, but were readily visible with a confocal microscope, which may also explain differences with previous studies. It is likely that the enhanced signal to noise provided by high-resolution microscopy enabled sensitive detection of the RNA foci, which are, therefore, likely to be small structures. Our use of z-stacks to image the entire volume of the cell allowed a more accurate estimation of RNA foci per cell and the percentage of neurons containing foci than using confocal images from a single plane, which, given the small size of foci, would lead to an underestimation of foci numbers.Sense RNA foci were initially reported in 25 % of cells in the frontal cortex of 2 C9FTLD cases and the spinal cord of 2 C9ALS cases , but a sWe identified sense and antisense RNA foci in astrocytes, microglia and oligodendrocytes, but at lower frequency than in neurons. This suggests neurons are the primary sites of foci pathology but also raises the possibility of impaired glial function due to RNA foci formation. It will be interesting to determine whether the same repertoires of RNA-binding proteins are sequestered in these different cell types, and to understand why neurons have a greater RNA foci burden. Although foci were present in all three brain regions, the highest burden of foci was consistently found in the frontal cortex, which is the region that suffers the greatest neuronal loss in FTLD. A previous study on the homozygous case described the clinical and pathological features as severe, but not completely outside of the usual disease spectrum . Our datThe majority of RNA foci in all cell types were located in the nucleus, consistent with a role in nuclear RNA-binding protein sequestration. In the different brain regions between 10 and 22 % of neuronal foci were located at the very edge of the nucleus, with the cerebellum having a significantly higher proportion than the frontal cortex. The significance of foci on the edge of the nucleus is unclear but it would be interesting to determine whether they are in proximity to nuclear pores and whether they have the potential to block nuclear RNA import or export. 9 % of sense RNA foci in the frontal cortex were cytoplasmic, consistent with RAN translation of the repeat transcripts, which would require the cytoplasmic translation machinery. Significantly fewer cytoplasmic antisense foci were found in the frontal cortex, which may reflect differential nuclear export or turnover. Neither sense nor antisense foci showed an obvious association with p62- or TDP-43-positive inclusions, although cytoplasmic foci were occasionally found within both types of inclusion in all brain regions where they are found. This suggests that the presence of RNA foci does not predict the presence of TDP-43- or p62-positive inclusions. Co-localisation analyses with antibodies specific for each RAN product will be required to determine whether individual sense or antisense RAN products are associated specifically with sense or antisense RNA foci.C9orf72 function or RAN translation products. The identification of proteins sequestered by RNA foci will be a key step in confirming the validity of the toxic RNA hypothesis. In conclusion, we have identified both GGGGCC sense and GGCCCC antisense RNA foci as a consistent and specific feature of C9FTLD in a series of cases and controls. These findings have important implications for understanding disease mechanism and may help inform the drug discovery process.Our identification of sense and antisense RNA foci, and the significant correlation between sense foci burden and age at onset, add weight to the possibility that toxic RNA gain of function is involved in C9FTLD pathogenesis, but does not rule out a role for loss of Supplementary material 1 (PDF 685 kb)Below is the link to the electronic supplementary material.


Many experiments in modern plant molecular biology require the processing of large numbers of samples for a variety of applications from mutant screens to the analysis of natural variants. A severe bottleneck to many such analyses is the acquisition of good yields of high quality RNA suitable for use in sensitive downstream applications such as real time quantitative reverse-transcription-polymerase chain reaction . Although several commercial kits are available for high-throughput RNA extraction in 96-well format, only one non-kit method has been described in the literature using the commercial reagent TRIZOL.We describe an unusual phenomenon when using TRIZOL reagent with young Arabidopsis seedlings. This prompted us to develop a high-throughput RNA extraction protocol (HTP96) adapted from a well established phenol:chloroform-LiCl method (P:C-L) that is cheap, reliable and requires no specialist equipment. With this protocol 192 high quality RNA samples can be prepared in 96-well format in three hours (less than 1 minute per sample) with less than 1% loss of samples. We demonstrate that the RNA derived from this protocol is of high quality and suitable for use in real time qRT-PCR assays.The development of the HTP96 protocol has vastly increased our sample throughput, allowing us to fully exploit the large sample capacity of modern real time qRT-PCR thermocyclers, now