Journal Article

Refining the transcriptome of the human malaria parasite Plasmodium falciparum using amplification-free RNA-seq

Berriman, Matthew, Russell, Timothy J., Rayner, Julian C., Otto, Thomas D., Llinás, Manuel, Chappell, Lia, Orchard, Lindsey, Ross, Philipp

2020

Overview

Background Plasmodium parasites undergo several major developmental transitions during their complex lifecycle, which are enabled by precisely ordered gene expression programs. Transcriptomes from the 48-h blood stages of the major human malaria parasite Plasmodium falciparum have been described using cDNA microarrays and RNA-seq, but these assays have not always performed well within non-coding regions, where the AT-content is often 90–95%. Results We developed a directional, amplification-free RNA-seq protocol (DAFT-seq) to reduce bias against AT-rich cDNA, which we have applied to three strains of P. falciparum (3D7, HB3 and IT). While strain-specific differences were detected, overall there is strong conservation between the transcriptional profiles. For the 3D7 reference strain, transcription was detected from 89% of the genome, with over 78% of the genome transcribed into mRNAs. We also find that transcription from bidirectional promoters frequently results in non-coding, antisense transcripts. These datasets allowed us to refine the 5′ and 3′ untranslated regions (UTRs), which can be variable, long (> 1000 nt), and often overlap those of adjacent transcripts. Conclusions The approaches applied in this study allow a refined description of the transcriptional landscape of P. falciparum and demonstrate that very little of the densely packed P. falciparum genome is inactive or redundant. By capturing the 5′ and 3′ ends of mRNAs, we reveal both constant and dynamic use of transcriptional start sites across the intraerythrocytic developmental cycle that will be useful in guiding the definition of regulatory regions for use in future experimental gene expression studies.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: 3' Untranslated Regions / 5' Untranslated Regions / Amplification / Animal Genetics and Genomics / Antisense RNA / Bias / Biomedical and Life Sciences / DNA microarrays / Erythrocytes / Eukaryote microbial genomics / Gene expression / Gene Expression Profiling - methods / Genes / Genomes / Genomics / Humans / Life Cycle Stages / Life Sciences / Malaria / Malaria, Falciparum - parasitology / Messenger RNA / Microarrays / Microbial Genetics and Genomics / Nucleic Acid Amplification Techniques - methods / Parasites / Plant Genetics and Genomics / Plasmodium falciparum / Plasmodium falciparum - classification / Plasmodium falciparum - genetics / Plasmodium falciparum - growth & development / Proteins / Proteomics / Protozoan Proteins - genetics / Regulatory sequences / Research Article / Ribonucleic acid / RNA / RNA, Messenger - genetics / Species Specificity / Transcription / Transcription (Genetics) / Vector-borne diseases