Eukaryotic mRNAs are extensively processed to generate functional transcripts, which are 5′ capped, spliced and 3′ polyadenylated. Accumulation of unprocessed (aberrant) mRNAs can be deleterious for the cell, hence processing fidelity is closely monitored by QC  mechanisms that identify erroneous transcripts and initiate their selective removal. Nucleases including Xrn2/Rat1 and the nuclear exosome have been shown to play an important role in the turnover of aberrant mRNAs. Recently, with the growing appreciation that mRNA processing occurs concomitantly with polII (RNA polymerase II) transcription, it has become evident that QC acts at the transcriptional level in addition to degrading aberrant RNAs. In the present review, we discuss mechanisms that allow cells to co-transcriptionally initiate the removal of RNAs as well as down-regulate transcription of transcripts where processing repeatedly fails. Most of the steps required to mature a nascent transcript to a functional mRNA take place while the transcript is still attached to elongating polymerase. This has been shown for mRNA capping –4, splicin vitro [Saccharomyces cerevisiae TRAMP (Trf4–Air2–Mtr4p polyadenylation) complex, which contains the poly(A) polymerases Trf4p or Trf5p, the RNA-binding proteins Air1p or Air2p, and the helicase Mtr4p. Taken together, these proteins add short poly(A) tails to RNA substrates, which is thought to make them more accessible [The prominent role of nucleases in removing aberrant mRNA species became evident after transcriptome analysis in various QC mutants –30. A cein vitro  and requcessible . TRAMP acessible .S. cerevisiae poly(A) polymerase and the SR (serine/arginine-rich) protein Npl3p (Srp2 in Schizosaccharomyces pombe) [in vivo and in vitro experiments indicate that Rrp6 is required for efficient transcription elongation [rrp6 in a cluster with transcription mutants rather than other decay factors [The role of the exosome in post-transcriptional QC is well established ,29,35,36s pombe) –45. In aongation ,47. Fina factors , indicatS. cerevisiae), together with its co-factor Rai1, is also involved in nuclear RNA decay and degrades 5′-monophosphorylated substrates [The 5′–3′ exonuclease Xrn2 (or Rat1p in bstrates . It is abstrates –53.7G (7-methylguanosine) cap occurs shortly after transcription initiation. The structure usually protects mRNAs from degradation and is required for efficient export and translation into protein [Addition of the m protein . Previou protein   that govern the expression of inducible genes are regulated by Dcp2 and Xrn2 [Premature termination by Xrn2 is also employed to regulate gene expression in a context of functional RNA processing. In and Xrn2 . In humaand Xrn2 . Promoteand Xrn2 ,67.Previous studies have demonstrated that eukaryotic promoters possess an intrinsic bidirectionality, but only transcription in one direction is productive, whereas PROMPTs (promoter upstream transcripts) are rapidly degraded ,26,66,68S. cerevisiae, Nrd1 has been found genome-wide at introns, potentially recruiting the exosome complex to unspliced RNAs [Budding yeast has evolved a different, but surprisingly analogous, solution to control productive elongation. Here, promoter-associated CUTs (cryptic unstable transcripts) are terminated by a specialized pathway, which involves the RNA-binding proteins Nrd1 and Nab3 ,74 FiguC. The Nrced RNAs . Also, Nced RNAs ,75. How ced RNAs ,88. RNA ced RNAs ,89. Inteced RNAs . AlthougDrosophila, Su(s) (suppressor of sable) co-transcriptionally recruits Rrp6 to aberrant RNAs that contain transposable elements [S. pombe to flies and mammals, and with it the principle of a degradative polyadenylation, although the range of substrates differs between species [S. cerevisiae is quite well understood, it is less clear how substrates are selected for degradation in other organisms. For example, the TRAMP-mediated destabilization of RNAs produced from heterochromatic regions in fission yeast appears to be sequence-independent [9 on histone H3) that recruit chromodomain protein 1 (Swi6/HP1) leading to heterochromatin formation. Both H3K9me and recruitment of Swi6 are required for rapid heterochromatic RNA turnover, but not sufficient to elicit decay on other RNAs [It is not clear whether there are functional homologues of Nrd1 and Nab3 in other species. Different proteins may be used for a similar purpose, e.g. in elements . The TRA species . Althougependent . Here, Rependent . RNAi diher RNAs ,93,94. Iher RNAs ,96. In cher RNAs .Cryptococcus neoformans [S. pombe [S. pombe, where failure to terminate can produce overlapping read-through transcripts [Besides the mechanisms outlined above, loci encoding RNAs that are intrinsically poorly processed, e.g. due to suboptimally positioned regulatory elements, can be targeted by another mechanism: transcriptional silencing. This was demonstrated for poorly spliced genes in oformans . Here, iS. pombe , indicatnscripts . These tDrosophila [To date, studies supporting a role for transcriptional silencing in mRNA QC have mostly been carried out in yeasts. However, comparable mechanisms may exist in metazoans. One report links exosome-dependent retention of unspliced pre-mRNAs to transcriptional silencing at the locus in osophila . Interesosophila , was alsosophila . Other eIt has become clear that cells do not only rely on their capacity to identify and clear up dysfunctional RNAs after they have been released into the nucleoplasm, but that mechanisms are in place that respond to processing errors while the transcript is still attached to chromatin. This has a dual advantage for the cell; first, it provides additional checkpoints for the removal of unprocessed and potentially deleterious transcripts, which are less likely to escape post-transcriptional RNA surveillance. Secondly, resources are mainly used to make correct RNA, thus avoiding wasteful transcription. Many of the important players in co-transcriptional QC, such as the exosome and Xrn2/Rat1, are widely conserved; however, their importance in dealing with different classes of substrates can vary between species . There a


Airn and Kcnq1ot1, whose length ranges between 80–118 kb. Transcripts show a more uniform read coverage when RNA is fragmented with RNA hydrolysis compared with cDNA fragmentation by shearing. Irrespective of the fragmentation method, all coding and non-coding transcripts longer than 8 kb show a gradual loss of sequencing tags towards the 3′ end. Comparisons to published RNA-Seq datasets show that the strategy presented here is more efficient in detecting known functional imprinted macro ncRNAs and also indicate that standardization of RNA preparation protocols would increase the comparability of the transcriptome between different RNA-Seq datasets.Imprinted macro non-protein-coding (nc) RNAs are cis-repressor transcripts that silence multiple genes in at least three imprinted gene clusters in the mouse genome. Similar macro or long ncRNAs are abundant in the mammalian genome. Here we present the full coding and non-coding transcriptome of two mouse tissues: differentiated ES cells and fetal head using an optimized RNA-Seq strategy. The data produced is highly reproducible in different sequencing locations and is able to detect the full length of imprinted macro ncRNAs such as  Airn, Kcnq1ot1 and Nespas have been shown to repress 1–10 genes in cisThe study of whole transcriptomes, which detects all RNAs expressed in one cell population, provides insights into mechanisms regulating gene expression in development and disease. The first large-scale mammalian transcriptome studies based on genome tiling array hybridization or cDNA sequencing, unexpectedly revealed that most RNAs excluding structural tRNAs, rRNAs and spliceosome RNAs were non-protein-coding (nc) RNAs The development of Next Generation Sequencing applied to RNA (referred to as RNA-Seq) provides an accurate, unbiased and quantifiable transcriptome The ease and cost effectiveness of RNA-Seq has led to a growing amount of publications that share their transcriptome studies with the scientific community The majority of cellular RNA is composed of ribosomal RNAs (rRNAs). As sequencing this part of the transcriptome is usually not the main research focus, the RNA sample is prepared either by selection of polyadenylated (polyA) RNAs or by selective removal of rRNAs (ribo-depletion). RNA-Seq using polyA selected RNA detects protein-coding mRNAs at a depth sufficient to determine tissue-specific gene expression differences and can also detect at least a subset of ncRNAs In order to address these issues we modified an established protocol for RNA-Seq using the Illumina/Solexa platform RNA for sequencing was obtained from retinoic acid differentiated mouse CCE embryonic stem cells and 14.5 dpc mouse fetal head (FH) and subject to ribosomal RNA depletion using either the RiboMinus Kit (Invitrogen) or the Ribo-Zero Kit . This comparison shows more unique and nomatch as well as less ribosomal/mitochondrial tags were obtained from RNA-hydrolysis compared to cDNA-shearing . The number of repeat tags did not change significantly between the two fragmentation methods . This indicates that RNA-hydrolysis performs better than cDNA-shearing in producing more unique and less ribosomal RNA tags and is therefore the favourable template fragmentation method.To optimise ribosomal RNA depletion we used an improved depletion method, Ribo-Zero, in combination with RNA-hydrolysis. For the RNA from both FH and CCE tissues, one Ribo-Zero treatment was performed, the RNA was hydrolysed and sequenced in Vienna-CeMM (Research Center for Molecular Medicine). This resulted in a more efficient removal of ribosomal RNAs as only 4–14% of all tags were of ribosomal origin. We also observed an increase in uniquely matching (36–54%) and multiple matching tags (28–30%)  values that are corrected for gene length and the total number of sequence tags. Although