The normal Q-Q plots of the calculated enrichment scores from Cappable-seq data.

<p>Q-Q plots of enrichment score quantiles calculated from Cappable-seq data (vertical axes) versus normally distributed theoretical quantiles (horizontal axes) are shown for scores before and after Box-Cox transformation for experimental replicate 1 (A) and replicate 2 (B). The critical value of enrichment score cutoff at p = 0.05 is indicated by the green horizontal line. The R² linear correlation coefficients are also shown on the plots. The Box-Cox lambda values used for transformation of enrichment scores are 0.1033 and 0.1136 for replicate 1 and 2 respectively.</p

Comparison of significantly enriched nucleotides detected by ToNER and TSSAR.

<p>Venn diagrams show overlaps of nucleotides detected as significantly enriched (p<0.05) from ToNER and TSSAR analyses for replicate 1 (cappable1), replicate 2 (cappable2), and the Fisher’s combined results from the two replicates (combine). The number of whole transcriptome significantly enriched positions are shown in black, whereas the number of enriched positions corresponding to known transcription start sites annotated in RegulonDB are shown in red.</p

Cappable-seq data and statistics of enrichment reported by ToNER and TSSAR for known transcription start sites (TSS).

<p>The Cappable-seq data and statistics of enrichment for 146 known <i>E</i>. <i>coli</i> TSS annotated in RegulonDB are shown in two parts. The first part on the left displays the normalized read depth as indicated by the green color gradient for the enriched library (E) and unenriched library (U), and the associated ToNER enrichment score (S) as indicated by the blue color gradient for each TSS in the Cappable-seq experimental replicate1 (cap1) and replicate 2 (cap2) datasets. The second part on the right displays nucleotide enrichment p-values obtained from ToNER and TSSAR analyses. For each software, the p-values for replicate 1 (cap1), replicate 2 (cap2), and Fisher’s combined p-values from both replicates (com) are shown. P-values of nucleotide enrichment are indicated in black color gradient for non-significant positions, whereas the significantly enriched positions (p<0.05) are shown in red color gradient. Positions in white have no reported p-value. This can occur because there are no mapped reads, and in case of TSSAR, positions with no statistic can also occur when there are more mapped reads in the unenriched library compared with the enriched library. The annotated TSS positions are grouped based on combined p-values, as indicated by the color bars in the ‘G’ column on the far right: positions detected with significant enrichment (p<0.05) by ToNER and TSSAR (pink); not detected by either software (black); detected by ToNER only (green) and detected by TSSAR only (red).</p

Example of an annotated TSS which is located in an unmodeled region by TSSAR in both Cappable-seq and dRNA-seq datasets.

<p>The plots of normalized read depth values of enriched and unenriched libraries including the corresponding enrichment scores reported by ToNER are shown for the 100 bp window (from -50 bp upstream to +50 bp downstream) of an annotated TSS position of <i>E</i>. <i>coli</i> (NC_000913.2 position 3,309,808; ‘-‘ strand). Data from the Cappable-seq protocol [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178483#pone.0178483.ref004" target="_blank">4</a>] are shown for Cappable-seq replicate 1 (A) and Cappable-seq replicate 2 (B). Data from the dRNA-seq protocol [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178483#pone.0178483.ref013" target="_blank">13</a>] are shown for dataset M63_0.4_B1_L1_GA (C) and dataset M63_0.4_B2_L1_HS2 (D). The ToNER calculated p-values of the annotated TSS position reported in Cappable-seq replicate 1, replicate 2, and combined result are 0.0043, 0.0126, and 0.0017, respectively. For dRNA-seq, the p-values reported in replicate B1, replicate B2, and combined result are 0.0549, 0.1046, and 0.0125, respectively.</p

The workflow of the ToNER software.

<p>The workflow of the ToNER software.</p

Inducible Knockdown of <i>Plasmodium</i> Gene Expression Using the <i>glmS</i> Ribozyme

<div><p>Conventional reverse genetic approaches for study of <i>Plasmodium</i> malaria parasite gene function are limited, or not applicable. Hence, new inducible systems are needed. Here we describe a method to control <i>P. falciparum</i> gene expression in which target genes bearing a <i>glmS</i> ribozyme in the 3′ untranslated region are efficiently knocked down in transgenic <i>P. falciparum</i> parasites in response to glucosamine inducer. Using reporter genes, we show that the <i>glmS</i> ribozyme cleaves reporter mRNA <i>in vivo</i> leading to reduction in mRNA expression following glucosamine treatment. Glucosamine-induced ribozyme activation led to efficient reduction of reporter protein, which could be rapidly reversed by removing the inducer. The <i>glmS</i> ribozyme was validated as a reverse-genetic tool by integration into the essential gene and antifolate drug target dihydrofolate reductase-thymidylate synthase (<i>Pf</i>DHFR-TS). Glucosamine treatment of transgenic parasites led to rapid and efficient knockdown of <i>Pf</i>DHFR-TS mRNA and protein. <i>Pf</i>DHFR-TS knockdown led to a growth/arrest mutant phenotype and hypersensitivity to pyrimethamine. The <i>glmS</i> ribozyme may thus be a tool for study of essential genes in <i>P. falciparum</i> and other parasite species amenable to transfection.</p></div

Ribozyme-mediated <i>Pf</i>DHFR-TS knockdown phenotype.

