No miRNA were found in Plasmodium and the ones identified in erythrocytes could not be correlated with infection

Abstract

Background: The transcriptional regulation of Plasmodium during its complex life cycle requires sequential activation and/or repression of different genetic programmes. MicroRNAs (miRNAs) are a highly conserved class of non-coding RNAs that are important in regulating diverse cellular functions by sequence-specific inhibition of gene expression. What is know about double-stranded RNA-mediated gene silencing (RNAi) and posttranscriptional gene silencing (PTGS) in Plasmodium parasites entice us to speculate whether miRNAs can also function in Plasmodium-infected RBCs.

Results: Of 132 small RNA sequences, no Plasmodium-specific miRNAs have been found. However, a human miRNA, miR-451, was highly expressed, comprising approximately one third of the total identified miRNAs. Further analysis of miR-451 expression and malaria infection showed no association between the accumulation of miR-451 in Plasmodium falciparum-iRBCs, the life cycle stage of P. falciparum in the erythrocyte, or of P. berghei in mice. Moreover, treatment with an antisense oligonucleotide to miR-451 had no significant effect on the growth of the erythrocytic-stage P. falciparum.

Methods: Short RNAs from a mixed-stage of P. falciparum-iRBC were separated in a denaturing polyacrylamide gel and cloned into T vectors to create a cDNA library. Individual clones were then sequenced and further analysed by bioinformatics prediction to discover probable miRNAs in P. falciparum-iRBC. The association between miR-451 expression and the parasite were analysed by Northern blotting and antisense oligonucleotide (ASO) of miR-451.

Conclusion: These results contribute to eliminate the probability of miRNAs in P. falciparum. The absence of miRNA in P. falciparum could be correlated with absence of argonaute/dicer genes. In addition, the miR-451 accumulation in Plasmodium-infected RBCs is independent of parasite infection. Its accumulation might be only the residual of erythroid differentiation or a component to maintain the normal function of mature RBCs.

Figure 1

Distribution of cloned short RNAs from P. falciparum-iRBC. One-hundred thirty-two short RNAs 18 to 26 nucleotides in length were isolated from P. falciparum-iRBCs and grouped according to sequence annotation (see Materials and Methods for details). Those with more than three mismatches to human or Plasmodium sequences were classified as ???.

Figure 2

Detection of expression of miR-451 by Northern blot. (A) Analysis of miR-451 expression in human WBCs and P. falciprum-iRBCs. Total RNA (2 μg) from WBCs and P. falciprum-iRBCs was separated by denaturing PAGE and blotted to a nylon membrane. Biotin-labeled miR-451 was used as a hybridization probe. Lane 1, infected RBC; Lane 2, isolated WBCs. (B) Analysis of miR-451 expression in different erythrocytic-stage P. falciparum-iRBCs. Total RNA (2 μg) from known numbers of cells were prepared at 24 h intervals from a single tightly synchronized culture. Parasite stage was determined by thin blood smears. Lane 1, healthy human RBCs; lane 2, 0-h culture after tight synchronization (ring-stage iRBCs); lane 3, 24-h culture (late trophozoite-stage iRBCs); lane 4, 48-h culture (schizont-stage iRBCs); lane 5, 72-h culture (new reproductive cycle of P. falciparum); lane 6, purified P. falciparum; lane 7, purified P. falciparum treated with RNase H to remove residual RNA contamination; lane 8, 72-h culture of P. falciparum-iRBCs loaded with equivalent numbers of erythrocytes as lane 2.

Figure 3

Analysis of miR-451 expression in the blood of P. berghei-infected KM mice at different parasitaemias. Female KM mice (6–8 weeks old) were infected with the blood stages of P. berghei (ANKA strain) by intraperitoneal injection of 10⁵ infected erythrocytes. The course of infection was monitored by examination of Giemsa-stained blood films. (A) Total RNAs from the whole blood of uninfected and infected mice with different parasitaemias were analysed by Northern blotting. Lane 1, whole healthy blood; lane 2, 0.5% parasitaemia; lane 3, 2% parasitaemia; lane 4, 12% parasitaemia; lane 5, 28% parasitaemia; lane 6, 43% parasitaemia; lane 7, 60% parasitaemia; lane 8, 86% parasitaemia. (B) Total RNAs from equivalent numbers of blood cells (10⁷) with different parasitaemias were analysed by Northern blotting. Lane 1, whole healthy blood; lane 2, 43% parasitaemia; lane 3, 60% parasitaemia.