FPKM (Fragments Per Kilobase of exon model per Million mapped reads) values are starting to replace RPKM, the latter are widely used to estimate expression levels of protein-coding genes as well as ncRNAs In the next step we analysed if the gene expression levels of protein-coding genes were comparable between the different fragmentation methods and locations. To obtain a comparable measure of gene expression we used RPKM  followed by cDNA synthesis produced a more equal representation of genes with sequencing tags than physically breaking up the cDNA (cDNA-shearing). In particular, this review noted an extreme 3′ positive bias for protein-coding genes from cDNA sheared templates in polyA RNA-Seq We next compared the gene coverage in the Ribo-Zero dataset to the RNA-hydrolysis RiboMinus dataset. We again observed a loss of sequence tags in both the 5′ and the 3′ UTRs. In comparison to the RiboMinus preparation, Ribo-Zero produces more tags in the 5′ and 3′ ends of small genes compared to the body of the genes  top, wheWe observed an under-representation of both 5′ and 3′ ends of genes of three size classes in sequencing tags from both RNA-hydrolysis and cDNA-shearing templates and for both ribosomal RNA depletion methods. We could directly link this phenomenon to 5′ and 3′ UTRs by analysing them separately from the coding regions. A similar drop of tag coverage at gene ends compared to gene bodies was previously reported, but was not connected to UTRs Based on the good correlation of all sequencing data , C we poalpha-crystallin A chain gene only in FH, which is specific for the developing eye and therefore absent from CCE cells (Pou5f1 (Oct4), still expressed in CCE and absent in FH  differentiated ES cells . The two ribo-depleted FH datasets generated here, detected five candidate ncRNAs (RPKM>3) that were not detectable to a reliable level in the Mortazavi et al. adult brain dataset  presented here in comparison to other RNA-Seq datasets. As no publicly available datasets directly match our datasets we could not compare gene expression levels directly. Therefore we chose to investigate the cDNA length distribution of differentially expressed genes between two datasets, as the length bias is a well established phenomenon distinguishing different template preparation protocols independently of biological differences We next tested the influence of the RiboMinus and the Ribo-Zero method on the comparability of our datasets to the published datasets introduced above. For determining differentially expressed genes we used a correction factor to maximise the number of genes showing no difference . We founIt is notable that the difference of the FH and CCE datasets to polyA RNA-Seq datasets that used a column-free RNA preparation protocol  was not as significant (minimum: p<0.003) as the differences to the Cloonan et al. dataset (p<0.0006) that used a column-based RNA preparation method. This indicates that independent of the polyA enrichment or ribosomal RNA depletion, a standardised RNA preparation protocol increases the comparability of the resulting transcriptomes markedly.Airn and Kcnq1ot1 macro ncRNAs. Finally we show that the use of a standardised RNA preparation, improves the comparability of the resulting transcriptome. With this awareness we propose the use of a limited number of tested protocols to allow the sharing of transcriptome data within the scientific community. A ribosomal RNA depletion approach, similar to the one presented here, could be one of these protocols as it is simple but powerful in detecting a large variety of protein-coding and non-protein-coding transcripts. This does not remove the necessity of additional data correction procedures but suggests that a closely matching experimental setup further improves the comparability of the resulting transcriptomes, allowing for even more accurate comparisons after data correction.In this study we investigated the use of ribo-depleted RNA-Seq for the study of whole transcriptomes. Using this approach we found differences in the coverage of genes with sequence tags when using different template fragmentation protocols, although not as dramatic as reported previously when comparing studies using different primers for the cDNA synthesis No animal experiments according to the Austrian Laboratory Animal Act were performed in this study, because humane killing of laboratory animals is not defined as animal experimentation under the Austrian Laboratory Animal Act . For this reason, approval of the study by an institutional ethics committee was not required. 14.5 days postcoitum mouse embryos were obtained after humane killing of wildtype FVB/N pregnant female mice by cervical dislocation by skilled qualified personnel. Mice were bred and housed at the Forschungsinstitut für Molekulare Pathologie GmbH, Dr. Bohr-Gasse 7, 1030 Vienna, Austria in strict accordance with national recommendations described in the “IMP/IMBA Common Institutional policy concerning the care and use of live animals” with the permission of the national authorities .CCE feeder-independent ES cells RNA-hydrolysis was done as described RNA fragmented by hydrolysis or intact RNA was used as template for cDNA synthesis using 5 µg random hexamers (Invitrogen) in a total volume of 20 µl according to the Superscript II Reverse Transcriptase (Invitrogen) standard protocol. Second-strand synthesis was performed by adding 91.8 µl water, 30 µl 5× Second strand buffer (Invitrogen), 3 µl 10 mM dNTP (Invitrogen), 4 µl DNA polymerase I (10 U/µl Invitrogen), 1 µl E. coli DNA ligase (10 U/µl Invitrogen) and 0.2 µl RNase H (2 U/µl Invitrogen), followed by incubation at 16°C for 2 hours. T4 DNA Polymerase (5 U/µl Invitrogen) (1 µl) was added followed by an additional 10 min at 16°C. The ds-cDNA was purified by using the MinElute Reaction Cleanup Kit (Qiagen), according to the manufacturer's protocol.2O.ds-cDNA prepared from intact RNA was fragmented in a volume of 130 µl by a Covaris S1 device according to the manufacturer's protocol using the following conditions: Duty cycle - 15.2%, Intensity - 6.0, Cycles/burst - 500, Duration - 60 sec, Cycle repeat - 5. After ethanol precipitation, the sheared cDNA was recovered in nuclease-free distilled HVienna-IMP and Nijmegen sequencing locations performed these steps independently using the same machines and the same ds-cDNA starting material. NGS in Nijmegen was done as described for libraries with a fragment size of 300 bp http://genome.ucsc.edu/, RiboMinus samples) and rRNA sequences . Remaining tags were aligned to the mouse genome  using BOWTIE High quality tags were aligned against mitochondrial sequences  entries were partitioned into a non-coding and protein-coding fraction: 1. non-coding RNA: NCBI was searched using “ncRNA RNA” AND “Mus musculus” 2. Protein-coding: all RefSeq entries occurring in the non-coding RNA list and all entries that start with “NR_” were removed. The resulting list was the RefSeq protein-coding gene set.The RefSeq  was calculated as described Pileup Per Kilobase of exon model per Millions of reads) is calculated as the RPKM but instead of the actual tags, the sum of the pileups of each base in the respective exons was used. RPKM and PPKM are highly correlated and a factor of 30 can be used to convert RPKM to PPKM (RPKM 3 = PPKM 90).RPKM . This data was not available for the Cui et al. and the Mortazavi et al. adult brain dataset Detailed information about the aligned sequence tags (pileups) was available from the Cloonan et al. dataset http://www.fon.hum.uva.nl/Service/Statistics/Wilcoxon_Test.html.t-tests were done in EXCEL: 1 tail, two-sample equal variance. For the comparison of the different tag types the percentages of tags were compared among all relevant, available replicates from the list shown in All RefSeq protein-coding genes that were expressed in the CCE and FH samples at an RPKM>3, were divided into 100 bins and the number of tags in each bin was determined separately for all sheared and all hydrolysed RiboMinus datasets and for the FH as well as the CCE Ribo-Zero datasets and normalised for the tag size and the total number of tags. Protein-coding genes were analysed as coding exons and UTRs separate, based on the RefSeq information. For the display UTRs bins were combined (averaged) into 10 bins. The bins were plotted relative to the bin with the highest tag count.The expression ratios of the RPKM/PPKM values . (A) Similar analysis as in B) Similar analysis as in C) Similar analysis as in (PDF)Click here for additional data file.Figure S2Determining correction factors for the calculation of differentially expressed genes. The number of genes showing expression differences as indicated by the bins on the x-axis was calculated for RefSeq protein-coding genes for the following RiboMinus (RM) datasets (shown in A) FH-RM – Cui et al. polyA, (B) CCE-RM – Cloonan et al. EBs (C) Mortazavi et al. adult brain - FH-RM . Note that the 0 bin contains all genes showing no expression in both datasets. Expression is defined by an expression value larger than RPKM 3 for all comparisons except for CCE-RM - Cloonan et al. EB, where a cutoff of PPKM 90 was used. All comparisons were corrected to maximize the number of genes in the bins with expression differences smaller than 8× except for (A) where no correction was necessary. Differential expression was defined as a larger than 8 fold expression difference. Grey bars show the number of genes before the correction, black bars show the number of genes after the correction. Note that the correction factor is shown in the figure and that the expression values for the tissue shown right were multiplied with this factor. For the Ribo-Zero comparisons (shown in shown in : (A) FH-shown in  the same(PDF)Click here for additional data file.Table S1Ribosome depletion, tag types and GEO accession numbers obtained in the individual sequencing reactions.(DOC)Click here for additional data file.