commonplace in many labs, and develop an effective high-throughput gene expression platform. We propose that the HTP96 protocol will significantly benefit any plant scientist with the task of obtaining hundreds of high quality RNA extractions. The scale of experiments conducted in modern plant molecular biology has grown such that hundreds or thousands of plant samples need to be processed by the researcher for use in a range of downstream applications, such as quantitative trait mapping, mutant screening and the analysis of gene expression in natural accessions-a rapidly growing resource for Arabidopsis research. Real time qRT-PCR is a common downstream application in such experiments and has become a major platform for high-throughput transcript profiling . A signiet al. [Conventional RNA isolation techniques are based on a 1.5 mL micro-centrifuge tube format (or larger) using commercially available spin/vacuum-column kits or organic solvents such as TRIZOL (Invitrogen) and phenol. Although effective for low-throughput applications, isolating RNA from thousands of samples in micro-centrifuge tube format is incompatible with modern demands for high-throughput applications. A large number of protocols have been published for isolating high quality RNA however, only one high-throughput 96-well protocol is apparent in the published literature. This uses the commercial reagent TRIZOL (Invitrogen) for 96-well format nucleic acid extraction from Arabidopsis tissues, where it was favoured for its ability to simultaneously extract both DNA and RNA in a small number of steps . Howeveret al.  showed tet al. [et al.[Cheap, reliable and with a proven track record for a wide range of plant tissues and species, phenol provides an excellent alternative to TRIZOL. We developed a 96-well RNA extraction protocol by adapting a simple phenol:chloroform-LiCl method (P:C-L) ,5 that wet al.  and Guti. [et al.) to contWe demonstrate that phenol is more suitable than TRIZOL for high-throughput RNA extraction from a broad range of developmental stages. Our phenol-based high-throughput RNA extraction protocol (HTP96) can simultaneously isolate good yields of high quality RNA from 192 samples in less than three hours, with minimal loss or degradation of samples . The efficacy of the HTP96 protocol is demonstrated by presenting the results of simultaneous RNA extraction from a large number of different Arabidopsis accessions sampled at various developmental stages, from newly germinated seedlings to mature 30-day old plants. Electrophoresis and UV-spectrometry are used to demonstrate the quality and yield of RNA. The suitability of HTP96 RNA for sensitive downstream applications was established by real time qRT-PCR without additional purification, quantitation or DNase treatment.To date we have used the HTP96 protocol to isolate total RNA from more than 3,000 Arabidopsis samples. The HTP96 RNA protocol has vastly improved our sample throughput, facilitating high-throughput screening of large numbers of transformants and transcript profiling in hundreds of Arabidopsis accessions. Although developed for Arabidopsis, we believe the HTP96 protocol will significantly benefit any plant scientist with the task of obtaining hundreds of high quality RNA extractions in a time and cost-effective manner.NOTE: If using alternative plastic ware, make certain it is resistant to phenol and chloroform.• 96-well racked 1.2 mL collection tubes and microtube caps .•  1.5 mL micro-centrifuge tubes.•  Powder funnels .• 3 mm tungsten-carbide grinding beads . NOTE: 3 mm steel beads provide a cheaper alternative.• 96-well PCR plates  and PCR seals .• Multi-channel pipette reagent reservoirs (60 mL).• Liquid nitrogen and dry ice.• RNA Extraction Buffer . Add 1% 2-mercaptoethanol before use.• Acidified phenol pH 4.3 ± 0.2 .• Chloroform.• Isopropanol (2-propanol).• 3 M sodium acetate (pH 5.2).• 70% ethanol.• Nuclease-free water.free™ .•  DNase e.g. TURBO DNA-• Fume cupboard for handling phenol, chloroform and 2-mercaptoethanol.• Bead-mill, preferably with a cryo-adapter capable of accommodating 96-well format collection tube racks and/or 1.5 mL micro-centrifuge tubes, e.g. GenoGrinder 2010 (SpexCertiprep) or similar.• Plate mixer, e.g. the Vortex Genie 2 mixer fitted with a 96-well plate adapter .• Plate centrifuge at room temperature capable of speeds ≥4000 × g, (e.g. Sigma 4-15 C centrifuge fitted with a QIAGEN plate rotor-Nr.09100).• Gel electrophoresis system.•  Agilent 2100 Bioanalyzer (Agilent Technologies).• NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies Inc.).