<p>Parasite growth in cultures of DHFR-TS-GFP_<i>glmS</i> integrant (A) or wild-type 3D7 (B) treated with varying levels of GlcN for up to 72 h was determined by counting 1000 infected erythrocytes from Giemsa-stained slides for each treatment time point. The data are the mean from triplicate experiments and error bars represent S.E.M. The morphology of treated parasites at different time points of treatment is shown for DHFR-TS-GFP_<i>glmS</i> integrant (C) or wild-type 3D7 (D). Panels are representative images from Giemsa-stained slides. Scale bars, 5 μm. Anti-malarial drug inhibition assays for pyrimethamine (E) and chloroquine (F) were performed in the presence (+ GlcN) or absence (-GlcN) of 2.5 mM GlcN in the parasite culture. The IC₅₀ values shown are the fitted values from four independent experiments and error bars represent the 95% C.I. Extra sum-of-squares F-test <i>P</i>-values comparing individual curve fits with the null hypothesis that slope and IC₅₀ are the same for both + GlcN and – GlcN: DHFR-TS-GFP_<i>glmS</i> integrant pyrimethamine <i>P</i><0.0001; wild-type 3D7 pyrimethamine <i>P</i> = 0.7138; DHFR-TS-GFP_<i>glmS</i> integrant chloroquine <i>P</i> = 0.4554; wild-type 3D7 chloroquine <i>P</i> = 0.7898.</p

<i>glmS</i> ribozyme cleavage and control of <i>P. falciparum</i> mRNA expression.

<p>(A) Schematic diagram of the DHFR-TS-GFP reporter gene with <i>glmS</i> ribozyme in the 3′-UTR position (reporter_<i>glmS</i>). The reporter gene is flanked by 5′-hsp86 and PbDT-3′ <i>Plasmodium</i> transcriptional regulatory sequences. The sequence regions analyzed in parts B and C are marked: FL probe, antisense RNA probe for RNase protection assay; 5′-P and 3′-P, 5′ and 3′ ribozyme cleavage products, respectively; RT-qPCR, amplicon for RT-qPCR analysis of reporter mRNA levels. (B) RNase protection assay revealed <i>glmS</i> ribozyme cleavage products (arrowed as 5′-P and 3′-P respectively) in <i>P. falciparum</i> expressing reporter_<i>glmS</i>. 10% ring-stage synchronized parasites were treated for 24 h in the presence of 10 mM GlcN prior to harvesting and extraction of total parasite RNA. The 3D7 wild-type parasite was used as a control to test for probe specificity. Control hybridizations and gel analysis without RNase (lanes 1 and 2 marked as -) were also performed to demonstrate integrity of the RNA probe. The migration of the full-length RNA probe complementary to the <i>glmS</i> RNA (FL probe) and small RNA ladder (New England Biolabs) bands are marked. (C) Analysis of reporter mRNA expression in response to treatment with 10 mM GlcN and Fru. The expression levels of reporter_<i>glmS</i> mRNA or reporter_M9 mRNA (bearing inactivating mutations in the <i>glmS</i> ribozyme cleavage site) in treated cultures relative to untreated were determined from RT-qPCR using the ΔΔCq method normalized to BSD mRNA. Starting with 10% ring-stage synchronized cultures, parasites were treated with 10 mM GlcN or Fru for 24 h prior to harvesting and RNA extraction. Error bars represent S.E.M. (<i>n</i> = 7 for GlcN experiments, <i>n</i> = 3 for Fru experiments). One sample two-tailed <i>t</i>-tests were performed to determine if change in mRNA expression was significant; NS denotes not significant, *** denotes highly significant. The calculated <i>P</i>-values comparing sample means against hypothetical mean = 1 were: reporter_<i>glmS</i> GlcN treatment, <0.0001; reporter_M9 GlcN treatment, 0.5136; reporter_<i>glmS</i> Fru treatment, 0.3986; reporter_M9 Fru treatment, 0.0619.</p

Genes showing significant changes in expression in DHFR-TS-GFP_<i>glmS</i> integrant parasites after GlcN treatment.

*<p>Changes in gene expression are shown as the log₂ mean normalized ratio of GlcN treated to untreated from 2 replicates.</p

Ribozyme-mediated control of endogenous <i>Pf</i>DHFR-TS expression.

<p>(A) Knockdown of <i>Pf</i>DHFR-TS expression in DHFR-TS-GFP_<i>glmS</i> integrant parasite clonal lines in response to GlcN. Parasitized erythrocytes expressing <i>Pf</i>DHFR-TS-GFP were enumerated by flow cytometry based on the level of GFP fusion partner. Extra sum-of-squares F- test comparing individual curve fits with the null hypothesis that slope and EC₅₀ are the same for both clone #1 and #2, <i>P</i> = 0.19. (B) Ribozyme-mediated knockdown of DHFR-TS-GFP protein is reversible. DHFR-TS-GFP_<i>glmS</i> integrant parasite clone #1 was cultured and treated with GlcN, and western immunoblotting to quantify DHFR-TS-GFP protein was performed as described in Fig. 3. Data are the mean from triplicate experiments and error bars represent S.E.M. (C) Representative images from Ponceau S staining of parasite lysates following electrophoresis and transfer to membrane, and chemiluminescent detection of GFP using specific antibodies. The pre-stained marker lane is marked M above the Ponceau S panel, and the sizes of two marker proteins are indicated.</p