Bacillus thuringiensis (Bt) toxins have been planted widely to control insect pests, yet evolution of resistance by the pests can reduce the benefits of this approach. Recessive mutations in the extracellular domain of toxin-binding cadherin proteins that confer resistance to Bt toxin Cry1Ac by disrupting toxin binding have been reported previously in three major lepidopteran pests, including the cotton bollworm, Helicoverpa armigera. Here we report a novel allele from cotton bollworm with a deletion in the intracellular domain of cadherin that is genetically linked with non-recessive resistance to Cry1Ac. We discovered this allele in each of three field-selected populations we screened from northern China where Bt cotton producing Cry1Ac has been grown intensively. We expressed four types of cadherin alleles in heterologous cell cultures: susceptible, resistant with the intracellular domain mutation, and two complementary chimeric alleles with and without the mutation. Cells transfected with each of the four cadherin alleles bound Cry1Ac and were killed by Cry1Ac. However, relative to cells transfected with either the susceptible allele or the chimeric allele lacking the intracellular domain mutation, cells transfected with the resistant allele or the chimeric allele containing the intracellular domain mutation were less susceptible to Cry1Ac. These results suggest that the intracellular domain of cadherin is involved in post-binding events that affect toxicity of Cry1Ac. This evidence is consistent with the vital role of the intracellular region of cadherin proposed by the cell signaling model of the mode of action of Bt toxins. Considered together with previously reported data, the results suggest that both pore formation and cell signaling pathways contribute to the efficacy of Bt toxins.Transgenic crops producing  Bacillus thuringiensis (Bt) kill some major insect pests, but are harmless to vertebrates and most other organisms The insecticidal proteins of Cry1A toxins bind to the extracellular domain of cadherin proteins that traverse the larval midgut membrane; disruption of this binding can cause resistance Heliothis virescens, Pectinophora gossypiella, and cotton bollworm, Helicoverpa armigeraH. armigerah, which varies from 0 for completely recessive resistance to 1 for completely dominant resistance Although data are limited on the genetic basis of field-evolved resistance to Bt crops H. armigera to Cry1Ac. We found this allele in three field-selected populations, each from a different province in northern China where Bt cotton that produces Cry1Ac has been grown intensively for many years Here we discovered a novel cadherin allele genetically linked with non-recessive resistance of H. armigera collected in 2009 from three field populations in northern China that had been exposed intensively to Bt cotton. To detect resistance alleles, we used an F1 screen of 572 single-pair families obtained by crossing moths derived from each of the three field populations with moths from the laboratory-selected resistant SCD-r1 strain  that carries a premature stop codon in the extracellular domain of HaCad1 progeny that survived exposure to a diagnostic concentration of Cry1Ac (1 µg Cry1Ac per cm2 diet) from 48 of the 112 single-pair families in which resistance was detected  HaCad resistance allele r15. We detected one r15 allele in each of the three field populations screened: Xiajin (Shandong Province), Anyang (Henan Province), and Anci (Hebei Province) . For these three populations pooled, the estimated r15 allele frequency is 0.0061   .r15 allele from each population has the same deletion in its cDNA sequence caused by loss of exon 32, the genomic DNA sequence in exon 32 varies among the three populations, indicating at least three independent origins for this allele  with 95% identity to part of the 3′ non-coding sequence of a carboxyl/choline esterase gene of H. armigera (GenBank no. FJ997310.1). The r15 allele from Anyang has a 92 bp deletion in exon 32 , one from Xiajin (XJ-r15) and the other from Anyang (AY-r15)  .1 progeny of crosses between the susceptible strain SCD and either XJ-r15 or AY-r15 indicate that the resistance associated with r15 is not recessive  of a strain divided by the LC50 of the susceptible SCD strain. Resistance ratios for Cry1Ac were 140 for XJ-r15, 82 for AY-r15, and 540 for SCD-r1 , which ranges from close to or <0 for resistance conferred by alleles at different loci to close to or >1 for resistance conferred by alleles at a shared locus To test for allelism between the 5×SCD-r1 . Based o strains . This imr15 allele (1 (XJ-r15×SCD) and a female SCD, the number of heterozygotes (r15s) relative to susceptible homozygotes (ss) was significantly higher for survivors on treated diet (68 to 5) than on untreated diet (27 to 23)  , all 16 survivors on diet treated with the highest toxin concentration used in the linkage analysis (0.5 µg Cry1Ac per cm2 diet) were r15s   . Whereas= 0.002) . Becauser15 in the mode of action of Cry1Ac, we expressed four cadherin alleles in Sf9 cells: susceptible (s), r15, and two chimeric alleles , Sf9 cells with the cytoplasmic domain encoded by the r15 allele (r15 and s/r15) were significantly less susceptible to Cry1Ac  is linked with cadherin and has amino acid substitutions in the putative toxin-binding region but not premature stop codons, deletions or insertions r15 allele considered together with earlier reports r15), non-recessive resistance linked with substitutions in the extracellular domain of cadherin (r12), non-recessive resistance not linked with cadherin (strain AY423), and recessive resistance associated with disruption of the extracellular domain of cadherin .Whereas non-recessive resistance to Bt toxins has been reported previously in r15 allele differs from the most common type of insect resistance to Bt toxins, which is called “Mode 1” and entails recessive inheritance, high levels of resistance to one or more Cry1A toxins, little or no cross-resistance to Cry1C, and reduced binding of one or more Cry1A toxins H. virescens, P. gossypiella and H. armigeraH. virescensPlutella xylostella and Trichoplusia niBombyx moriB. moriThe resistance conferred by the r15 allele of HaCad is as follows: In r15s heterozygotes, Cry1Ac binds to the mutant cadherin protein encoded by the r15 allele , but this binding does not lead to toxicity because the r15 mutation interferes with post-binding events. This binding also reduces the amount of toxin available to cause toxicity by binding with the susceptible cadherin . Thus, sensitivity to Cry1Ac is substantially lower for r15s heterozygotes than susceptible homozygotes, which yields non-recessive resistance. By contrast, when reduced binding of toxin is the primary mechanism of resistance as with the r1 allele of HaCad, Cry1Ac does not bind to the mutant cadherin in r1s heterozygotes. This increases the amount of toxin available to bind with the susceptible cadherin and ultimately to cause toxicity via post-binding events. Therefore, even though only half of the cadherin protein binds Cry1Ac in r1s heterozygotes, their net sensitivity to Cry1Ac similar to that of susceptible homozygotes, yielding recessive resistance.We hypothesize that the mechanism of non-recessive resistance to Cry1Ac associated with the H. armigera host plants, which may be a key factor slowing resistance r15 allele on evolution of resistance in the field.The non-recessive inheritance detected here and previously in field-selected populations of cotton bollworm from China may have important practical implications for resistance management H. armigera was started with insects from the Côte d'Ivoire (Ivory Coast), Africa over 30 years ago and was maintained in the laboratory without exposure to insecticides or Bt toxins r1 allele of the cadherin gene (HaCad) . All of these field-collected male moths were crossed individually to virgin female moths from the SCD-r1 strain that were homozygous for the r1 allele (r1r1). In addition, we collected fourth instars surviving on Bt cotton plants from Xiajin in July. We reared these field-collected larvae to pupation in the laboratory on artificial diet without Bt toxin and allowed moths to emerge. Each resulting field-derived male or female moth was crossed individually with a virgin moth of the opposite sex from the SCD-r1 strain.We used the F1 offspring from each of 572 single-pair families (n = 48 second instars per family) with a discriminating concentration of activated Cry1Ac (1 µg toxin per cm2 diet) We screened the FActivated Cry1Ac, Cry1Aa, and Cry1Ab were provided by Dr. Marianne P. Carey . Cry2Ab protoxin was provided by the Institute of Plant Protection, Chinese Academy of Agricultural Sciences (CAAS), China.H. armigera from China, we used the same bioassay method for Cry1A toxins that was employed in previous studies H. armigeraWe used a diet surface contamination bioassay 1 screen from each of 48 resistant single-pair families using the SV total RNA isolation system according to the manufacturer's instructions . We performed reverse transcription with the Moloney murine leukemia virus reverse transcriptase (Promega). We used four primer pairs to amplify four overlapping fragments that completely cover the cDNA of HaCadWe extracted total RNA from larval midguts of a subset of survivors from the Fr15 , and 2.5 µl of 10× PCR buffer. The PCR amplification protocol included denaturation at 94°C for 3 min, followed by 30 cycles  and a final extension at 72°C for 10 min. PCR products were analyzed by 1.5% agarose gel electrophoresis and ethidium bromide staining.We prepared genomic DNA from individual larvae or adults using a genomic DNA extraction kit according to the manufacturer's instructions . We designed a forward primer (Cyto-F in Exon32) and a reverse primer (Cyto-R in Exon 33) to amplify the genomic DNA flanking the mutation site of ss) had only one fragment of 556 bp, resistant homozygotes (r15r15) had only one fragment of 2,014 bp, and heterozygotes (r15s) had two fragments  (We used the banding patterns of PCR products to determine the cadherin genotype for the XJ-r15 strain. Susceptible homozygotes ( 556 bp) .r15 (XJ-r15), we started with the first single-pair F1 family in which r15 was identified (r15s) and a female moth from SCD-r1 (r1r1). The F1 progeny from this family that survived exposure to the diagnostic concentration of Cry1Ac were r1r15. These survivors were crossed with susceptible SCD adults (ss) to produce F2 progeny consisting of a mixture of r1s and r15s. Single-pair matings were made among the F2 progeny to generate F3 progeny consisting of a mixture of three genotypes including r15s . Survivors of this screen were used to establish the XJ-r15 strain. We used diagnostic PCR . Starting with the single-pair F1 family in which r15 was identified from Anyang, we used parallel procedures to establish resistant strain AY-r15, which was also homozygous for r15.To establish the first resistant strain homozygous for entified . The parg r15s . After >stic PCR  to verif1 progeny between a susceptible strain (SCD) and each of three resistant strains: XJ-r15 and AY-r15 with resistance allele r15 and SCD-r1 with resistance allele r1. For each of the three resistant strains, the F1 progeny were generated with reciprocal crosses as follows: We crossed 30 virgin female moths from the resistant strain with 30 male moths of the SCD strain and vice versa for