Additional file 22: Figure S8. of Genome-wide transcriptome profiling reveals functional networks involving the Plasmodium falciparum drug resistance transporters PfCRT and PfMDR1

Cluster heatmap of gene expression data for pfcrt/pfmdr1 and all other genes. The hierarchical clustering was generated using PCC values calculated using log2-transformed and normalized expression values of 2,600 genes across 110 pairwise comparisons of 11 parasite transcriptome data sets, corresponding to all possible combinations in both directions (i.e. A vs. B and B vs. A). Squares indicate areas containing genes that are all strongly correlated or strongly anti-correlated with the expression of both pfcrt and pfmdr1. (PDF 499 kb

Zcchc11 deficiency decreases IGF-1 expression <i>in vivo</i>.

<p>(A) IGF-1 mRNA, measured by qRT-PCR, was decreased by Zcchc11 deficiency in 8-day-old livers. *p<0.05 <i>vs</i> Zcchc11^+/+ by paired student's t-test. (B) Growth hormone (GH) in serum, measured by ELISA, was unaffected by genotype in 8-day-old mice, based on no significant difference using student's t-test. (C) STAT5 content and phosphorylation, as detected by immunoblot, were unaffected by Zcchc11 deficiency in the 8-day-old liver. (D) Growth factor PCR array of the livers from 8-day-old mice revealed IGF-1 to be the only transcript in this set to be substantially expressed and diminished by Zcchc11 deficiency. Numerical values for each genotype indicate dCT of transcript expression normalized against the mean expression of three separate housekeeping genes. The “fold” column represents the fold-change expression in Zcchc11^−/− compared to Zcchc11^+/+ livers. (E) Histone H3 content, as detected by immunoblot, was unaffected by Zcchc11 deficiency in the 8-day-old liver. (F) IGF-1 protein was decreased in the serum of 8-day-old Zcchc11^−/− mice. *p<0.05 <i>vs</i> Zcchc11^+/+ by student's t-test.</p

Zcchc11 is essential for the uridylation of miRNAs.

<p>The miRNA libraries from Zcchc11-defficent and wild type livers at 8 days of age were analyzed for differences in sequence diversity. (A) Histogram of the number of sequences of a given read length across the libraries, showing fewer 23 nucleotide-long reads in the mutant samples. *p<0.05 by two-way ANOVA with Bonferroni <i>post hoc</i> test. (B) End modifications, identified as any nucleotide one base beyond the length of the miRNA published in miRBase, were quantified for each library. Most miRNAs ending in uridine in the control mice did so less frequently in Zcchc11-deficient mice. The mean of the fold change in the percent of sequences adenylated or uridylated between the knockout and wild type were graphed as waterfall plots, with every bar representing the geometric mean of (percent of miR-X sequences modified in the mutants)/(percent of miR-X sequences modified in the WT) for the three separate libraries. Only miRNAs with >10,000 reads and >0.1% sequences end-modified in the wild type libraries were included. Data were expressed as fold-change in Zcchc11^−/− mice compared to Zcchc11^+/+ mice. Light grey bars indicate miRNAs with a seed sequence complementary to a portion of the IGF-1 3′ UTR. (C) Average fold change across all miRNA species shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003105#pgen-1003105-g003" target="_blank">Figure 3B</a>, showing that uridines but not adenines were less frequent at miRNA termini due to Zcchc11 deficiency. Error bars indicate 95% c.i. *p<0.05 <i>vs.</i> 0 by student's t-test. (D) Immunoblots for the indicated non-canonical poly(A) polymerases in tissue homogenates prepared from the livers of 8 day-old mice, showing no effect of genotype. (E) qRT-PCR for Zcchc6 expression in the livers of 8 day-old mice, showing no effect of genotype.</p

Zcchc11 enhances growth and fitness through the perinatal period.

<p>(A) Immunoblots of organs from C57BL/6 and Zcchc11^−/− mice show deletion of Zcchc11 protein. (B) Fraction of homozygous Zcchc11-deficient offspring from Zcchc11^+/− parents at day E14, P1, P8 and P21, indicating decreased survival by day 8. *p<0.05 <i>vs.</i> 0.25 by Chi-squared test, N as indicated. (C) Body weights of Zcchc11^+/+ and Zcchc11^−/− littermates at day 1 and 8 (*p<0.05), showing poor growth in mutants. (D) Let-7 content in primary embryonic stem cell (ESC) cultures that were wild type (+/+), heterozygous (+/−), or deficient (−/−) in Zcchc11 expression, showing no significant effects of genotype (by two-way ANOVA). (E) Proportion of organ weight to body weight in 8-day-old C57BL/6 and Zcchc11^−/− mice, showing no difference between genotypes across tissues and suggesting a system-wide growth defect rather than organ-specific effects. (F) Age-dependent expression of Zcchc11, as shown by immunoblots of tissues from C57BL/6 mice at 2 days, 2 weeks, and 10 weeks of age, reveals strongest expression in most organs at young ages. GAPDH is provided as a loading control.</p

Zcchc11 stabilizes the IGF-1 3′ UTR.

