G-quadruplex RNA motifs influence gene expression in the malaria parasite Plasmodium falciparum

Abstract

G-quadruplexes are non-helical secondary structures that can fold in vivo in both DNA and RNA. In human cells, they can influence replication, transcription and telomere maintenance in DNA, or translation, transcript processing and stability of RNA. We have previously showed that G-quadruplexes are detectable in the DNA of the malaria parasite Plasmodium falciparum, despite a very highly A/T-biased genome with unusually few guanine-rich sequences. Here, we show that RNA G-quadruplexes can also form in P. falciparum RNA, using rG4-seq for transcriptome-wide structure-specific RNA probing. Many of the motifs, detected here via the rG4seeker pipeline, have non-canonical forms and would not be predicted by standard in silico algorithms. However, in vitro biophysical assays verified formation of non-canonical motifs. The G-quadruplexes in the P. falciparum transcriptome are frequently clustered in certain genes and associated with regions encoding low-complexity peptide repeats. They are overrepresented in particular classes of genes, notably those that encode PfEMP1 virulence factors, stress response genes and DNA binding proteins. In vitro translation experiments and in vivo measures of translation efficiency showed that G-quadruplexes can influence the translation of P. falciparum mRNAs. Thus, the G-quadruplex is a novel player in post-transcriptional regulation of gene expression in this major human pathogen.

The in vivo RNA structurome of the malaria parasite Plasmodium falciparum, a protozoan with an A/U-rich transcriptome

“…On the contrary, late trophozoites and schizonts are more translationally active and negative correlations between translation efficiency and complex structures like hairpins, multi-loop stem and bulges were observed. Recently, we studied a particularly stable RNA structure in P. falciparum, the RNA G-quadruplex, and demonstrated that it likewise had a negative impact on translation [64]. We also observed a positive correlation between translation efficiency and a simpler RNA structure, stems.…”

“…Recently, we studied a particularly stable RNA structure in P . falciparum , the RNA G-quadruplex, and demonstrated that it likewise had a negative impact on translation [ 64 ]. We also observed a positive correlation between translation efficiency and a simpler RNA structure, stems.…”

Recent advances in RNA structurome

“…In vitro PDS HeLa, HEK293T, Arabidopsis, E. coli, P. aeruginosa, plasmodium falciparum (Dumetz et al, 2021;Kwok et al, 2016a;Shao et al, 2020;Yang et al, 2020b;Yeung et al, 2019) Pros: Long flanking sequence (250-300 nt), without an input of known PQSs, high resolution. Cons: In vitro rG4 mapping only RT Stop profiling In vitro DMS Mouse embryonic stem cells (mESCs) (Guo and Bartel, 2016) Pros: 60-80 nt RNA fragments were selected Cons: Should ideally be performed under single-hit kinetics conditions, CircLigase-mediated intramolecular ssDNA ligation has more bias, in vitro rG4 mapping only…”

“…The associated bioinformatics analysis tool, rG4-seeker, was developed to allow for better false-positive discrimination and improved sensitivity to non-canonical rG4s, enabling high-confidence identification of novel and non-canonical rG4 motifs from rG4-seq experiments (Chow et al, 2020). So far, rG4-seq has been applied to many other species (Dumetz et al, 2021;Shao et al, 2020;Yang et al, 2020b).…”

Enhanced transcriptome-wide RNA G-quadruplex sequencing for low RNA input samples with rG4-seq 2.0

“…Recently, we have refined a few steps of the rG4-seq experimental procedures [ 23 ] and also developed a bioinformatics pipeline known as rG4-seeker [ 24 ]. To date, rG4-seq has been successfully applied to study the rG4s in human [ 22 ], plants [ 25 ], bacteria [ 26 ], and plasmodium [ 27 ].…”