the reciprocal cross. We used bioassays to determine the responses to Cry1Ac of F1 larvae from each reciprocal cross.To evaluate dominance, maternal effects, and sex linkage, we used bioassays (as described above) to determine responses to Cry1Ac of the Fr1 cadherin resistance allele in strain SCD-r1 and any major resistance alleles in strains XJ-r15 and AY-r15, we crossed SCD-r1 with XJ-r15 and AY-r15 and tested the F1 progeny using bioassays with Cry1Ac as described above. If resistance is not completely dominant in two resistant strains crossed in an interstrain complementation test for allelism, the F1 progeny from the cross will be more resistant if the resistance alleles occur at the same locus in both strains than if they occur at different loci in each strain To test for allelism between the 1 offspring. Next, we crossed an F1 male with a female from SCD to produce an F2 backcross family. Using the bioassay method described above, we reared larvae from the backcross family for five days on diet with either 0 (control), 0.3 or 0.5 µg Cry1Ac per cm2 diet. After five days, we transferred survivors to untreated diet and reared them until they reached the final instar. From each survivor to the final instar, we obtained genomic DNA that was used to determine the cadherin genotype of each individual using diagnostic PCR  and resistant strain XJ-r15 (r15) with high fidelity PrimeSTAR® DNA HS polymerase  using the primers HaCad-Not1-F and HaCad-Xba1-R, respectively. With these two primers, Not1 and Xba1 restriction sites were introduced to the amplified fragments for subsequent in-frame cloning.We amplified the coding sequence of HaCad from SCD and XJ-r15 strain using the forward primer HaCad-Not1-F and the reverse primer TMR. The second PCR amplified the cytoplasmic domain of HaCad from SCD and XJ-r15 strain using the forward primer TMF and the reverse primer HaCad-Xba1-R. Using the mixture of the first PCR amplification products of HaCad from SCD (ss) and the second PCR product from XJ-r15 (r15r15) as template, a chimeric cDNA (s/r15) was amplified with the primers HaCad-Not1-F and HaCad-Xba1-R. Analogously, a complementary cDNA (r15/s) was amplified using the mixture of the first PCR amplification products of HaCad from XJ-r15 (r15r15) and the second PCR product from SCD (ss) as template.Overlap extension PCR was used to construct the two chimeric alleles. The first PCR amplified the ectodomain of ss, r15r15, s/r15, and r15/s) was transfected into E. coli TOP10 and selected for ampicillin-resistant transformants. We confirmed the presence and correct orientation of the inserts in the plasmids by restriction analysis and sequencing.The four resulting PCR products of the expected sizes were excised and purified using the Wizard SV Gel and PCR Clean-Up System (Promega), and the four genes were ligated into the expression vector pFastBac1 . Each of the four pFastBac1-HaCad plasmids . The recombinant bacmids were purified and used to transfect Spodoptera frugiperda (Sf9) cells according to the manufacturer's protocol (Invitrogen). Transfection was achieved using Cellfectin reagent according to the manufacturer's instructions (Invitrogen). Viral supernatants from the initial P1 transfected cultures were harvested five days after transfection. Virus titers were improved through serial infections as recommended by the manufacturer (Invitrogen). P3 viral stocks containing the highest viral titer  were stored at 4°C and used for further transfections.Each of the four pFastBac1-HaCad plasmids was purified and transfected into 7 cells/ml and were subcultured every 3–4 days. For infections, log phase cells were seeded at a density of 2×106 cells/ml and infected with P3 recombinant baculovirus stocks in low serum media containing 2% fetal bovine serum. Sf9 cells were infected with recombinant viruses for each of the four recombinant cadherin alleles in six-well plates containing glass coverslips. Control Sf9 cells were either infected with an empty bacmid or were not infected.Sf9 cells (ATCC 1711-CRL) were cultured at 27°C in Sf-900 II SFM medium supplement (Invitrogen) with 10% fetal bovine serum and 10 µg/ml gentamycin. The cells were maintained at a density of 1×10Two days after infection, cells were harvested and washed twice with PBS (pH 7.4) and fixed in 4% parafomaldehyde solution for 30 min at room temperature (RT). Fixed cells were washed three times with PBS (pH 7.4) and then blocked with 1% BSA for 1 h at RT. After blocking, they were incubated with rabbit polyclonal anti-Cadherin IgG antibody (1∶100 dilution) for 1 h at RT. The anti-cadherin antibody was raised against the toxin-binding portion of HaCad For immunolocalization of Cry1Ac binding, after BSA blocking the coverslips were washed in PBS and incubated with 10 nM Cry1Ac toxin for 2 h at 25–28°C. Coverslips were then washed as described above and incubated with rabbit polyclonal anti-Cry1Ac antibody (1∶100 dilution) for 1 h at 25–28°C.After 1 h, the cells were rinsed three times with PBS, followed by incubation with FITC-conjugated goat IgG secondary antibodies  at 1∶100 dilution for 1 h. The unbound conjugate was removed by washing with PBS. The coverslips were sealed and examined immediately in a LSM Zeiss laser scanning confocal microscope, using excitation at 488 nm and 20× objective with additional zooming. Image acquisition of the controls and data processing were performed under the same conditions.6 cells were incubated for 1 h with or without Cry1Ac toxin. All incubations were performed in sterile 96-well cell culture plates at 27°C with pipetting repeated every 10 min to maintain homogenization. After incubation, two samples of 10 µl were transferred separately from each well into two new wells and thoroughly mixed with 10 µl of Trypan Blue solution (0.4% in 1× PBS). The number of live (unstained) and dead (stained blue) cells were counted in a hemacytometer. About 10,000 cells were counted for each replicate of each treatment. The experiment was repeated with three independent transfections for each of the six types of Sf9 cells .Sf9 cells were infected with recombinant viruses in six-well plates with a multiplicity of infection of three. Three days after infection, cells were harvested and washed twice in PBS and centrifuged for 5 min at 500 g at 4°C. Cells were re-suspended in PBS buffer and counted in a hemacytometer. One hundred microliter aliquots containing 5×1050), the 95% fiducial limits (FL) of each LC50 value, as well as the slope of each concentration-mortality lines and the standard error (SE) of the slope. We calculated the resistance ratio of each strain or group of F1 progeny as its LC50 divided by the LC50 of the susceptible SCD strain. We considered LC50 values significantly different if their 95% fiducial limits did not overlap. We adjusted for control mortality (range = 0 to 6%) to estimate LC50 values, but not in the bioassays at only the diagnostic concentration, which had lower control mortality (<5%).We analyzed mortality data from diet bioassays with larvae and cytoxicity assays with Sf9 cells using probit analysis  as described previously h based on survival at the diagnostic concentration of Cry1Ac and via estimation of D50 values of Cry1Ac. D ranges from −1 (completely recessive) to 1 (completely dominant) while h ranges from 0 (completely recessive) to 1 (completely dominant). For comparison, we converted D to the same scale as h as follows: h = (D+1)/2 We estimated dominance , SCD-r1 was one resistant strain (R1), and either XJ-r15 or AY-r15 was the second resistant strain (R2). To calculate C, we used data from To quantify the results of interstrain complementation tests for allelism, we used the index of commonality (C) Figure S1Genomic DNA sequence of exon 32 of HaCad in susceptible and resistant strains. SCD: susceptible strain with wild type sequence. AY-r15: resistant strain with a 92 bp deletion. XJ-r15: resistant strain with a 1,459 bp insertion. Both mutations yield a predicted HaCad protein lacking 55 amino acids near the 5′-end of the cytoplasmic domain.(TIFF)Click here for additional data file.Figure S2Marker-assisted selection used to produce resistant strains XJ-r15 and AY-r15, which are homozygous for the r15 allele.(TIFF)Click here for additional data file.Figure S3Cadherin in Sf9 cells transfected with four HaCad alleles. As in s), resistant (r15) causing a 55 amino acid deletion in C, chimeric allele s/r15 with C from r15 and the other components from s, and complementary chimeric allele r15/s with C from s and the other components from r15.(TIFF)Click here for additional data file.Figure S4Detection of HaCad protein in Sf9 cells transfected with four alleles of HaCad by immunochemical analysis under confocal microscopy. Cells were probed sequentially with anti-HaCad antiserum (1∶100) and FITC-conjugated anti-rabbit antibody (1∶100). HaCad was detected in Sf9 cells transfected with each of the four alleles of HaCad, but not in control Sf9 cells that were either transfected with an empty bacmid (EB) or not transfected. See (TIFF)Click here for additional data file.Figure S5Diagnostic PCR for the r15 allele of HaCad in the XJ-r15 strain. The primer pair Cyt-F/Cyt-R was used for PCR amplifications with genomic DNA as templates. The susceptible homozygote (ss) had one fragment of 556 bp, the resistant homozygote (r15r15) had one fragment of 2014 bp, and the heterozygote (r15s) had two fragments (2014 and 556 bp).(TIFF)Click here for additional data file.Table S1Frequency of cadherin resistance allele r15 in F1 screens of three field populations of H. armigera sampled in northern China during 2009.(DOCX)Click here for additional data file.Table S2Cross-resistance of strain XJ-r15 of H. armigera, which had a resistance ratio of 140 against Cry1Ac relative to the susceptible strain SCD (see ).(DOCX)Click here for additional data file.Table S3Primers used for amplifying the cadherin alleles of H. armigera.(DOCX)Click here for additional data file.Table S4PCR amplification protocols for recombinant HaCad alleles of H. armigera.(DOCX)Click here for additional data file.


