Functional roles for the terminal uridyltransferase enzymes Zcchc6 and Zcchc11 in mammalian biology

Diverse species of RNA can be post-transcriptionally uridylated on 3’ termini to modulate their biological function. Key cellular enzymes involved in the catalysis of RNA uridylation have been identified as two closely related terminal uridyltransferase (TUT) family members known as Zcchc6 and Zcchc11. To date, most reports on the function of TUTs have been gleaned from reductionist in vitro systems, however the biological function of these enzymes in integrated animal models remains unknown. The goal of this work was to investigate physiological roles for the TUT proteins during normal homeostasis and during infectious stress. To achieve this, we generated two constitutive, animal models of TUT-deficiency for both Zcchc6 and Zcchc11. The first knockout animal of the two TUTs to be examined was Zcchc11. Given previous reports that Zcchc11 played essential roles in stem cell biology, it was anticipated that Zcchc11-null mice would be result in embryonic lethality. We observed decreased survival and organismal growth following birth. The hepatic small RNA transcriptome revealed reduced sequence lengths and terminal uridylation across mature microRNAs and the expression of IGF-1 was enhanced by Zcchc11 expression in vitro. MiRNA silencing of IGF-1 was alleviated by the uridylation of IGF-targeting miRNA. We concluded that the Zcchc11-mediated terminal uridylation of mature microRNAs is pervasive and physiologically significant. In the second model examined, we observed that Zcchc6 deficiency did not impact perinatal mortality or litter size, but in contrast to Zcchc11-null mice, Zcchc6-null animals exhibited enhanced organismal growth following birth. A survey of tissue Zcchc6 mRNA expression showed enrichment in the lungs and upon further analysis we observed Zcchc6 to be uniquely and highly expressed in mouse and human primary alveolar and bone marrow derived macrophages, increased during monocyte-to-macrophage differentiation and regulated the expression of select cytokines including IL-6 and CXCL1 during inflammation. These studies indicated that Zcchc6 was required for the development of immunocompetent macrophages and calibrated macrophage-mediated innate immune responses in the airspaces. Taken together, these data provide evidence of independent and overlapping roles for TUT proteins in diverse physiological systems in living animal models

Genome-wide transcriptome profiling reveals functional networks involving the Plasmodium falciparum drug resistance transporters PfCRT and PfMDR1

Background The acquisition of multidrug resistance by Plasmodium falciparum underscores the need to understand the underlying molecular mechanisms so as to counter their impact on malaria control. For the many antimalarials whose mode of action relates to inhibition of heme detoxification inside infected erythrocytes, the digestive vacuole transporters PfCRT and PfMDR1 constitute primary resistance determinants. Results Using gene expression microarrays over the course of the parasite intra-erythrocytic developmental cycle, we compared the transcriptomic profiles between P. falciparum strains displaying mutant or wild-type pfcrt or varying in pfcrt or pfmdr1 expression levels. To account for differences in the time of sampling, we developed a computational method termed Hypergeometric Analysis of Time Series, which combines Fast Fourier Transform with a modified Gene Set Enrichment Analysis. Our analysis revealed coordinated changes in genes involved in protein catabolism, translation initiation and DNA/RNA metabolism. We also observed differential expression of genes with a role in transport or coding for components of the digestive vacuole. Interestingly, a global comparison of all profiled transcriptomes uncovered a tight correlation between the transcript levels of pfcrt and pfmdr1, extending to dozens of other genes, suggesting an intricate regulatory balance in order to maintain optimal physiological processes. Conclusions This study provides insight into the mechanisms by which P. falciparum adjusts to the acquisition of mutations or gene amplification in key transporter loci that mediate drug resistance. Our results implicate several biological pathways that may be differentially regulated to compensate for impaired transporter function and alterations in parasite vacuole physiology

Zcchc11 Uridylates Mature miRNAs to Enhance Neonatal IGF-1 Expression, Growth, and Survival