<p>(A) Northern blotting was used to identify the predominant IGF-1 isoform expressed in the livers of wild type and Zcchc11-deficient mice at 8 days old. (B) The 3′ UTR from this isoform, cloned onto the end of a firefly luciferase reporter and co-transfected, along with a <i>Renilla</i> Luciferase containing a minimal promoter for normalization, decreased reporter expression in H1299 cells. (C) These same constructs were transfected in H1299 cells along with plasmids encoding EGFP, Zcchc11, catalytically inactive Zcchc11 (DADA), or the N-terminal half of Zcchc11. The full-length Zcchc11 increased expression of the IGF-1 3′ UTR reporter, which was significantly inhibited by selective mutation of the catalytic domain or complete deletion of the C terminal half. *p<0.05 <i>vs.</i> EGFP <i>†</i>p<0.05 <i>vs</i> Zcchc11 by one-way ANOVA.</p

Zcchc11 deficiency does not affect quantities of mature miRNAs or miRNA–related proteins in the liver.

<p>Three deep sequencing libraries were created from livers of sex- and littermate-matched 8-day-old Zcchc11^+/+ or Zcchc11^−/− mice. (A) Mature miRNA content, expressed as reads per million (RPM) was compared for wild type and Zcchc11-deficient livers (correlation coefficient, r = 0.975). (B) Quantitative RT-PCR was used to measure the expression of several miRNAs including some implicated in Zcchc11 pre-miRNA uridylation (Let-7), those highly expressed in the livers (miR-122), and those showing trends towards change in the deep sequencing data (miR-139 and miR-379), revealing no difference between genotypes. (C) As another approach to examining Let-7 content, Let-7a in the livers of 8-day old mice was measured by Northern blotting, and did not increase due to Zcchc11 deficiency. An adult wild type mouse was also included for comparison. (D) Immunoblots for RISC-related proteins in tissue homogenates were prepared from the livers of 8 day-old mice, showing no differences due to genotype. (E) Immunoblots for Lin-28 family members in the livers and skeletal muscles of 8 day old Zcchc11^+/+ and Zcchc11^−/− mice revealed no effects of Zcchc11 deficiency. Actin was measured as a loading control. For all blots, each lane represents the RNA or protein from a separate individual of the indicated age and genotype.</p

Zcchc6 and Zcchc11 expression in adult mouse tissues.

<p>(A) Total RNA was prepared from C57BL/6 mouse tissues and was assayed for Zcchc6 and Zcchc11 mRNA expression by qRT-PCR. Data are normalized to 18S rRNA and displayed as fold induction over Zcchc11 expression (n = 3,3). *p<0.05 ***p<0.001 by two-way ANOVA with Bonferroni post-test. (B)(C) Zcchc6 protein expression was measured by immunoblot in primary human monocyte derived macrophages (4 donors), human monocytes (5 donors), and human alveolar macrophages (3 donors). Lung lysates generated from WT or Z6 deficient mouse lungs were used as a control in (C). Zcchc6 protein expression was assessed in (D) PMA or vehicle treated U937 or THP-1 human monocytic cell lines and in (E) mouse bone marrow-derived macrophages during a 7-day time-course of differentiation.</p

Top 20 GO Biological process terms.

<p>Top 20 GO Biological process terms.</p

Zcchc6 minimally directs neutrophil emigration during pneumococcal pneumonia.

<p>(A) Total BAL cell counts (B) alveolar macrophage numbers and (C) neutrophil emigration were assessed by bronchoalveolar lavage from <i>Zcchc6</i>^<i>+/+</i> or <i>Zcchc6</i>^<i>-/-</i> mice infected with of Sp19 i.t. for 4 hours. **p<0.01 by Student’s t-test. (D) Bacterial lung burdens were determined from <i>Zcchc6</i>^<i>+/+</i> or <i>Zcchc6</i>^<i>-/-</i> mice 30 hours post intratracheal instillation of Sp19. (E) Cytokine mRNA expression was measured by qRT-PCR on total lung left lobe RNA isolated from Sp19-infected <i>Zcchc6</i>^<i>+/+</i> and <i>Zcchc6</i>^<i>-/-</i> mice at 4 hours post infection. Data are normalized to 18S rRNA levels and expressed as fold induction over <i>Zcchc6</i>^<i>+/+</i>. n = 6,8; *p<0.05 by Student’s t-test. (F) CXCL1 protein levels were measured by ELISA in supernatants collected from cultured BMDMs (n = 4,3) stimulated with 1 x 10⁶ CFU/mL Sp19.</p

Establishment of a mouse model of Zcchc6 deficiency.

<p>(A) Gene rearrangement in Zcchc6^-/- mice at the DNA level was confirmed by PCR of tail genomic DNA. (B) Zcchc6 mRNA expression was measured by qRT-PCR from total left lobe lung RNA and normalized to 18S rRNA. Data expressed as mean and SEM, ***p<0.001 by Student’s t-test (C) Immunoblot analysis of Zcchc6 protein expression across multiple tissue cell lysates. (D) Genotypes of pups weaned from Zcchc6^+/- x Zcchc6^+/- crosses. Dotted line indicated expected frequency of homozygous animals.</p