Isolated influenza A virus nucleoprotein exists in an equilibrium between monomers and trimers. Samples containing only monomers or only trimers can be stabilized by respectively low and high salt. The trimers bind RNA with high affinity but remain trimmers, whereas the monomers polymerise onto RNA forming nucleoprotein-RNA complexes. When wild type (wt) nucleoprotein is crystallized, it forms trimers, whether one starts with monomers or trimers. We therefore crystallized the obligate monomeric R416A mutant nucleoprotein and observed how the domain exchange loop that leads over to a neighbouring protomer in the trimer structure interacts with equivalent sites on the mutant monomer surface, avoiding polymerisation. The C-terminus of the monomer is bound to the side of the RNA binding surface, lowering its positive charge. Biophysical characterization of the mutant and wild type monomeric proteins gives the same results, suggesting that the exchange domain is folded in the same way for the wild type protein. In a search for how monomeric wt nucleoprotein may be stabilized in the infected cell we determined the phosphorylation sites on nucleoprotein isolated from virus particles. We found that serine 165 was phosphorylated and conserved in all influenza A and B viruses. The S165D mutant that mimics phosphorylation is monomeric and displays a lowered affinity for RNA compared with wt monomeric NP. This suggests that phosphorylation may regulate the polymerisation state and RNA binding of nucleoprotein in the infected cell. The monomer structure could be used for finding new anti influenza drugs because compounds that stabilize the monomer may slow down viral infection. The RNAs of negative strand RNA viruses are encapsidated by their specific viral nucleoproteins, forming helical nucleoprotein-RNA structures that are the template for transcription and replication. All these nucleoproteins have two activities in common: RNA binding and self-polymerisation, and it is likely that these activities are coupled. All these viruses have to keep their nucleoprotein from binding to cellular RNA and from polymerisation before viral RNA binding. The non-segmented viruses solve this by coding for a phosphoprotein that binds to the nucleoprotein, blocking both activities. The segmented viruses, such as influenza and Bunyaviruses, do not code for a phosphoprotein and need to solve this problem differently. Here we present the atomic structure of monomeric influenza virus nucleoprotein. Although the structures of the influenza virus and the Rift Valley Fever Virus (Bunya virus) nucleoproteins are different, there are functional similarities when the monomer and polymer structures are compared. Both nucleoproteins have a core structure that is identical in the monomer and the polymer. They contain a flexible arm that moves over to a neighbouring protomer in the polymer structure but that folds onto the core in the monomer structure, hiding the RNA binding groove in the Rift valley Fever Virus nucleoprotein and modifying the electrostatic potential of the RNA binding platform of the influenza virus protein. Negative strand RNA viruses have an RNA genome in the opposite sense of that of messenger RNA. Therefore, the first viral activity after entering the host cell is transcription by the viral RNA-dependent RNA polymerase. The template for transcription is a complex between the viral RNA and the nucleoprotein (NP) that binds to the RNA sugar-phosphate backbone Rhabdoviridae (ex. vesicular stomatitis virus (VSV) and rabies virus) and the Paramyxoviridae (ex. Sendai and measles virus) and segmented viruses like the Arenaviridae (Lassa fever virus), the Bunyaviridae ) and the Orthomyxoviridae (influenza viruses). When expressed in a transfected cell in the absence of other viral components, the nucleoproteins of most of these viruses bind to cellular RNA and form nucleoprotein-RNA complexes that are indistinguishable from the viral complexes 00P complex of VSV shows how the P binding site overlaps with the RNA binding groove on the nucleoprotein and with one of the sites involved in nucleoprotein polymerisation, thus blocking both activities Negative strand RNA viruses include non-segmented viruses like the The segmented viruses do not code for an equivalent of a phosphoprotein and solve the problem in different ways. The nucleoprotein of RVFV has been crystallised in two forms; as a monomer and as a hexameric ring The nucleoproteins of influenza A H1N1 and H5N1 crystallised as trimers Here we show the structure of the monomeric R416A mutant and describe the RNA binding characteristics of monomeric wild type NP. In the infected cell NP may be kept monomeric by post translational modification. Mass spectroscopy analysis on NP isolated from viral RNP showed phosphorylation of serine 165. By mutagenesis we generated the S165D mutant to mimic this phosphorylation. S165D was monomeric with the same biophysical characteristics as the R416A mutant but showed high cooperativity for RNA binding at concentrations above the Kd.et al.We previously showed that influenza virus NP can be stabilised as monomers in 50 mM salt and as trimers and tetramers in 300 mM salt 1 diffracted to 2.7 Å . Escherichia coli BL21 (DE3) cells carrying the plasmids were induced 4 hours by adding 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C or 12 h at 28°C (mutant proteins) and collected by centrifugation. The pellet was resuspended and sonicated in a lysis buffer composed of 50 mM Tris at pH 7.4 with 300 mM NaCl, 15 to 30 mM imidazole, 1 M NDSB201 (Sigma), 5 mM β-ME and 5 mM MgCl2. The protein was purified by Ni2+ affinity chromatography  followed by a Heparin column . The protein was then dialyzed against 20 mM Tris pH 7.4 and 50 mM or 300 mM NaCl. The last purification step was size-exclusion chromatography using a Superdex S200 column. The protein was eluted in high or low salt according to the required polymerisation state. The protein concentration was determined by using the extinction coefficient ε = 55500 M−1.cm−1 at 280 nm.The full-length NP gene of the H1N1 strain A/WSN/33 with a 6-His-tag at its C-terminal end was cloned in the pET22 vector (Novagen) under the control of a T7 promotor. The R416A, Y148A and S165D mutations were introduced by using The R416A mutant protein was crystallized by vapor diffusion using the sitting drop method. The crystals were obtained in 0.1 M Hepes pH 7.5, 1.2 M potassium sodium tartrate with a protein concentration of about 10 mg/ml. Data were collected at the ESRF (beamline ID14-4) and processed with the XDS package Samples were applied between a carbon and a mica layer and negatively stained with 2% (w/v) sodium silicotungstate (pH 7.0). The carbon film was covered by a copper grid and air dried. Micrographs were recorded with a JEOL 1200 EX II microscope at 100 kV with a nominal magnification of 40,000×. Micrographs were taken on a 2K×2K CCD camera (Gatan Inc.). The RNA binding kinetics was performed with a protein solution at 100 µM and an RNA of 51 nucleotides The measurements were performed on a Malvern nanosizer instrument thermostated at 20°C, in 20 mM Tris pH7.5, 50 or 300 mM NaCl according to the sample. The protein concentrations were in a range of 5 to 50 µM and for each sample, 12 to 18 scans were averaged. All experiments were repeated at least 3 times. The scattering intensity data were processed using the instrumental software to obtain the hydrodynamic radius and the size distribution of scatterers in each sample.Protein samples were centrifuged at 16000× g and the protein concentration in the supernatant was adjusted to 0.5 mg/ml. CD experiments were performed on a JASCO-810 spectrometer at 20°C with a 1 mm path-length quartz cell using a bandwidth of 1 nm, an integration time of 1 second and a scan rate of 50 nm/min. Each spectrum is the average of 10 scans. All spectra were corrected by subtracting the buffer spectrum acquired under the same conditions. All data were normalized to mean residue ellipticity. The melting temperature was obtained for each protein sample by measuring the CD signal at 208 nm and 222 nm from 20°C to 80°C every 2°C. At 80°C all proteins precipitated. The value of the denaturation midpoint (°C) is the average of at least 3 measurements on independent protein preparations.The RNA binding kinetics were performed on a Biacore 3000 apparatus using streptavidin coated sensor chips  prepared as indicated by the manufacturer. Immobilization of the biotinylated 24 nt oligoribonucleotide The filter binding experiments were performed with a radiolabelled 81 nt panhandle RNA as described NP from egg grown influenza A/PR/8/34 virus was cut out from a Coomassie-stained 1D SDS-PAGE gel and digested with Trypsin (Roche Applied Science). The digest was analyzed by LC-ESI-MS/MS with an Ion trap MS HCT ultra PTM Discovery System  coupled with a Nano-LC 2D HPLC system . A CapRod monolithic C18 column  was used to separate the peptides. The gradient was 10–70% ACN within 20 min at 300 nl/min flow rate. The top two precursor ions were selected over m/z range from 400 to 1200 for fragmentation. Fragmentation was performed subsequently for 60 ms over 300 to 2000 m/z. The raw data were processed with DataAnalysis . The extracted MS/MS data were submitted to MASCOT  in-house server via Biotools . Proteins were identified by searching the peptide lists against SwissProt. The following parameters were used: Taxonomy: Viruses; Enzyme: trypsin; Max Missed Cleavages: 2; Variable modifications: oxidation (M); carbamidomethyl (C); phospho (ST), phospho(Y); Peptide Mass Tolerance: ±0.5 Da: Fragment Mass Tolerance: ±0.5 Da.Text S1Supporting Figures S1, S2, S3, S4.(PPTX)Click here for additional data file.


Enterovirus 71 (EV-71) is the main etiological agent of hand, foot and mouth disease (HFMD). Recent EV-71 outbreaks in Asia-Pacific were not limited to mild HFMD, but were associated with severe neurological complications such as aseptic meningitis and brainstem encephalitis, which may lead to cardiopulmonary failure and death. The absence of licensed therapeutics for clinical use has intensified research into anti-EV-71 development. This review highlights the potential antiviral agents targeting EV-71 attachment, entry, uncoating, translation, polyprotein processing, virus-induced formation of membranous RNA replication complexes, and RNA-dependent RNA polymerase. The strategies for antiviral development include target-based synthetic compounds, anti-rhinovirus and poliovirus libraries screening, and natural compound libraries screening. Growing knowledge of the EV-71 life cycle will lead to successful development of antivirals. The continued effort to develop antiviral agents for treatment is crucial in the absence of a vaccine. The coupling of antivirals with an effective vaccine will accelerate eradication of the disease. Picornaviridae. The EV-71 genome is a single-stranded, positive sense RNA with approximately 7411 nucleotides, and consists of an open reading frame flanked by 5′ and 3′ untranslated regions (UTRs)  pyrimidine derivative, DTriP-22, was shown to effectively target the RdRP of EV-71 with IC50 values of 0.15 – 0.98 μM, and suppress the accumulation of both positive and negative strands of viral RNA during EV-71 infection. DTriP-22-resistant mutants had mutations in the RdRP, implying that DTriP-22 interacts with RdRP and inhibits poly (U) elongation activity, but not VPg uridylylation [Ribavirin  is a conventional nucleoside analogue that targets the RdRP of picornaviruses . Ribavir in mice . Recentllylation .50. Only bovine lactoferrin, pleconaril, shRNA, siRNA, rupintrivir, ribavirin and 17-AAG have been tested in vivo. Ribavirin and amantadine are already in clinical use for other viruses, and rupintrivir and pleconaril are in clinical development.Figure The availability of a suitable animal model carrying all the required receptors and attachment factors for testing of the antivirals will accelerate the development of antivirals. The clinical use of other antiviral agents has been hampered by the potential adverse effects to the host and emergence of drug resistance mutants. Combination therapy targeting different replication steps of EV-71 infection cycle has shown synergistic activity  and coultrans-acting factor; MAVS: Mitochondrial anti-viral signaling; MDA-5: Melanoma differentiation associated gene; ORF: Open reading frame; PV: Poliovirus; RdRP: RNA-dependent RNA polymerase; VPg: Viral protein genome-linked.EV-71: Enterovirus 71; HFMD: Hand, foot and mouth disease; IRES: Internal ribosome entry site; ITAF: IRES-specific CWT and YFC have a pending patent on SP40 peptide.CWT, JKFL, ICS and YFC drafted the manuscript. All authors read and approved the final manuscript.


