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. 2016 Jan 20;15:30.
doi: 10.1186/s12936-015-1081-9.

UTR Introns, Antisense RNA and Differentially Spliced Transcripts Between Plasmodium Yoelii Subspecies

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Free PMC article

UTR Introns, Antisense RNA and Differentially Spliced Transcripts Between Plasmodium Yoelii Subspecies

Jian Li et al. Malar J. .
Free PMC article

Abstract

Background: The rodent malaria parasite Plasmodium yoelii is an important animal model for studying host-parasite interaction and molecular basis of malaria pathogenesis. Although a draft genome of P. yoelii yoelii YM is available, and RNA sequencing (RNA-seq) data for several rodent malaria species (RMP) were reported recently, variations in coding regions and structure of mRNA transcript are likely present between different parasite strains or subspecies. Sequencing of cDNA libraries from additional parasite strains/subspecies will help improve the gene models and genome annotation.

Methods: Here two directional cDNA libraries from mixed blood stages of a subspecies of P. yoelii (P. y. nigeriensis NSM) with or without mefloquine (MQ) treatment were sequenced, and the sequence reads were compared to the genome and cDNA sequences of P. y. yoelii YM in public databases to investigate single nucleotide polymorphisms (SNPs) in coding regions, variations in intron-exon structure and differential splicing between P. yoelii subspecies, and variations in gene expression under MQ pressure.

Results: Approximately 56 million of 100 bp paired-end reads were obtained, providing an average of ~225-fold coverage for the coding regions. Comparison of the sequence reads to the YM genome revealed introns in 5' and 3' untranslated regions (UTRs), altered intron/exon boundaries, alternative splicing, overlapping sense-antisense reads, and potentially new transcripts. Interestingly, comparison of the NSM RNA-seq reads obtained here with those of YM discovered differentially spliced introns; e.g., spliced introns in one subspecies but not the other. Alignment of the NSM cDNA sequences to the YM genome sequence also identified ~84,000 SNPs between the two parasites.

Conclusion: The discoveries of UTR introns and differentially spliced introns between P. yoelii subspecies raise interesting questions on the potential role of these introns in regulating gene expression and evolution of malaria parasites.

Figures

Fig. 1
Fig. 1
Correlation plot of gene expression levels (reads per kilobase per million or RPKM) between NSM_1 and NSM_2. Only genes with RPKM ≥10 and ≤6000 in NSM_1 were plotted; seven genes with PRKM ≥6000 were excluded in order to show genes with lower expression. Hsp70 heat shock protein 70, H4 histone H4, H2A histone H2A, H2B histone H2B, H3 histone H3, Eno enolase, Ldh l-lactate dehydrogenase
Fig. 2
Fig. 2
Types of mismatches in gene models detected after alignment of directional cDNA sequences to the YM genome sequences. a Alternatively-spliced introns at 5′ UTR; b an intron at the 3′ UTR; c an intron within coding region; d an intron smaller than the one predicted, possibly an alternatively-spliced intron, too; e overlapping transcripts in two neighboring genes; f a putative new transcript with an intron. The figures are images from Integrative Genomics Viewer (IGV). Purple sense reads; pink antisense reads. The blue bars are predicted gene models from the Plasmodium y. yoelii YM genome
Fig. 3
Fig. 3
Examples of differentially (alternatively) spliced introns between Plasmodium y. yoelii YM and P. y. nigeriensis NSM. cDNA reads from YM (Otto et al. [19]) and NSM were compared using IGV. Gene IDs are on top of each panel. Reads in pink are sense, and those in purple are antisense. a A coding intron present in NSM, but not in YM; b an intron in YM, but not in NSM; c, d 5′ UTR introns present in both YM and NSM, but the proportions of reads with spliced forms are different. For PYYM_0502300, most of the reads from NSM are spliced, but not those of YM (c). e additional sliced forms of 5′ UTR introns are present in YM, but not NSM; f an additional alternatively spliced coding intron in YM, but not NSM. YM may also have an extra 3′ UTR intron; g one intron in NSM, but multiple introns in antisense transcripts in YM, which may generate a large number of variants (>10)
Fig. 4
Fig. 4
Distribution of genes with antisense/sense (AS:S) ratio ≥1 across the 14 parasite chromosomes. Each horizontal line represents a chromosome. The relative positions of the genes with antisense reads ≥sense reads on the chromosomes are as marked, with longer vertical lines representing higher antisense/sense ratios
Fig. 5
Fig. 5
Verification of differentially spliced introns between Plasmodium y. nigeriensis NSM and Plasmodium y. yoelii 17XNL. DNA and mRNA samples were prepared from NSM and 17XNL parasites; 17XNL was used because YM parasite was not available in the laboratory in China. a, b RNA-seq read alignments showing a predicted intron and primer positions (arrows) from the PYYM_0205900 gene that has the same spliced intron in both NSM and YM (a) and amplification products using primers 5′-TGTCCATCAAATAATAAAGCTAAAATATATTCCTCTCA-3′ and 5′-TATAGTTAGATGTGTTTAATATTTAAGG-3′. Parasite names with ‘g’ indicate amplification products from genomic DNA, and those with ‘c’ were from cDNA. The results showed no DNA band in the cDNA preparations, suggesting lack of DNA contamination in the cDNAs. c, d RNA-seq alignments (c) and amplification products (d) from gene PYYM_0406600 using primers 5′-GTAAGAAATATACAACAATACTATTCCTTGGCAA-3′and 5′-CTCTCCCATTTTTAGGTATAAAAAATAACTAAATATG-3′, showing a much stronger sliced product (arrowhead) in 17XNL than in NSM. e, f RNA-seq alignments (e) and amplification products from gene PYYM_0710900 (f) showing differential spliced bands (arrowheads) between NSM and 17XNL parasites (primers: 5′-GATTTCTATTAGCTTTGTGAAGTC-3′ and 5′- TGTAATATATTATCGAAAGACGTG-3′). In addition to differential splicing, size polymorphism in genomic DNA between NSM and 17XNL was also detected

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