• Suitable multi-channel pipette . NOTE: Avoid using electronic pipettes for sensitive pipetting steps.Arabidopsis seeds were sown on soil in plastic pots (7 cm × 7 cm) and stratified for three days in a cold room . Seedlings were grown in a controlled environment growth room . In some cases seeds were stratified for three days and the seedlings pre-grown for seven days and subject to cold treatment for up to six weeks in a CR. Sowings for different treatments were planned such that seedlings were sampled at equivalent developmental stages.Typically, RNA was isolated from 200 mg of ground Arabidopsis tissue powder prepared under liquid nitrogen. For TRIZOL, RNA was extracted from tissue powder according to the manufacturer's instructions and resupended in 50 μL of nuclease-free water. P:C-L RNA extraction was conducted using a scaled-down version of protocols previously described ,5 and re32-dCTP β-TUBULIN (At1g20010) and 18S rDNA probes prepared using Klenow fragment. Blots were exposed on phosphor screens (Kodak) and imaged with a Typhoon 9200 Variable Mode Imager (Amersham Biosciences).10 μg RNA was loaded on a denaturing 1.2% agarose gel and transferred to Hybond™ N+ membranes (GE Amersham) by capillary transfer . The RNAFor each RNA sample, an equal volume of RNA was prepared with a 2× denaturing RNA loading buffer . The RNA was denatured by heating to 70°C for ten minutes with the RNA loading buffer and then run on a 1.2% agarose gel as described by [free™ DNase (Ambion) according to manufacturer's instructions. 4 μL of DNase treated RNA (300 ng μCL-1) were run on an Agilent 2100 Bioanalyzer microfluidic electrophoresis chip according to the manufacturer's instructions.RNA was first of all treated with TURBO DNA-FLOWERING LOCUS C  was normalised to UBIQUITIN CONJUGATING ENZYME1  using the comparative Cq (quantification cycle) method [FLC; 5'-CTG CGA CTC AGG GAA TCT TCT AA-3' and 5'-TTG TGC CAT TGA ATT GAA CCC-3' for UBC.1 μg of total RNA preparation was used in a first strand cDNA synthesis reaction  using Superscript III and oligo(dT)20 (Invitrogen) following the manufacturer's instructions. After cDNA amplification, the 10 μL reaction was diluted with 60 μL of nuclease-free water, 5 μL of which was used in a 20 μL real time qRT-PCR reaction using SYBR Green Jumpstart Taq Ready Mix  and a Roche Lightcycler 480II instrument. Expression of the MADS-box transcription factor ) method -16. PrimIn deciding to develop a high-throughput RNA extraction method appropriate for the isolation of RNA from hundreds of Arabidopsis samples, over a broad range of developmental stages, we first examined the suitability of TRIZOL, as it had been described in previous high-throughput nucleic acid extractions  where itβ-TUBULIN expression grew as a proportion of total RNA in the TRIZOL-extracted RNA (as indicated by 18S rRNA expression). However, when an equal quantity of the same tissue was used for RNA extraction by P:C-L [To explore this observation we extracted RNA from Arabidopsis seedlings grown at ambient temperature for three, six or twelve days followed by six weeks of cold treatment before immediately harvesting entire seedlings after the cold Figure . Northerby P:C-L  no such Closer inspection of the RNA isolated from the youngest Arabidopsis seedlings using TRIZOL reveal several interesting differences when compared with RNA isolated from an equal quantity of the same tissue using P:C-L extraction. RNA yield did not vary greatly, UV-spectrometry indicated high yields of total RNA for both methods. From 200 mg of ground plant material the TRIZOL method isolated, on average, 52 ± 35 μg total RNA, whereas from the same starting tissue the P:C-L method isolated an average of 120 ± 32 μg total RNA. Inspection of TRIZOL and P:C-L extracted RNA by gel electrophoresis revealed several differences in the total RNA profile Figure . Firstlyet al. [230/260 ratio. The authors presented high-resolution microfluidic electrophoresis of soybean RNA extracted by several methods including TRIZOL, but there were no similar differences in the banding profile to what we asterisked in our gel image . However, if sampling more mature plants it may be convenient to collect and homogenise tissue using 1.5 mL micro-centrifuge tubes but this requires additional handling time. To obtain the best quality RNA it is essential that harvested material is frozen rapidly upon sampling and that the material is not allowed to thaw.The HTP96 RNA extraction protocol has been developed for sampling tissues of all stages of Arabidopsis development including just germinated seedlings (three to five-days old) and mature plants. Whenever possible it is advisable to sample and homogenise tissue directly in 96-well format racked 1.2 mL collection tubes (hereafter 1. Prepare racks of 1.2 mL collection tubes (or 1.5 mL micro-centrifuge tubes) with 3 mm tungsten-carbide grinding beads (1 per sample).2. On dry ice, harvest no more than 100 mg of fresh tissue into each collection tube. If using 1.5 mL micro-centrifuge tubes up to 200 mg of tissue can be collected and frozen immediately with liquid nitrogen. NOTE: Do not overfill tubes and ensure plant material is free of surface water for optimal tissue homogenisation.3. Firmly secure 8-strip collection tube caps and allow them to cool at -80°C before homogenising the tissue. Samples are homogenised directly in collection tube racks using a commercial bead-mill. For most Arabidopsis tissues a fine powder can be produced in around 30 seconds on a moderate to high setting, we use a GenoGrinder 2010 at 1500 rpm for 30 seconds to prepare up to 384 samples simultaneously. NOTE: Avoid excessive homogenisation to prevent thawing of tissue powder.4. . If using 1.5 mL micro-centrifuge tubes, homogenise samples in pre-cooled sample holders or specially manufactured cryo-adapters to