Dictyostelium discoideum, we have deleted the encoding genes rrpA, rrpB and rrpC in all possible combinations. In these strains, expression of either antisense or hairpin RNA constructs against the transgene lacZ resulted in a 50% reduced β-Galactosidase activity. Northern blots surprisingly revealed unchanged lacZ mRNA levels, indicative of post-transcriptional regulation. Only in rrpC knock out strains, low levels of β-gal small interfering RNAs (siRNAs) could be detected in antisense RNA expressing strains. In contrast to this, and at considerably higher levels, all hairpin RNA expressing strains featured β-gal siRNAs. Spreading of the silencing signal to mRNA sequences 5′ of the original hairpin trigger was observed in all strains, except when the rrpC gene or that of the dicer-related nuclease DrnB was deleted. Thus, our data indicate that transitivity of an RNA silencing signal exists in D. discoideum and that it requires the two enzymes RrpC and DrnB.RNA interference (RNAi) is a gene-regulatory mechanism in eukarya that is based on the presence of double stranded RNA and that can act on both, the transcription or post-transcriptional level. In many species, RNA-dependent RNA polymerases (RdRPs) are required for RNAi. To study the function of the three RdRPs in the amoeba  Double stranded RNA is the central nucleic acid entity in the gene regulatory mechanism RNA interference (RNAi) C. elegans RdRP EGO-1 Paramecium tetraureliaThe third class of proteins, RdRPs, can synthesize primer-dependent or -independent complementary RNAs on a single stranded RNA template, and they are required for several mechanisms of RNA-mediated gene regulation, including RNAi C. elegans in investigations of chimeric unc-22/gfp transgenes, from which the term transitive silencing was coined Because of the directionality of nucleic acids polymerases, secondary siRNA can also be observed 5′ of the original silencing trigger. This 5′ spreading has been observed operational in D. discoideum encodes three RdRP, named RrpA-C D. discoideum correspond to the retrotransposon DIRS-1 rrpC gene. Since the genome of D. discoideum harbors many copies of this retrotransposon, and siRNAs appear to be covering its entire sequence, it is very difficult to study on this endogenous target, whether transitive silencing D. discoideum. To allow for the investigation of this 5′ spreading of a silencing signal, we have established here an artificial system based on the lacZ gene, encoding β-D-Galactosidase. We observe post-transcriptional gene silencing upon expression of a hairpin or an antisense RNA in various rrp gene deletion strains. Transitivity of the silencing signal is observed in the majority of investigated strains but not when the rrpC gene is deleted, or that of the dicer-related nuclease DrnB.Dictyostelium discoideum strain A×2 was grown axenically in HL5 medium containing 50 µg/mL ampicillin, 250 ng/mL amphotericin, 10 µg/mL penicillin and 10 µg/mL streptomycin at 22°C in shaking suspension. In strains transformed with pDneo2a lacZ, the medium was supplemented with 10 µg/mL geniticin (G418) and for strains transformed with constructs of the pDM326 series, additionally with 10 µg/mL blasticidin.The DNA oligonucleotides were purchased from Sigma and are listed in Genomic DNA was isolated as described previously Taq polymerase and 2 µL Pfu polymerase in a total volume of 50 µL.PCR reactions were carried out on 10–100 ng template DNA in 1×PCR buffer (Thermo Scientific), using 0.2 mM dNTPs, 0.5 µM each primer and 1 µL rrp genes are listed in the online resource www.dictybase.org with their accession numbers DDB_G0289659 (rrpA), DDB_G0291249 (rrpB) and DDB_G0280963 (rrpC). The latter gene previously had been named dosAdrnB– strain was described recently rrp genes was performed as described using constructs generated by combinatorial cloning D. discoideum cells by electroporation Klebsiella aerogenes and analyzed initially by PCR and deletions were confirmed by Southern blot ((r) cassette by transient expression of the Cre recombinase rrpA–:rrpB–, rrpA–:rrpC– and rrpB–:rrpC–. Again the BS(r) cassette was excised and the strain rrpA–:rrpB– was used for the generation of the rrpA–:rrpB–:rrpC– (“triple–”) knock out strain, from which the resistance cassette was also removed.The ern blot , as descE. coli lacZ gene from the plasmid pET-14b lacZ, which was a kind gift of Science Bridge , using primers #0182 and #0183. These primers add a Pst I site at the 5′ end and an Xho I site at the 3′ end for subsequent cloning in pDneo2a. Furthermore, primer #0182 featured an “A run” sequence between the Pst I site and the Start codon, which increases expression levels in D. discoideumlacZ was confirmed by sequencing. Using the introduced restriction sites, the A-run modified lacZ gene was digested from the cloning vector and cloned in the expression vector pDneo2a Pst I and Xho I, yielding construct pDneo2a lacZ.For the generation of ß-Galactosidase expressing strains, we PCR amplified the coding sequence of the sense, a 985 bp fragment, representing positions 1143–2127 of the lacZ gene was PCR amplified from vector pJET1.2 lacZ, using primers #1196 and #1197, which add 5′ Bgl II or 3′ Spe I restriction sites. Upon cloning in the vector pGEM-T easy, the correct insert was confirmed by sequencing. Using the added restriction sites, the insert was excised from the cloning vector and inserted in the vector pDM326 Bgl II and Spe I. Analogously, pDM326 ß-gal antisense was generated, by initially PCR amplifying a 985 bp fragment, representing positions 1143–2127 of the lacZ gene. The used primers #0190 and #0191 localize to the same position as #1196 and #1197, however, with a mirrored positioning of the added Spe I and Bgl II sites. Upon cloning in the vector pGEM-T easy, the correct insert was confirmed by sequencing. Directed cloning in pDM326 resulted in the generation of the antisense construct.For generation of pDM326 ß-gal hairpin construct, initially the sense part of the hairpin, representing positions 1359–1858 of the lacZ gene, was PCR amplified from vector pJET1.2 lacZ using primers #0184 / #0185 that add Dra III and Spe I sites to the PCR product at the 5′ end and the 3′ end, respectively. Analogously, an antisense fragment, representing positions 1200–1858 of the lacZ gene, was PCR amplified from vector pJET1.2 lacZ using primers #0186 / #0187, which add Spe I and Dra III sites to the 5′ end and a Bgl II site to the 3′ end of the PCR product. The sense and antisense PCR products were cloned in the vector pGEM-T easy and confirmed by sequencing. The GFP loop fragment was PCR amplified from vector pDneo2a GFP Ava I site to the 5′ end and both, Dra III and Spe I sites to the 3′ end of the PCR product. To combine these three fragments, initially, the antisense fragment in the pGEM-T easy vector was digested with Spe I and Ava I, of which the latter targets an endogenous restriction site in that fragment, resulting in the removal of a 162 bp fragment from the 3′ end of the antisense fragment. Using the same restriction enzymes, also the PCR product of the GFP loop was digested and the resulting fragment ligated in the shortened pGEM-T easy ß-gal antisense construct. The resulting construct pGEM-T easy ß-gal antisense-GFP loop was verified by sequencing. From this construct, the insert representing ß-gal antisense-GFP loop was cloned in vector pDM304 Bgl II and Spe I sites in both vectors. Upon verification of the resulting construct pDM304 ß-gal antisense-GFP loop by restriction digest, this vector was digested with Dra III and Spe I, which allowed for the directed insertion of the sense fragment from pGEM-T easy ß-gal sense by ligation, yielding the construct pDM304 ß-gal hairpin. Finally, by a double digest with Bgl II / Spe I the ß-gal hairpin construct was inserted in vector pDM326 by directional cloning, resulting in vector pDM326 ß-gal hairpin. Transformation of expression and silencing constructs was carried out by electroporation as described earlier For cloning of the ß–gal 7 cells of axenically grown D. discoideum strains using TRIzol® (Invitrogen) according to the manufacturer's instructions. Finally the isolated total RNA was re-suspended in dH20 and the concentration was determined spectro-photomectrically by using a NanoDrop (Peqlab).Total RNA was isolated from 5×102). Pre-hybridization and hybridization were carried out in Church buffer (500 mM sodium phosphate (pH 7.2), 1 mM EDTA, 7% w/v SDS, 1% w/v BSA) using DNA oligonucleotides end-labeled with [γ-32P] ATP by T4-PNK (Thermo Scientific) or PCR products that were random-primed-labeled using Klenow Fragment (Thermo Scientific) as described recently For total RNA blots, 10 µg were separated by gel electrophoresis in a 1,2% GTC-agarose gel in 1×TBE (pH 8.0). Upon capillary transfer to nylon membranes (Roti®-Nylon plus), UV cross-linking was carried out  for 10 min at 20 V in dH2O (semi-dry), RNA was either UV cross-linked as described above or chemically cross-linking as described recently For small RNA blotting, 20 µg total RNA were separated by gel electrophoresis in a 11% polyacrylamide gel in 20 mM MOPS (pH 7.0). Upon electro-blotting onto a nylon membrane . Cells were pelleted again by centrifugation under identical conditions and resuspended in 200 µL ONPG lysis buffer  and incubated for 10 min on ice. For the photometric ONPG assay, 5 µL of this cell lysate was mixed with 800 µL Z buffer  and 200 µL ONPG solution . Absorption measurements were carried out at room temperature at a wavelength of 405 nm. For standardisation, the total protein content in the cell lysate was determined in duplicate by a Bradford Assays -1 • mg-1), ΔE405 the change in absorption (in min-1), Vtotal the reaction volume (in mL), Vprotein the volume of the cell lysate in the reaction (in mL), cprotein the protein concentration in the cell lysate (in mg • mL-1), d the cuvette path length (in cm) and ε the molar extinction coefficient  assay D. discoideum and if so, whether any of the three RNA dependent RNA polymerases (RdRPs) are involved in this process. The three large proteins are encoded by the genes rrpA, rrpB and rrpC, respectively , and subsequently all possible combinations of rrp gene deletions. All strains were verified by extensive PCR analysis (data not shown) and Southern blotting  doubling times, which, however, disappeared with time. Thus, none of the gene deletion strains displayed significant defects under axenic growth or during development.The aim of this study is the investigation whether transitivity of a silencing signal exists in ectively  and featectively . With thblotting , Table 2Escherichia coli lacZ gene that encodes β-D-Galactosidase . For the ectopic expression of this transgene in D. discoideum, earlier studies have shown that the use of endogenous promoter and terminator sequences results in no undesired obvious changes in the phenotype rrp gene deletion strains as well as a strain, in which the drnB gene is disrupted To investigate the silencing effect of different RNA constructs, we have generated a heterologous reporter system based on the To induce PTGS, these strains were subsequently transformed with a second vector encoding for one of two different RNA silencing constructs, either an antisense RNA or a hairpin RNA directed against lacZ mRNA . Both thThe silencing constructs were cloned in the pDM326 vector — and the seven rrp gene deletion strains with pDneo2a lacZ and either the β-gal hairpin RNA expressing vector or the empty pDM326 vector. The ONPG assays revealed a uniform reduction of the enzymatic activity of β-Gal by approximately 50% in either strain , that did not appear to be additionally increased by the presence of the β-gal hairpin RNA.To investigate the silencing potential of the β-gal hairpin RNA, we transformed A×2 wild type cells, drnBr strain . We presand O148 . This anand O148 . For exaIn analogy to the experiments carried out for the hairpin RNA constructs, also the transformants of the sense and antisense RNA expressing constructs were analyzed. We observe in ONPG assays no significant influence by the sense RNA expressing construct. The antisense RNA, however, led to a reduction in the enzymatic activity of β-Gal similar to that seen for the hairpin RNA . Again, lacZ mRNA stability, we analyzed next whether siRNAs could be observed at all in the presence of the silencing constructs. For this purpose, we employed β-gal siRNA probes (lacZ mRNA was present (Since the presence of neither the β-gal hairpin RNA nor the β-gal antisense RNA had apparently an effect on A probes  for dete present . As expe present . The lev present .lacZ mRNA, which should allow us to monitor secondary siRNAs, in case they do appear (rrpA and rrpB gene deletion strains, but not in either strain, in which the rrpC or drnB genes were deleted (D. discoideum and that for the hairpin RNA, RrpC and DrnB are involved in transitivity. While we observed antisense RNA derived siRNAs using the probes β-gal siRNA and 5′ upstream (To investigate whether