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Plasmodium falciparum

ABSTRACT: 5CAPture-seq: a method for enriching full-length cDNA and identifying 5′ capped nucleotides

PROVIDER: PRJNA399851 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR12404029_1.fastq.gz	Fastqsanger.gz
	SRR12404029_2.fastq.gz	Fastqsanger.gz
	SRR12404030_1.fastq.gz	Fastqsanger.gz
	SRR12404030_2.fastq.gz	Fastqsanger.gz
	SRR12404031_1.fastq.gz	Fastqsanger.gz

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5CAPture-seq: a method for enriching full-length cDNA and identifying 5′ capped nucleotides

Project description:Current transcriptomic methods for mapping 5′-ends of eukaryotic RNA employ only 5′-end enriched samples. The absence of a matched unenriched control limits the ability to distinguish genuine 5′ ends from incomplete cDNAs. We developed a transcriptomic method based on sequencing of cDNA enriched for full-length fragments and an unenriched control. From the mapped sequence reads, enrichment scores are calculated for all transcribed nucleotides and a statistical model of the enrichment constructed. We tested the method in the human malaria parasite Plasmodium falciparum. Data were obtained from ring, trophozoite and schizont stages of the parasite intra-erythrocytic development cycle. Two groups of 5′ capped nucleotides were assigned by unsupervised clustering. The first group contains sites located mostly outside of annotated protein-coding exons in regions of high local nucleosomal occupancy, but low occupancies of ribosome and elongating RNA polymerase II. The majority of sites in the second group are intra-exonic and show different patterns, most notably a peak of ribosome occupancy centered on the 5′ end position and a biased representation among codons possibly prone to ribosome stalling. Our method can be used to annotate 5′ capped nucleotides, including intra-exonic nucleotides that can be distinguished from incomplete cDNA artifacts.

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Characterization of human malaria parasite Plasmodium falciparum eIF4E homologue and mRNA 5′ cap status

Project description:The mRNA 5′ cap is normally essential for eukaryotic mRNA translation, stabilization and transport and both the cap and eIF4E are important elements of post-transcriptional gene regulation. To further our understanding of mRNA translation in the human malaria parasite Plasmodium falciparum, we have investigated the parasite translation initiation factor eIF4E and its interaction with 5′ capped mRNA. We have purified P. falciparum eIF4E as a recombinant protein and demonstrated that it has canonical mRNA 5′ cap binding activity. We used this protein to purify P. falciparum full-length 5′ capped mRNAs from total parasite RNA. Microarray analysis comparing total and eIF4E-purified 5′ capped mRNAs shows that a subset of 34 features were more than two-fold under-represented in the purified RNA sample, including 19 features representative of nuclear transcripts. The uncapped nuclear transcripts may represent a class of mRNAs targeted for storage and cap removal. Keywords: total RNA vs purified capped mRNA

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Project description:The mRNA 5â² cap is normally essential for eukaryotic mRNA translation, stabilization and transport and both the cap and eIF4E are important elements of post-transcriptional gene regulation. To further our understanding of mRNA translation in the human malaria parasite Plasmodium falciparum, we have investigated the parasite translation initiation factor eIF4E and its interaction with 5â² capped mRNA. We have purified P. falciparum eIF4E as a recombinant protein and demonstrated that it has canonical mRNA 5â² cap binding activity. We used this protein to purify P. falciparum full-length 5â² capped mRNAs from total parasite RNA. Microarray analysis comparing total and eIF4E-purified 5â² capped mRNAs shows that a subset of 34 features were more than two-fold under-represented in the purified RNA sample, including 19 features representative of nuclear transcripts. The uncapped nuclear transcripts may represent a class of mRNAs targeted for storage and cap removal. Keywords: total RNA vs purified capped mRNA The microarray data were obtained from four hybridizations using RNA from two independent GST-PfeIF4E purifications from separate malaria cultures.

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