The Zcchc11 enzyme is implicated in microRNA (miRNA) regulation. It can uridylate let-7 precursors to decrease quantities of the mature miRNA in embryonic stem cell lines, suggested to mediate stem cell maintenance. It can uridylate mature miR-26 to relieve silencing activity without impacting miRNA content in cancer cell lines, suggested to mediate cytokine and growth factor expression. Broader roles of Zcchc11 in shaping or remodeling the miRNome or in directing biological or physiological processes remain entirely speculative. We generated Zcchc11-deficient mice to address these knowledge gaps. Zcchc11 deficiency had no impact on embryogenesis or fetal development, but it significantly decreased survival and growth immediately following birth, indicating a role for this enzyme in early postnatal fitness. Deep sequencing of small RNAs from neonatal livers revealed roles of this enzyme in miRNA sequence diversity. Zcchc11 deficiency diminished the lengths and terminal uridine frequencies for diverse mature miRNAs, but it had no influence on the quantities of any miRNAs. The expression of IGF-1, a liver-derived protein essential to early growth and survival, was enhanced by Zcchc11 expression in vitro, and miRNA silencing of IGF-1 was alleviated by uridylation events observed to be Zcchc11-dependent in the neonatal liver. In neonatal mice, Zcchc11 deficiency significantly decreased IGF-1 mRNA in the liver and IGF-1 protein in the blood. We conclude that the Zcchc11-mediated terminal uridylation of mature miRNAs is pervasive and physiologically significant, especially important in the neonatal period for fostering IGF-1 expression and enhancing postnatal growth and survival. We propose that the miRNA 3′ terminus is a regulatory node upon which multiple enzymes converge to direct silencing activity and tune gene expression

The RNA uridyltransferase Zcchc6 is expressed in macrophages and impacts innate immune responses

<div><p>Alveolar macrophages orchestrate pulmonary innate immunity and are essential for early immune surveillance and clearance of microorganisms in the airways. Inflammatory signaling must be sufficiently robust to promote host defense but limited enough to prevent excessive tissue injury. Macrophages in the lungs utilize multiple transcriptional and post-transcriptional mechanisms of inflammatory gene expression to delicately balance the elaboration of immune mediators. RNA terminal uridyltransferases (TUTs), including the closely homologous family members Zcchc6 (TUT7) and Zcchc11 (TUT4), have been implicated in the post-transcriptional regulation of inflammation from studies conducted <i>in vitro</i>. <i>In vivo</i>, we observed that Zcchc6 is expressed in mouse and human primary macrophages. Zcchc6-deficient mice are viable and born in Mendelian ratios and do not exhibit an observable spontaneous phenotype under basal conditions. Following an intratracheal challenge with <i>S</i>. <i>pneumoniae</i>, Zcchc6 deficiency led to a modest but significant increase in the expression of select cytokines including IL-6, CXCL1, and CXCL5. These findings were recapitulated <i>in vitro</i> whereby Zcchc6-deficient macrophages exhibited similar increases in cytokine expression due to bacterial stimulation. Although loss of Zcchc6 also led to increased neutrophil emigration to the airways during pneumonia, these responses were not sufficient to impact host defense against infection.</p></div

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

A tetracycline-repressible transactivator system to study essential genes in malaria parasites

A major obstacle in analyzing gene function in apicomplexan parasites is the absence of a practical regulatable expression system. Here, we identified functional transcriptional activation domains within Apicomplexan AP2 (ApiAP2) family transcription factors. These ApiAP2 transactivation domains were validated in blood-, liver-, and mosquito-stage parasites and used to create a robust conditional expression system for stage-specific, tetracycline-dependent gene regulation in Toxoplasma gondii, Plasmodium berghei, and Plasmodium falciparum. To demonstrate the utility of this system, we created conditional knockdowns of two essential P. berghei genes: profilin (PRF), a protein implicated in parasite invasion, and N-myristoyltransferase (NMT), which catalyzes protein acylation. Tetracycline-induced repression of PRF and NMT expression resulted in a dramatic reduction in parasite viability. This efficient regulatable system will allow for the functional characterization of essential proteins that are found in these important parasites

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 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