avoid thawing. Powdered plant material must then be transferred (on dry ice) to pre-cooled 96-well racks of collection tubes. This must be done for each sample individually using liquid nitrogen cooled funnels. To prepare two duplicate plates in this way takes approximately 90 minutes (one minute per sample) providing samples are organized appropriately, we typically do this by sowing and collecting samples in a specified order to minimise excessive handling of fresh and frozen samples.Typically two 96-well format racks of RNA are prepared simultaneously 192 samples) but this may be increased without significant difficulty if appropriate equipment is available. All steps of the protocol, including centrifugation, are carried out at room temperature. Use a fume hood and wear protective clothing and eyewear when handling 2-mercaptoethanol, phenol and chloroform. A bench summary of the HTP96 RNA extraction protocol is presented in Figure  samples 5. For 192 RNA extractions prepare 60 mL of RE buffer (add 600 μL of 2-mercaptoethanol to 60 mL RE buffer from a stock solution) and heat to 60°C in a waterbath. Prepare 60 mL of 1:1 acidic phenol:chloroform pH 4.3 ± 0.2 (P:C) and an excess (e.g. 1 L) of 70% ethanol. NOTE: In the absence of LiCl precipitation we use acidified phenol as it has previously been reported to reduce gDNA contamination .6. Working carefully but quickly, add 300 μL of RE buffer to each collection tube of frozen tissue powder using a suitable multi-channel pipette. Firmly secure 8-strip collection tube caps and mix the contents vigorously using a plate mixer until the mixture thaws.7. Briefly centrifuge each plate to collect the contents of each tube and prevent cross contamination during removal of collection tube caps. Add 300 μL of P:C to each collection tube, apply fresh tube caps and mix the contents vigorously for ten minutes.8. Separate the aqueous (upper) and organic phases by centrifugation for 15 minutes at maximum speed (>4000 × g).9. (During step 8) prepare fresh racks of collection tubes each containing 240 μL isopropanol and 30 μL 3 M sodium acetate (pH 5.2).10. Carefully transfer up to 300 μL of the aqueous phase to the freshly prepared racks of collection tubes, apply a PCR plate seal and briefly mix using a plate mixer at low speed. NOTE: Remove the aqueous phase carefully; this is best done using a manual rather than electronic multi-channel pipettor, providing greater control over the pipetting action thereby avoiding disturbance of the interphase layer.11. Precipitate the nucleic acids at -80°C for 15 minutes. If small amounts of tissue debris are transferred at this stage they are usually removed in the remaining steps.12. Collect the nucleic acid precipitate by centrifugation for 30 minutes at maximum speed (>4000 × g). Do not be alarmed if the samples are frozen after precipitation at -80°C, they will thaw rapidly during centrifugation.13. Ensure all collection tube strips are intact, secure the collection tubes to the rack with masking tape and discard the supernatant by inverting the plate over a suitable container. As the nucleic acids are precipitated at this stage the possibility of cross-contamination is minimal (a pipette can be used if preferred). NOTE: If the 8-strip collection tube format is damaged, separated collection tubes can be lost during inversion of the tube rack. This is easily avoided by careful handling.14. Wash the nucleic acid pellets with 600 μL of 70% ethanol to remove SDS, EDTA and other contaminants, apply a PCR plate seal, mix gently for a short time and centrifuge at maximum speed (>4000 × g) for five minutes. Discard the ethanol as described in step 13 and wash a second time.15. Remove any remaining traces of ethanol by inverting the racked collection tubes onto absorbent paper (or by using a pipette) and allow the pellets to air dry. Drying is facilitated by periodically knocking the racks firmly, but gently, several times onto absorbent paper. The RNA pellet is considered dry when no further drops of liquid appear, this typically requires 30 minutes with three or four rounds of knocking onto absorbent paper. NOTE: Be careful not to lose the RNA pellets, they are securely attached to the tube but rough handling can dislodge them.16. Dissolve the nucleic acid pellet (mostly RNA) with 50-100 μL of nuclease-free water, apply a PCR plate seal and gently mix the contents using a plate mixer. Briefly centrifuge the dissolved RNA to remove any unwanted solid contaminants, e.g. tissue debris and transfer to a 96-well PCR plate. The samples can be stored at -20°C short term but should be stored long-term at -80°C.  