Directional gene expression and antisense transcripts in sexual and asexual stages of Plasmodium falciparum

María J López-Barragán,Kim C Williamson,Jacob Lemieux,Carolina Barillas-Mury,Xin-Zhuan Su,Keji Zhao,Kairong Cui,Alvaro Molina-Cruz,Mariam Quiñones

doi:10.1186/1471-2164-12-587

Abstract

BackgroundIt has been shown that nearly a quarter of the initial predicted gene models in the Plasmodium falciparum genome contain errors. Although there have been efforts to obtain complete cDNA sequences to correct the errors, the coverage of cDNA sequences on the predicted genes is still incomplete, and many gene models for those expressed in sexual or mosquito stages have not been validated. Antisense transcripts have widely been reported in P. falciparum; however, the extent and pattern of antisense transcripts in different developmental stages remain largely unknown.ResultsWe have sequenced seven bidirectional libraries from ring, early and late trophozoite, schizont, gametocyte II, gametocyte V, and ookinete, and four strand-specific libraries from late trophozoite, schizont, gametocyte II, and gametocyte V of the 3D7 parasites. Alignment of the cDNA sequences to the 3D7 reference genome revealed stage-specific antisense transcripts and novel intron-exon splicing junctions. Sequencing of strand-specific cDNA libraries suggested that more genes are expressed in one direction in gametocyte than in schizont. Alternatively spliced genes, antisense transcripts, and stage-specific expressed genes were also characterized.ConclusionsIt is necessary to continue to sequence cDNA from different developmental stages, particularly those of non-erythrocytic stages. The presence of antisense transcripts in some gametocyte and ookinete genes suggests that these antisense RNA may play an important role in gene expression regulation and parasite development. Future gene expression studies should make use of directional cDNA libraries. Antisense transcripts may partly explain the observed discrepancy between levels of mRNA and protein expression.

Highlights

It has been shown that nearly a quarter of the initial predicted gene models in the Plasmodium falciparum genome contain errors
Several recent studies have reported large-scale sequencing of transcriptome and epigenome from P. falciparum, including samples from clinical isolates [17,18,19,20]. These studies identified large numbers of additional intronexon splicing junctions missed by the initial genome annotation, alternative splicing events, and antisense transcripts, and have greatly improved expressed sequence tag (EST) coverage and genome annotation; these studies primarily focused on genes expressed in asexual stages
R, ET, LT, Sc, gametocyte stage II (GII), gametocyte stage V (GV), and Oo correspond to ring, early trophozoite, late trophozoite, schizont, gametocyte II, gametocyte V, and ookinete stages, respectively

Summary

Introduction

It has been shown that nearly a quarter of the initial predicted gene models in the Plasmodium falciparum genome contain errors. The 5’ and 3’ UTRs of the parasite–including the polyA tail–are usually very AT rich and are difficult to sequence These difficulties have prevented the cloning and sequencing of many P. falciparum cDNA sequences, transcripts larger than 2 kb. Several recent studies have reported large-scale sequencing of transcriptome and epigenome from P. falciparum, including samples from clinical isolates [17,18,19,20] These studies identified large numbers of additional intronexon splicing junctions missed by the initial genome annotation, alternative splicing events, and antisense transcripts, and have greatly improved EST coverage and genome annotation; these studies primarily focused on genes expressed in asexual stages. No systematic cDNA sequence analysis or verification of gene models has been done for gametocyte (G) and mosquito stages, which are expected to have many stage-specific expressed genes

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