The mRNA-bound proteome of the human malaria parasite Plasmodium falciparum

Bunnik, Evelien M.; Batugedara, Gayani; Saraf, Anita; Prudhomme, Jacques; Florens, Laurence; Roch, Karine G. Le

doi:10.1186/s13059-016-1014-0

Cited by 95 publications

(86 citation statements)

References 78 publications

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“…One explanation is that they are a normal byproduct of promoter activity, similar to that described previously for higher eukaryotes (57) . A second explanation is that these transcripts have some function in gene regulation, which we speculate could include binding by regulatory proteins containing an RNA recognition motif (RRM), many of which are encoded in the P. falciparum genome (66) , but which are largely uncharacterised (67) . Additionally, our identification of independently regulated ncRNAs may help future studies of gene regulation, as seen with an ncRNA antisense to GDV1 (68) (which can be observed in our data, Figure S27 ), which is involved in the regulation of sexual commitment.…”

Section: Discussionmentioning

confidence: 99%

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

Chappell

Ross

Orchard

et al. 2019

Preprint

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Plasmodium parasites undergo several major developmental transitions during their complex lifecycle, which are enabled by precisely ordered gene expression programs. Transcriptomes from the 48-hour 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%. 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 75% of the genome transcribed into mRNAs. These datasets allowed us to refine the 5' and 3' untranslated regions (UTRs), which can be variable, long (>1,000 nt), and often overlap those of adjacent transcripts. We also find that transcription from bidirectional promoters frequently results in non-coding, antisense transcripts. By capturing the 5' ends of mRNAs, we reveal both constant and dynamic use of transcriptional start sites across the intraerythrocytic developmental cycle resulting in an updated view of the P. falciparum transcriptome. Materials and MethodsParasite culture and RNA extraction P. falciparum clone 3D7 was cultured in O+ human erythrocytes and 10% human serum in RPMI-based media, using standard methods (29) . RNA extractions used the TRIzol reagent as previously described (30) . RNA was quality controlled and quantified using an Agilent Bioanalyzer 2100 Nano RNA chip.Directional, Amplification-Free RNA-seq ( DAFT-seq) libraries PolyA+ RNA (mRNA) was selected using magnetic oligo-d(T) beads. Reverse transcription using Superscript II (Life) was primed using oligo d(T) primers, then second strand cDNA synthesis included dUTP. The resulting cDNA was fragmented using a Covaris AFA sonicator. A "with-bead" protocol was used for dA-tailing, end repair and adapter ligation (NEB) using "PCR-free" barcoded sequencing adaptors (Bioo Scientific, similar to Korarewa et al.). After 2 rounds of SPRI cleanup the libraries were eluted in EB buffer and USER enzyme mix (NEB) was used to digest the second strand cDNA, generating directional libraries. The libraries were quantified by qPCR and sequenced on an Illumina HiSeq2000, generating 100bp paired-end reads. Reads were mapped using TopHat2 (31) , using directional parameters and a maximum intron size of 5,000 nt. 5UTR-seq librariesPolyA+ RNA was isolated using oligo d(T)-coated magnetic beads. Superscript II reverse transcriptase was used to synthesise first strand cDNA using oligo-d(T) primers and in the presence of template switching oligos, which had the same sequence as those in the Smart-seq2 protocol (32, 33) . Template-switching oligos (TSOs) were used to "tag" the end of the cDNA sequences; this tag is used to prime second str...

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Section: Discussionmentioning

confidence: 99%

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

Chappell

Ross

Orchard

et al. 2019

Preprint

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“…Applied to HeLa and HEK293 cells, RNA interactome capture identified more than a thousand RBPs, hundreds of which were previously unknown to bind RNA. This method was successfully used to determine the poly(A)-RNA-binding proteome of different human cell types, including human (Baltz et al 2012;Castello et al 2012;Beckmann et al 2015) and murine (Kwon et al 2013;Liao et al 2016;Liepelt et al 2016) cells, as well as from organisms such as S. cerevisiae (Mitchell et al 2013;Beckmann et al 2015;Matia-González et al 2015), C. elegans (Matia-González et al 2015), D. melanogaster (Sysoev et al 2016;Wessels et al 2016), A. thaliana (Marondedze et al 2016;Reichel et al 2016), and the parasites plasmodium and trypanosoma (Bunnik et al 2016;Lueong et al 2016). However, the identification of the RBPs bound to specific RNAs remains challenging.…”

Section: Introductionmentioning

confidence: 99%

Specific RNP capture with antisense LNA/DNA mixmers

Rogell

Fischer

Rettel

et al. 2017

RNA

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RNA-binding proteins (RBPs) play essential roles in RNA biology, responding to cellular and environmental stimuli to regulate gene expression. Important advances have helped to determine the (near) complete repertoires of cellular RBPs. However, identification of RBPs associated with specific transcripts remains a challenge. Here, we describe "specific ribonucleoprotein (RNP) capture," a versatile method for the determination of the proteins bound to specific transcripts in vitro and in cellular systems. Specific RNP capture uses UV irradiation to covalently stabilize protein-RNA interactions taking place at "zero distance." Proteins bound to the target RNA are captured by hybridization with antisense locked nucleic acid (LNA)/DNA oligonucleotides covalently coupled to a magnetic resin. After stringent washing, interacting proteins are identified by quantitative mass spectrometry. Applied to in vitro extracts, specific RNP capture identifies the RBPs bound to a reporter mRNA containing the Sex-lethal (Sxl) binding motifs, revealing that the Sxl homolog sister of Sex lethal (Ssx) displays similar binding preferences. This method also revealed the repertoire of RBPs binding to 18S or 28S rRNAs in HeLa cells, including previously unknown rRNA-binding proteins.

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“…In a related study, it was further shown that a messenger ribonucleoprotein (mRNP) from P. berghei gametocytes contains DOZI and the Sm‐like factor CITH (homolog of worm CAR‐I and fly Trailer Hitch) . The composition analysis of mRNP complex indicated that it contains 16 major protein factors including eIF4E and poly(A)‐binding protein . During the intraerythrocytic developmental cycle, numerous RNA‐binding proteins including DOZI have been reported to be implicated in post‐transcriptional regulation of gene expression .…”

Section: Dead‐box Helicases Of Plasmodium Falciparummentioning

confidence: 99%

Unraveling the importance of the malaria parasite helicases

Tuteja

2017

The FEBS Journal

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Malaria is a human parasitic disease caused by infection from Plasmodium species, particularly Plasmodium falciparum. Each year millions of people are infected with malaria and large numbers of deaths result due to this deadly infection. P. falciparum contains 14 chromosomes, nearly 5400 genes and a multistage life cycle in humans and mosquitoes. The control of malaria is still a challenge as the parasite is continuously developing resistance to available antimalarial drugs and the mosquito vector is developing resistance to insecticides. The availability of P. falciparum genome has resulted in the identification of parasite‐specific proteins that can be targeted without harmful effects to the human host. Toward this goal, we have been working on the identification and characterization of helicases in order to find parasite‐specific helicases, which can be used as novel drug targets to tackle the rising problem of drug resistance. Helicases are ATP‐dependent nucleic acid unwinding enzymes. The P. falciparum genome analysis depicts that it contains some parasite‐specific helicases and homologs to most of the human helicases. Here, we present an overview of P. falciparum helicases and their importance in parasite growth and survival.

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The mRNA-bound proteome of the human malaria parasite Plasmodium falciparum

Cited by 95 publications

References 78 publications

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

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

Specific RNP capture with antisense LNA/DNA mixmers

Unraveling the importance of the malaria parasite helicases

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