At this stage aliquots of RNA may be treated with DNase as required. NOTE: In most cases DNase treatment is not necessary if HTP96 RNA is to be used for real time qRT-PCR.RNA quality was assessed using standard agarose gel electrophoresis Figure  and Bioa260) is in the region of 40 to 100 μg of RNA per 100 mg of starting tissue. The purity of the RNA is measured by calculating the ratio A260/A280, whereas the level of organic contaminants, e.g. polysaccharides and polyphenolics, is measured by the ratio A260/A230. Typically HTP96 RNA preparations have an A260/A280 ratio of ~2.0 and A260/A230 ratio >2.2 indicating that HTP96 RNA is suitable for immediate use in downstream applications without further purification.RNA concentration and purity can be determined spectrophotometrically  by measuFLOWERING LOCUS C (FLC). Several Arabidopsis accessions with well characterised responses of FLC to cold treatment  were as FRI-SF2  and whic FRI-SF2 .FLC expression in the Col FRI-SF2 accession [FLC expression consistent with this from both P:C-L and HTP96 extracted RNA  of RNA preparation without prior quantification and compared this to real time qRT-PCR results obtained with precisely quantified RNA (1 μg). Simplification of reverse transcription did not cause any significant bias in the linearity of mRNA amplification to cDNA as seen by real time qRT-PCR  and is of comparable quality to RNA isolated using a standard P:C-L extraction protocol but with improved throughput .Here we demonstrate concern over the use of TRIZOL for the extraction of RNA from the youngest Arabidopsis seedlings and suggest that phenol provides a cheaper and more reliable alternative. We describe an optimised, 96-well format high-throughput RNA extraction protocol (HTP96) based on a simple, cheap and reliable P:C-L method ,5 that iWe have shown that HTP96 RNA is suitable for immediate use in reverse transcription and real time qRT-PCR. Quantification of RNA can be omitted due to the high level of consistency in RNA yield and quality when using a similar input of tissue. Furthermore, we have shown that the use of exon-bridging real time qRT-PCR primers controlled the moderate gDNA contamination present in HTP96 RNA preparations. This removes the need for DNase treatment and LiCl precipitation, facilitating high-throughput analyses of gene expression. However, in cases where primers cannot be designed to bridge exons, e.g. when working with intronless transcripts (21.7% of Arabidopsis genes lack introns , or whenThe HTP96 protocol is a novel application of a well-established method of isolating high quality RNA. By adopting a 96-well format we have developed an improved protocol that allows phenol:chloroform extraction to be used to isolate large numbers of RNA samples using commonly available laboratory equipment. This method provides a cheap alternative to expensive 96-well format kits or effective, but low-throughput methods. We have since used the HTP96 protocol to assess gene expression in thousands of samples from hundreds of Arabidopsis accessions in conjunction with 96 and 384-well real time qRT-PCR thermocyclers, now commonplace in many laboratories.We propose that the HTP96 protocol is of broad significance to the wider plant science community and will be fruitful for other users wishing to conduct high-throughput transcript profiling in a time and cost effective manner.The authors declare that they have no competing interests.JSM compared TRIZOL with P:C-L. MSB, VC and JSM developed the HTP96 extraction protocol. VC performed the gene expression analysis. MSB and JSM wrote the manuscript. All authors contributed to the preparation and approval of the final manuscript.


Montastraea cavernosa (a sediment resistant species that may benefit from low light levels). An overall negative relationship between fleshy macroalgae and slow-growing reef-building organisms  was recorded, suggesting competition between these organisms. The opposite trend (i.e. positive relationships) was recorded for turf algae and the two reef-building organisms, suggesting beneficial interactions and/or co-occurrence mediated by unexplored factors. Turf algae cover increased across the region between 2006 and 2008, while scleractinian cover showed no change. The need of a continued and standardized monitoring program, aimed at understanding drivers of change in community patterns, as well as to subsidize sound adaptive conservation and management measures, is highlighted.The Abrolhos Bank (eastern Brazil) encompasses the largest and richest coral reefs of the South Atlantic. Coral reef benthic assemblages of the region were monitored from 2003 to 2008. Two habitats  were sampled per site with 3–10 sites sampled within different reef areas. Different methodologies were applied in two distinct sampling periods: 2003–2005 and 2006–2008. Spatial coverage and taxonomic resolution were lower in the former than in the latter period. Benthic assemblages differed markedly in the smallest spatial scale, with greater differences recorded between habitats. Management regimes and biomass of fish functional groups  had minor influences on benthic assemblages. These results suggest that local environmental factors such as light, depth and substrate inclination exert a stronger influence on the structure of benthic assemblages than protection from fishing. Reef walls of unprotected coastal reefs showed highest coral cover values, with a major contribution of  Worldwide, reef ecosystems are declining rapidly due to multiple disturbances such as climate change, overfishing, emerging diseases, pollution and sedimentation, implying severe losses of biodiversity and ecosystem services The balance between abundance of relatively slow-growing reef building organisms  and fast-growing non-building