transitivity can be observed in the β-Gal system introduced here, we have located the β-gal hairpin RNA centrally on the o appear . Since ao appear . Our ana deleted . From thupstream  that botupstream , our attdrnB deletion strain (In the hairpin construct, we had cloned a GFP-derived loop sequence. To analyze the fate of this loop sequence, we re-probed this blot , top witn strain , middle.D. discoideum encodes three RdRPs that have been shown earlier to be involved in the silencing of endogenous retrotransposons D. discoideum. Although we did not trigger full RNAi with the positioning of our hairpin RNA construct, we could show that this 5′ spreading of small RNAs indeed exists in the amoeba, as monitored by the appearance of secondary siRNAs that cannot be derived of the original trigger (C. elegansThe social amoeba  trigger  and 5B.  trigger . If the rrp gene products, but they depend on DrnB. Formally, the GFP loop is single-stranded. An inspection of predicted secondary structures of this sequence by the Mfold program D. discoideum microRNA precursors that also feature imperfect base complementarity The siRNAs that are derived of the GFP loop appear to be independent of any of the rrpC gene, alone or in combination with either of the other two rrp genes, but, surprisingly, not in the triple knock out strain. An enrichment of small RNAs in a deletion strain of an RdRP has also been reported for example for the Arabidopsis thaliana enzyme RDR2 In terms of enzymatic activity of β-Galactosidase, the β-gal antisense construct seemed to have the same effect than the β-gal hairpin construct . Our anaA. thaliana RdRP Rrp6 Neurospora crassa enzyme QDE-1 rrpC gene deletion strains would be unused substrates rather than products. This, however, does not explain why they disappear in the triple knock out strain. One explanation for this latter observation could be that RrpA and RrpB be responsible for the siRNAs that are seen in the antisense RNA expressing rrpC gene deletion strain (P. tetraureliarrp deletion strain. Alternatively, this latter observation might indicate some kind of compensatory mechanism in that triple knock out strain.Why should siRNAs accumulate in a strain that lacks an enzyme that is supposed to synthesize RNA? One explanation might be that the responsible RrpC acts in an RNA-primer dependent fashion, a mode of action that has been shown to be operational in the n strain , similarD. discoideum in strains transformed with antisense and hairpin RNA silencing constructs. We report only for the hairpin RNA, but not for the antisense RNA silencing construct, the appearance of a transitive silencing signal that localizes 5′ to the original trigger. These transitive RNAs appear, however, dispensable for the post-transcriptional silencing effect, for which the presence of the original trigger might suffice.In summary, our data indicate distinct, but partially overlapping activities of RdRPs in the generation of siRNAs in


Following inoculation, the mice were randomly divided into two groups. The control group (n=6) was fed a regular mouse chow diet and the NM 1% group (n=6) the same diet, but supplemented with 1% NM. Four weeks later the mice were sacrificed and the abdominal cavity was opened. Mice in the control group exhibited extensive metastasis in the peritoneal cavity and severely enlarged right testes and necrotic seminiferous tubules. By contrast, in the NM 1% fed group there was no evidence of peritoneal metastasis in 50% of the animals and mild metastasis in the remaining 50%. The right testes were enlarged and seminiferous tubules in the area of invasion showed evidence of degeneration. No metastasis to the liver, kidney or spleen were evident in either group. However, severe lung metastasis was observed in 2 of 6 mice in the control group and mild metastasis in 2 of 6 mice in the NM 1% group. In conclusion, these results confirm earlier studies and verify the anti-metastatic potential of NM.Metastasis, commonly to the lung, is the major cause of mortality from testicular cancer. The aim of the present study was to examine the effect of a novel nutrient mixture (NM) containing ascorbic acid, amino acids and green tea extract on the inhibition of melanoma growth and metastasis using a model of intratesticular inoculation of B16FO cells into nude mice. Male athymic mice (n=12), 10–12 weeks of age, were inoculated with 5×10 Approximately 8,590 new cases are likely to be diagnosed in the United States in 2012, of which 360 individuals are expected to succumb to the disease  containing lysine, proline, ascorbic acid and green tea extract has demonstrated anticancer activity in a number of human cancer cell lines, inhibiting cancer cell growth, MMP secretion, invasion, metastasis and angiogenesis . In a prThe aim of this study was to investigate the effect of NM on the experimental metastasis of melanoma cells by the intratesticular injection of B16FO cells into male nude mice. This experimental model of metastasis was selected to study the effectiveness of nutrients against metastasis to the lungs and other organs from the testes since melanoma cells are aggressive enough to result in significant metastasis in mice, particularly pulmonary metastasis, a common end organ of metastasis for testicular cancer. Furthermore, the intratesticular model was found to be an effective model for studying mechanisms of metastasis and evaluating treatment strategies due to the stable formation of tumors with metastatic potential .Murine melanoma B16FO cells obtained from ATCC  were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin and 100 μg/ml streptomycin. The media and sera used were obtained from ATCC, while the antibiotics (penicillin and streptomycin) were purchased from Gibco-BRL .Male nude mice, approximately 9–11 weeks of age on arrival, were purchased from Simonsen Laboratories , and kept in microisolator cages under pathogen-free conditions on a 12-h light/dark schedule for a week. The animals were cared for in accordance with institutional guidelines for the care and use of experimental animals.The regular rodent diet was obtained from Purina Mills . The NM 1% supplemented diet mix was milled and pressed by Purina Mills, LLC, and generated by Vitatech . The NM 1% diet comprised the following in the ratio indicated: vitamin C  700 mg; L-lysine 1000 mg; L-proline 750 mg; L-arginine 500 mg; N-acetyl cysteine 200 mg; standardized green tea extract (80% polyphenol) 1000 mg; selenium 30 μg; copper 2 mg; manganese 1 mg.5 B16FO melanoma cells in 100 μl of PBS into the right testis, while the left testis was left untreated. The cavities were sutured and clamped. After inoculation, the mice were randomly divided into 2 groups. The control group (n=6) was fed regular Purina mouse chow diet, while the mice in the NM 1% group (n=6) were fed the same diet, but supplemented with 1% NM. The quantity of diet provided to the mice was unrestricted, however, the mice consumed, on average, 4 g of their respective diets/day. Thus, the supplemented mice received ∼40 mg of NM/day, indicating that they received the following amounts of NM components/day: ascorbic acid 7 mg, L-lysine 10 mg, green tea extract 10 mg, L-proline 7.5 mg, L-arginine 5 mg, N-acetyl cysteine 2 mg, selenium 0.3 μg, copper 0.02 mg, manganese 10 μg. Four weeks later the mice were sacrificed, the abdominal cavity was opened and testes, lungs, kidneys, livers and spleens were excised from all the animals and examined. Since growth of testes in the control animals expanded profoundly into the peritoneum making it difficult to determine the limits of the organs, measurements of volume and weight were not carried out. Growth of melanoma colonies in testicles were evaluated by sectioned tissue. Lung metastasis was evaluated by observation of the melanoma colonies. A control mouse was sacrificed and examined at 1 week. All procedures were performed according to humane and customary care and use of experimental animals and conducted under protocols approved by the Internal Animal Care and Use Committee (IACUC).Male athymic mice (n=12), 10–12 weeks of age, were anesthetized by inhalation utilizing isofluorane USP . The right side of the abdomen overlying the testis was sterilely prepped and a skin incision of 1 cm was made to expose the right testis. Mice were inoculated with 5×10Testicular tissues were fixed in 10% buffered formalin, embedded in paraffin and cut at 4–5 microns. Sections were deparaffininzed through xylene and graduated alcohol series to water, and stained with hematoxylin and eosin (H&E) for microscopic evaluation by IDEXX Reference Laboratories.The mice (6/6) in the control group exhibited extensive metastasis in the peritoneal cavity, which was totally masked by B16FO melanoma cells, in contrast to the NM 1% group, which showed no peritoneal metastasis in 3 mice and mild metastasis in 3 mice. Representative images of the peritoneum in the two groups are shown in The right testes in the two groups showed overgrowth of melanoma cells. All of the control mice showed testicular metastasis, while in the NM diet group, only 3 mice showed mild metastasis and 3 mice showed no metastasis. A week after the melanoma injection, the left testis (untreated) of the control mouse was normal, while the right testis showed a focal area of melanoma invasion . The magNo metastasis to the liver, kidney or spleen was detected in either group. Lung metastasis was observed in 2 of 6 mice in each group. However, severe lung metastasis was observed in the control group, while mild metastasis was detected in the NM 1% group .No significant difference was found between the initial and final mean weights within the two groups. The mean initial weight of the control group was 37.2±1.3 g and the mean final weight was 38.4±1.6 g. The mean initial weight of the NM 1% group was 36.6±1.5 g and the final weight was 36.7±1.1 g.The aim of the present study was to investigate the effect of a nutrient mixture on melanoma B16FO growth and metastasis from intratesticular injection into nude mice, representing the lymphatic and hematogenous dissemination of melanoma malignancy. In our study, supplementation with the nutrient mixture suppressed B16FO melanoma cell growth in the testes and metastasis to the peritoneum and lungs after intratesticular injection. All of the mice receiving the control diet exhibited extensive metastasis in the peritoneal cavity, in contrast to the NM 1% diet group, which showed no metastasis in 50% of mice and mild peritoneal metastasis in the remaining mice. No metastasis to liver, kidney or spleen was evident in either of the two groups. Lung metastasis was observed in 2 of 6 mice in each group, with severe lung metastasis being observed in the control group and mild metastasis in the NM 1% group.in vitro studies have demonstrated significant inhibition of melanoma B16FO and A2058 cell proliferation and strong induction of apoptosis at 500 μg/ml NM, suggesting that inhibition of tumor growth was due probably to induction of apoptosis , but did not metastasize to the left testes (untreated), suggesting the testes are not common sites for melanoma B16FO cell metastasis. Previously, we showed that intraperitoneal injection of B16FO melanoma cells into C57BL/6 mice demonstrated intraperitoneal growth and ascites, but did not result in metastasis to other organs . In regapoptosis . These fin vitro studies have shown that NM significantly inhibited melanoma and other cancer cell MMP-2 and -9 secretion and Matrigel invasion (Degradation of the extracellular matrix (ECM) by matrix metalloproteinases (MMPs) plays a critical role in the formation of tumors and metastasis . Findinginvasion . In addiinvasion .in vitro and in vivo (Rath and Pauling  suggeste in vivo .NM is a mixture of nutrients that addresses critical physiological targets in cancer progression and metastasis, such as ECM integrity and MMP activity. Optimal ECM formation and structure is dependent upon adequate supplies of ascorbic acid and the amino acids lysine and proline, which ensure proper synthesis and hydroxylation of collagen fibers. Manganese and copper are also essential for collagen formation. Lysine, a natural inhibitor of plasmin-induced proteolysis, is crucial in supporting ECM stability ,24. Greein vivo safety studies. During an in vivo study on possible toxicity from NM, we found that NM did not have any adverse effect on vital organs, such as the heart, liver and kidney, nor on the associated functional serum enzymes (In conclusion, the results of the present study have shown that the nutrient mixture was effective in significantly reducing melanoma B16FO cell testicular tumor growth and peritoneal and lung metastasis in male nude mice injected with melanoma cells intratesticularly. These findings together with our earlier results clearly indicate the anticancer potential of NM. Furthermore, use of the nutrient mixture is not likely to pose any toxic effect clinically, especially in the relevant doses, as demonstrated by  enzymes .


