Abstract
The glms ribozyme system has been used as an amenable tool to conditionally control expression of genes of interest. It is generally assumed that insertion of the ribozyme sequence does not affect expression of the targeted gene in the absence of the inducer glucosamine-6-phosphate, although experimental support for this assumption is scarce. Here, we report the unexpected finding that integration of the glms ribozyme sequence in the 3′ untranslated region of a gene encoding a HECT E3 ubiquitin ligase, termed Plasmodium falciparum ubiquitin transferase (PfUT), increased steady state RNA and protein levels 2.5-fold in the human malaria parasite P. falciparum. Overexpression of pfut resulted in an S/M phase-associated lengthening of the parasite’s intraerythrocytic developmental cycle and a reduced merozoite invasion efficiency. The addition of glucosamine partially restored the wild type phenotype. Our study suggests a role of PfUT in controlling cell cycle progression and merozoite invasion. Our study further raises awareness regarding unexpected effects on gene expression when inserting the glms ribozyme sequence into a gene locus.
Highlights
Many proteins are post-translationally modified by the covalent attachment of a small polypeptide, termed ubiquitin
In an effort to elucidate the biological function of Plasmodium falciparum ubiquitin transferase (PfUT), we generated a conditional knock-down mutant in the P. falciparum line 3D7, by inserting a triple hemagglutinin (HA) tag followed by the glmS ribozyme sequence in the 3′ untranslated region of pfut[27,30], via CRISPR-Cas[9] genome editing technology[31] (Fig. 1a)
There have been conflicting results of whether or not pfut is essential for intraerythrocytic development of P. falciparum
Summary
Many proteins are post-translationally modified by the covalent attachment of a small polypeptide, termed ubiquitin. The human malaria parasite P. falciparum encodes 8 ubiquitin-activating enzymes (E1s), 14 ubiquitin-conjugating enzymes (E2s), and 54 ubiquitin ligases (E3s)[7]. The role of these enzymes in the biology and pathology of P. falciparum is only partly understood. We have recently associated polymorphisms in a HECT (homologous to E6AP C-terminus) E3 ubiquitin ligase, termed PfUT (MAL7P1.19 or PF3D7_0704600), with altered responsiveness to the antimalarial drug quinine and its enantiomer quinidine[15]. Insertion of the ribozyme sequence into the pfut gene locus was not inert, but instead resulted in 2.5-fold higher steady state transcript levels and associated with it 2.4-fold increased protein amounts, compared with the parental strain. Our data suggest that PfUT partakes in the regulatory network that controls merozoite invasion and cell cycle progression during schizogony
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