Nascent RNA sequencing reveals mechanisms of gene regulation in the human malaria parasite Plasmodium falciparum

Abstract

Gene expression in Plasmodium falciparum is tightly regulated to ensure successful propagation of the parasite throughout its complex life cycle. The earliest transcriptomics studies in P. falciparum suggested a cascade of transcriptional activity over the course of the 48-hour intraerythrocytic developmental cycle (IDC); however, the just-in-time transcriptional model has recently been challenged by findings that show the importance of post-transcriptional regulation. To further explore the role of transcriptional regulation, we performed the first genome-wide nascent RNA profiling in P. falciparum. Our findings indicate that the majority of genes are transcribed simultaneously during the trophozoite stage of the IDC and that only a small subset of genes is subject to differential transcriptional timing. RNA polymerase II is engaged with promoter regions prior to this transcriptional burst, suggesting that Pol II pausing plays a dominant role in gene regulation. In addition, we found that the overall transcriptional program during gametocyte differentiation is surprisingly similar to the IDC, with the exception of relatively small subsets of genes. Results from this study suggest that further characterization of the molecular players that regulate stage-specific gene expression and Pol II pausing will contribute to our continuous search for novel antimalarial drug targets.

Fluorescence‐based assay for accurate measurement of transcriptional activity in trypanosomatid parasites

“…For example, run-on in association with RNA-seq, called global run-on sequencing assay (GRO-seq) in other organisms (21,69,70), would allow the purification of nascent RNA and further sequencing to map and identify the genomic regions that are being transcribed. Additionally, the methodology can be analyzed using a highcontent image system.…”

“…Other methodologies can be applied, as deep sequencing RNA (RNAseq) and in vitro transcription with radiolabeled nucleotide (20). Differently, in vitro transcription assay allows to quantify the transcriptional activity through the measurement of nascent RNA, that represents a fraction of the total RNA (21). Differently, in vitro transcription assay allows to quantify the transcriptional activity through the measurement of nascent RNA, that represents a fraction of the total RNA (21).…”

“…RNA-seq allows quantifying the steady-state levels of RNA, which is the balance between transcription, stability and degradation. Differently, in vitro transcription assay allows to quantify the transcriptional activity through the measurement of nascent RNA, that represents a fraction of the total RNA (21).…”

Characterization of the Catalytic Subunits of the RNA Exosome‐like Complex in Plasmodium falciparum

“…, Lu et al. ). Previous studies revealed that P. falciparum had a very unique mRNA metabolism reflected by the prolonged mRNA decay with the parasite development inside the erythrocytes (Shock et al.…”

“…Of the five species of malaria parasites that infect humans, Plasmodium falciparum is the most prevalent pathogen and responsible for most deaths (Murray et al 2012, WHO 2014. RNA profilling analysis suggests that there may be an exosome-like complex for RNA metabolism for P. falciparum to achieve its complicated life cycle (Sim et al 2009, Hughes et al 2010Zhang et al 2014a,b;Clayton and Estevez 2011;Bunnik et al 2016, Lu et al 2017. Previous studies revealed that P. falciparum had a very unique mRNA metabolism reflected by the prolonged mRNA decay with the parasite development inside the erythrocytes (Shock et al 2007).…”