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

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 in vitro. In vivo, 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 S. pneumoniae, 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 in vitro 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.

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.

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 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.