organisms  is one of the most widely used metric to evaluate reef condition, with dominance of the former indicating a healthy ecosystem The general perception of decline in coral cover is mostly based on long-term datasets and meta-analytical studies from the Caribbean and Indo-Pacific regions Protection from fishing through establishment of no-take marine reserves may influence reef benthic assemblages via habitat protection and trophic cascading effects Mussismilia braziliensis) are of special concern Brazilian coral reefs represent a priority area for biodiversity conservation in the Atlantic Ocean due to their relatively high endemism levels  concentrated in a small reef area (5% of West Atlantic reefs) This study aims to describe spatial and temporal patterns in reef benthic assemblages of the Abrolhos Bank, eastern Brazil, as well as to infer possible anthropogenic and natural processes/disturbances responsible for the observed patterns. The Abrolhos region encompasses the largest and richest coral reef complex in the South Atlantic Ocean and the oldest among the few networks of marine protected areas in the country 2), with depths rarely exceeding 30 m and a shelf edge at about 70 m. Reefs and rhodolith beds are the most prominent benthic features in the region The Abrolhos Bank  is a wide portion of the continental shelf . A large estuary formed by River Caravelas and River Peruípe is a remarkable feature of the coastline in the central portion of the Bank, near the main reefs in situ and classified as follows: turf algae, crustose calcareous algae, fire-corals (milleporids), fleshy macroalgae, live corals, octocorals and zoanthids. The “live coral” category includes only scleractinians, with no species distinction. During this first period, monitoring was performed in four areas (Area 1) No-take reserve of Timbebas Reef (three sampling sites) – Located within the National Marine Park of Abrolhos (NMPA). Created by the Brazilian government in 1983, the NMPA comprises two discontinuous portions, one closer to the coast and poorly enforced (Timbebas Reef), and another farther from the coast and more intensively enforced (Abrolhos Archipelago and Parcel dos Abrolhos Reef). Areas 2 and 3) Multiple-use and no-take zones of Itacolomis Reef – Itacolomis Reef is the largest reef complex (∼50 km2) within the Marine Extractive Reserve of Corumbau (MERC) Area 4) Unprotected coastal reefs (five sampling sites) – It encompasses the Parcel das Paredes Reef and Sebastião Gomes Reef, both subjected to the highest fishing pressure in the region The long-term monitoring program of coral reef benthic assemblages of the Abrolhos Bank started in 2003, through engagement of scientists and members of governmental and non-governmental organizations related to coastal management. Surveys were always carried out in the summer (January–March), thus avoiding seasonal artifacts. Each site was about 300 m in diameter and composed by 1–3 interconnected reef pinnacles, except for the rocky reefs of the Abrolhos Archipelago (see below). Spatial coverage and sampling methodologies varied through time, with two main periods. From 2003 to 2005 point-intercept lines  ur areas , as foll2. Quadrats were permanently delimited by fixed metal pins and set at haphazardly distances along 20–50 m axes. Relative cover of different benthic organisms was estimated through the identification of organisms (lowest taxonomic level possible) below 300 randomly distributed points per quadrat  using the Coral Point Count with Excel Extensions Software Between 2006 and 2008 benthic assemblages were characterized using fixed photo-quadrats http://datadryad.org/).Logistical support and research permits were provided by Parque Nacional Marinho de Abrolhos and Reserva Extrativista Marinha de Corumbau/ICMBio . Data from this work was made available for public access through the Dryad platform , in which data was obtained with a higher taxonomic resolution and a greater spatial coverage (see above). Inferences for the entire sampling period  were performed by making separate analyses for the two sampling periods: 2003–2005 and 2006–2008, and by considering only the same sampling sites and benthic categories (i.e. by standardizing data obtained in the two sampling periods). Long-term changes were taken into account only when similar trends were recorded for both sampling periods.Canistrocarpus spp., Dictyota spp. and Dictyopteris spp.) were difficult to distinguish in the images, thus being pooled into a single category . All scleractinians were identified to the species level, except for Siderastrea spp., a genus for which three morphologically similar species are recorded for Brazil  Millepora brasiliensis and M. alcicornis), but treated separately for the small-sized and conspicuous Millepora nitida.Some metal pins marking the fixed photo-quadrats were lost during the sampling period. These samples were excluded from the analyses in order to assure that exactly the same photo-quadrats were used for the temporal comparisons. Final sample size ranged between 7–10 quadrats per habitat per site per year. Three common genera of fleshy macroalgae  was used to evaluate spatial and temporal variations