The sense of agency is an intriguing aspect of human consciousness and is commonly defined as the sense that one is the author of their own actions and their consequences. In the current study, we varied the number of action alternatives  that participants could select from and determined the effects on intentional binding which is believed to index the low-level sense of agency. Participants made self-paced button presses while viewing a conventional Libet clock and reported the perceived onset time of either the button presses or consequent auditory tones. We found that the binding effect was strongest when participants had the maximum number of alternatives, intermediate when they had medium level of action choice and lowest when they had no choice. We interpret our results in relation to the potential link between agency and the freedom to choose one’s actions. The sense of agency is one of the most pervasive aspects of human consciousness and is commonly defined as the sense that one is author of their own actions and sensory consequences  and higher order (reflective) levels of processing  and thus ascertain the effect that the number of choice alternatives has on intentional binding. The fundamental question is, do more action alternatives produce greater levels of intentional binding than a more constrained choice set, where the agent is less involved in selecting which action to make?By extension and reducing the general idea of a link between freedom and agency to a testable laboratory task, intentional binding might also be expected to vary with differences in the degree of freedom. Again, agency and freedom are often talked about together and the feeling of freedom has been linked to choice  took part in the study. All participants had normal or corrected-to-normal vision and received partial course credits for their participation. The study was approved by the Research Ethics Board of Wilfrid Laurier University and all participants gave written informed consent prior to beginning the study. One participant’s data was not included in the analyses due to not following the experimental instructions.The experiment was programmed in Superlab 4.5  and ran on a Dell personal computer (3.07 GHz). The stimuli were presented on a 20 inch monitor (1600 × 1200). Participants sat approximately 60 cm away from the computer monitor and the responses were recorded on a laptop by the experimenter. The experiment consisted of baseline and operant conditions in which the number of keys to press  and the critical event  that participants judged the timing were manipulated. Similar to Haggard et al.  study, tEach trial began with a warning signal noting that a new trial will begin, which remained on the screen for 1 s. The fixation cross was then presented for 500 ms and followed by the display of the Libet clock (1.8 cm in diameter) with a minute hand pointing to one of 12 positions marked at 5-minute intervals. Participants were told to report their judgments between 0 (12 O’clock position) and 59, including the intermediate values. The minute hand remained stationary at the center of the screen for 500 ms and then started rotating clockwise at a 2.5 s period. In the baseline—where the single event was the key press only—and in the operant conditions, participants were told to make the key press at their own pace using their right index finger after the clock started rotating. They were instructed not to give stereotyped responses in the high and medium level of choice conditions and not to press the key at predetermined minute hand positions. In the baseline tone-only condition, participants did not make any key press but reported the onset of the tone occurred at a random time (jittered between 200 and 2000 ms) after the clock hand rotation started. The clock continued rotating for about 2000 ms after the participants reported the timing of the critical event. The perceptual times were verbally reported as minute hand positions and recorded by the experimenter on a laptop. At the end of the experiment, participants were debriefed and thanked for their participation in the study  × 3  × 2  repeated measures design. After converting the clock hand judgments to time values in milliseconds, we calculated the judgment errors for each condition as the difference between perceived and actual times of events Table . Trials We then obtained the perceptual shifts in terms of the difference between judgment errors between operant and the corresponding single event baseline conditions for both key press and tone judgments. For example, the perceptual shift for the high level action choice condition was calculated as the difference between the judgment errors in the operant-high-level condition from the baseline-high-level condition. Similarly, the perceptual shifts for the tone judgments were calculated as the difference between the judgment errors in each choice level-tone judgment condition and baseline-tone only condition. The positive shifts in the key press judgments and the negative shifts in the tone judgments relative to the corresponding baseline conditions demonstrate the temporal attraction, i.e., the intentional binding effect, between actions and effects Figure .F = 3.359, p < .05) and a significant main effect of critical event  = 5.148, p < .05). The interaction between these factors was also significant  = 3.389, p < .05). We predicted that binding would be least for the no choice condition, strongest for the high level of choice condition and intermediate for the medium level condition. We thus conducted one-tailed Paired Samples t tests to examine the 2-way interaction in more detail.We ran a 3  × 2  repeated measures ANOVA to examine the effect of having different number of action choices on the perceptual shifts. The analysis revealed a significant main effect of key press choice  = 2.287, p < .05) and to when they had no choice (t(22) = 1.792, p < .05). The difference between medium level of choice condition and no choice condition was not significant (p > .05).The t(22) = −2.186, p < .05) and also between medium level of choice and no choice conditions (t(22) = −2.260, p < .05). The difference in the perceptual shifts between high and medium level of choices was not significant (p > .05).With respect to the tone judgments, the perceptual shifts moved toward the perceived action onsets for both medium and high levels of choices. The size of the shift was greater for the medium level than the high level and it was in the opposite direction for the no choice condition. We found a significant difference in the perceptual shifts between high level of choice and no choice conditions  = 3.389, p < .05). As expected, we found that overall binding was strongest in the high level of action choice condition, intermediate for the medium level of choice condition and lowest for the no choice condition  = 1.998, p < .05) condition. However, the difference between high level of choice and medium level of choice condition as well as the difference between medium level of choice and no choice conditions were not significant (p > .05).We sought further the effect of choice levels on the mean overall binding by calculating the absolute value of subtraction of the mean key press shift in each condition from the tone shift (Wenke et al., Previous research focusing on different forms of the sense of agency has examined the contribution of various factors including predictive and retrospective processes (see Moore and Obhi, We measured the perceived times of individual key press and tone events separately in both baseline and operant conditions which allowed us to compare the size of the perceptual shift between each level of action choice. First, we found that perceived times of key presses for all levels of choices were shifted forward in time. In the medium level and high level conditions, the direction of the perceived time of the tones was shifted toward the key press whereas, somewhat surprisingly, this was not the case for the no-choice condition. Importantly though, as Figure What could be driving our observed effects of choice on intentional binding and by extension, the sense of agency? Given that all possible actions in the set of alternatives produced the same auditory event, our method could be construed as a true test of action selection on the sense of agency. That is, there is no obvious reason why an individual participant may have chosen one action over another, given that the outcome, or reward value of each possible action was fixed. Several explanations are possible.First, the results we report here are consistent with the finding that intentional binding is stronger when participants specify both the “what” and the “when” component of a pending action, compared to when they specify just one of these dimensions (i.e., “when” or “what”—Brass and Haggard, Another interesting framework within which to consider our results is based on the affordance competition hypothesis that models behavior as resulting from competition between different representations of potential actions Cisek, . In thisWithin this framework, we suggest that high, medium and no choice conditions differ in the degree of this dynamic activation and inhibition process that is ultimately responsible for action selection. Specifically, the no-choice condition may not involve the same degree of this dynamic inhibitory and excitatory activity as the high choice condition. We suggest that this difference might result in stronger activation of the representation of the action selected among many, such as in the high choice condition of the present experiment.This is akin to more endogenous processing being linked to greater agency, as suggested above, with the endogenous activity being specifically the dynamic interplay between excitatory and inhibitory processes during action selection. This explanation also predicts greater binding for the medium choice condition compared to the no choice condition as reported in our study, although the difference was not significant. From the present study, it appears that when seven alternative actions are available, this is sufficient to change the subjective experience of actions compared to when there is no alternative. However three alternatives demonstrate no difference from seven or no alternatives. Clearly, more work is required to determine if this suggestion is tenable, but at the very least, our data do indicate that high choice affects binding in a way that no choice does not.One might argue that the cognitive load varied across three levels of action choices in our study, which could have contaminated our results. However, as previous studies discussed this concern in detail, (e.g., Haggard et al., Having demonstrated that a high degree of choice is linked to increased binding, it is important to consider that there are limitations to the present study. For example, we did not assess the explicit sense of agency in this study and so cannot speak to how the number of action choice alternatives might affect the explicit feeling of agency. In addition, we did not manipulate the outcome of the different action alternatives. This is an obvious extension of the current work and would allow for determining the influence of reward on intentional binding and the sense of agency.Despite these limitations, showing that intentional binding is influenced by the degree of action choice is an important finding and we believe the current study provides a new set of questions relating to how choice affects the sense of agency, which could apply to many domains that extend beyond a fundamental consideration of how the sense of agency arises.why and precisely how the sense of agency and binding are affected by these kinds of manipulations. For now though, the current results reinforce the suggestion that increased personal choice increases agency which could form the foundation for a sense of freedom.Finally, the current results, along with other recent results from our and other labs, bolster the notion that intentional binding is linked, in some complex way to agentic experience. Specifically, we have previously shown that priming low power reduces binding and activating memories of depression reduces binding, whereas others have shown that less versus more control of an aircraft, when control is shared with an automatic pilot, reduces binding (Berberian et al., 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.