in benthic cover. Two separate groups of ANOVA were calculated, the first one focusing on differences between tops and walls (considering reef pinnacles only) and the second one focusing on differences between reefs while ignoring between-habitat variability, this latter including the shallow rocky reefs of the Abrolhos Archipelago . Because data could not be collected in the tops of three reefs see , betweenNon-metric multidimensional scaling (MDS) ordination was used to summarize spatial and temporal similarities (Bray-Curtis) on the structure of benthic assemblages, and separate one-way analyses of similarities (ANOSIM) were used to evaluate significant differences according to reef areas, habitats and years Canonical correspondence analysis Palythoa caribaeorum) on the abundance of key reef-building organisms . Percentage cover data are compositional and thus subjected to constant sum constraint. Because this may mask true relationships among variables, analyses were performed using the centered log-ratio transformation Multiple linear regression analyses Palythoa caribaeorum (6.6%), the scleractinian coral Monstastrea cavernosa (4.1%) and the category composed by the fleshy macroalgae Canistrocarpus spp., Dictyota spp. and Dictyopteris spp. (2.9%) , belonged to different functional groups. Turf algae were by far the most abundant benthic organisms (56.1%), followed by crustose calcareous algae (12.1%), the zoanthid . (2.9%) . In ANOS. (2.9%) . Benthicavernosa .1% and targassum  and S3. avernosa .1% and tbacteria  and S3.Montastraea cavernosa and Mussismilia hispida. The reef coral Mussismilia harttii, octocorals, the algae Halimeda spp. and zoanthids of genus Zoanthus were also common at Timbebas, while the fire-coral Millepora nitida was also abundant at unprotected coastal reefs. The colonial zoanthid P. caribaeorum showed highest cover values at coastal reefs (both protected and unprotected) and at the mid-shelf fully-protected reefs of Parcel dos Abrolhos. Turf algae and fleshy macroalgae, particularly Sargassum spp., were more prevalent in the no-take and multiple-use zones of Itacolomis Reef  and unprotected coastal reefs, with major contributions of mis Reef  and S3. sum spp. .Caulerpa spp. and cyanobacteria was not consistent among reef areas, with significant interactions being recorded . However, significant variations were recorded for several individual organisms/categories. Most noticeable was the increase in turf algae cover in most reef areas between 2006 and 2008  and S3. recorded  and S3.2 = 0.57) than for crustose calcareous algae (r2 = 0.13). Negative relationships were recorded between fleshy macroalgae and reef-building organisms, the opposite trend (i.e. positive relationships) being recorded between turf algae and reef-building organisms. The zoanthid P. caribaeorum showed a negative effect on the cover of crustose calcareous algae and a weak yet significant positive effect on the cover of scleractinian corals , the four most important variables affecting the structure of benthic assemblages. Latitude and distance offshore were positively correlated with each other, with southern reefs (Parcel dos Abrolhos Reef) more distant from the coast than northern ones (Itacolomis Reef), making difficult to disentangle the effect of these two explanatory variables. The first two axes explained 75.3% of the relationship between environmental characteristics and benthic assemblages' structure. Inclusion of the remaining three explanatory variables  increased the power of explanation of the model by less than 1%. Most importantly, these latter three variables were not significant (P>0.05) according to the Monte Carlo test. There was a clear distinction of samples obtained in different habitats and reef areas in the two-dimensional ordination diagram . Four maWhen considering data pooled for all habitats and sites, temporal variations between 2003 and 2008 (i.e. for both sampling periods) were recorded for CCA only, with no clear overall trend of increase or decrease through time . A signiThis is one of the most comprehensive characterizations of shallow coral reef benthic assemblages of the Abrolhos Bank performed to date and one of the few studies focusing on a relatively long temporal scale Montastraea cavernosa) were recorded in reef walls of unprotected coastal reefs  that do not have great importance as reef builders, they were not assessed”. This limitation in the sampling design of previous studies is particularly important considering the results obtained here showing that differences between habitats are more important than differences between reefs. Results from this study indicate that M. cavernosa is a major reef-building species in SW Atlantic coral reefs, highlighting the importance of studies focusing particularly on the healthy and dynamics of this species. In the Caribbean, habitats dominated by Montastraea spp. have the highest biodiversity and support the largest number of ecosystem processes and services Agaricia fragilis, Mussismilia hispida and Scolymia wellsi) may have also been underestimated in previous studies, but see P. caribaeorum) between tops and walls.In the present study, highest coral cover values  reef communities A. fragilis, M. hispida and S. wellsiM. cavernosa on inshore reefs of the Abrolhos Bank is the relatively high availability of nutrients in inshore reefs, which may lead to high coral growth rates and reproductive output The high coverage of Mussismilia braziliensis was an exception to the abovementioned pattern, as this species was relatively more abundant inside no-take zones (Timbebas Reefs and the Abrolhos Archipelago) (see M. braziliensis inside no-take zones within the Abrolhos Bank (Parcel dos Abrolhos Reef and Timbebas Reefs). A recent study The strong between-habitat differences recorded here suggest that factors such as light, depth and bottom inclination are the main drivers of benthic assemblages' structure in the Abrolhos region. Other studies performed elsewhere have highlighted the importance of such environmental variables for coral reef benthic assemblages ago) see . [52] alPalythoa caribaeorum in the Abrolhos Bank is noteworthy (up to 25% in some sites). This species is an aggressive competitor for space, killing or inhibiting the growth of nearly all other sessile reef invertebrates, including corals P. caribaeorum and that of crustose calcareous algae suggests competition between these organisms. P. caribaeorum cover, the opposite trend being recorded for scleractinians. Such a pattern was not recorded here, with highest values of P. caribaeorum cover recorded in tops of both inshore unprotected coastal reefs and tops of mid-shelf fully-protected reefs (Parcel dos Abrolhos Reef). P. caribaeorum and scleractinians, with high sedimentation levels favoring the former. Sedimentation, desiccation and predation levels are some of the environmental and ecological drivers that may influence P. caribaeorum abundance P. caribaeorum in the Abrolhos Bank and elsewhere.The relatively high cover of the zoanthid per se due to diseases A moderate increase in turf algae cover was recorded across the Abrolhos Bank between 2006 and 2008. The lack of relationship between biomass of herbivorous fish and algae abundance reported here indicate that other factors, such as coral mortality Results from the multiple regression models obtained here indicate that competition with fleshy macroalgae is important for both scleractinian corals and crustose calcareous algae. Negative effects of fleshy macroalgae on corals have been widely reported The lack of historical ecological data impedes the understanding of processes underlying community-level dynamics and the evaluation of the actual degree of conservation/degradation of reef communities Figure S1Decreasing order of abundance of benthic organisms in the Abrolhos Bank. Species codes: First three letters of genus name followed by first three letters of specific epithet (see full names in (TIF)Click here for additional data file.Table S1Summary of environmental characteristics of sampling reefs and sites.(DOC)Click here for additional data file.Table S2Analyses of Variance (ANOVA) testing the effect of reef areas (R), habitats (H) and years (Y) in cover of different benthic organisms with data obtained between 2006 and 2008. The Abrolhos Archipelago area (rocky reef) was excluded from these analyses in order to allow a more comprehensive comparison between pinnacles' tops and walls .(DOC)Click here for additional data file.Table S3Significant differences in benthic cover according to reef areas (R), habitats (H) and years (Y), as determined by Student-Newman-Keuls (SNK) post-hoc comparisons. Reefs arranged in decreasing order of benthic cover, with homogeneous groups linked by an equal sign. Reef areas: IN – Itacolomis Reef (no-take), IT – Itacolomis Reef (multiple-use), PB – Parcel dos Abrolhos (no-take), TI – Timbebas Reef (no-take), UP – Unprotected coastal reefs. Habitats: TP – tops and WA – walls. Years: 2006–2008. The Abrolhos Archipelago area  was excluded from these analyses in order to allow a more comprehensive comparison between pinnacles' tops and walls .(DOC)Click here for additional data file.Table S4Analyses of Variance (ANOVA) testing the effect of reef areas (R) and years (Y) in cover of different benthic organisms with data obtained between 2006 and 2008. Reef areas: AR – Abrolhos Archipelago, IN – Itacolomis Reef (no-take), IT – Itacolomis Reef (multiple-use), PB – Parcel dos Abrolhos (no-take), TI – Timbebas Reef (no-take), UP – Unprotected coastal reefs.(DOC)Click here for additional data file.Table S5Multiple regression results showing the relative influence of fast growing non-reef building organisms  on abundance of key reef-building organisms . Levels of significance for full model and partial 2r: * P<0.05; ** P<0.01; *** P<0.001.(DOC)Click here for additional data file.Table S6Analyses of Variance (ANOVA) testing the effect of reef areas, habitats and years in cover of different benthic organisms for the two sampling periods (2003–2005/2005–2008).(DOC)Click here for additional data file.


Affiliation number 1 for all authors is incorrect. The correct affiliation is: Shanghai Tenth People's Hospital, Department of Bioinformatics, Shanghai Key Laboratory of Signaling and Disease Research, the School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China.