Fig. 3

Characterization of 1134 unannotated P. falciparum lncRNAs reveals global trends as well as intriguing outliers. (A) Without annotation assistance, at least 4707 out of 5777 (81.5 %) annotated transcripts could be contiguously assembled in our blood stage samples. 696 annotated transcripts could not be contiguously assembled, and we excluded 374 short and/or structural RNAs from assembly. Given this high reassembly rate of known transcripts, it is possible that the 660 intergenic lncRNAs and 474 antisense RNAs described here represent the majority of lncRNAs transcribed in P. falciparum. (B) Comparative inspection of non-clustering heatmaps showed that predicted lncRNAs were developmentally regulated in a similar periodic fashion to annotated mRNAs. However, it was also apparent that a subset of lncRNAs strongly peaked in expression during parasite invasion, and that there was a paucity of antisense transcript levels during parasite invasion. The 48 hpi invasion time-point is indicated with purple arrows. Transcripts are ordered by their angular position in the MDS plot of transcript expression profiles, and samples are ordered by time. Mean-centered expression is in units of log2(FPKM + 1). (C) The distribution of maximum expression levels for each transcript class suggested that both intergenic lncRNAs (red) and antisense RNAs (blue) were robustly expressed, albeit they typically reached lower maximum expression levels than annotated mRNAs (black). (D) Pearson correlation during the 56-hour time course between 50,000 random mRNA gene pairs (orange) as compared to 5251 mRNA-neighboring gene pairs (black), 498 intergenic lncRNA-neighboring gene pairs (red), and 445 antisense-sense gene pairs (blue). To be consistent, we defined the neighboring gene used in both the mRNA and intergenic lncRNA pairings as the more correlated neighboring mRNA. (E) The distribution of GC content for each transcript class indicated that intergenic lncRNAs (red) and antisense RNAs (blue) typically had lower GC content than annotated transcripts (green), though a handful of intergenic lncRNAs had unusually high GC content (purple arrow). (F) The distribution of transcript length for each transcript class showed that intergenic lncRNAs (red) and antisense RNAs (blue) were comparable in length to annotated transcripts (green), with the average of each class being longer than 1 kb. Markedly long intergenic lncRNAs (>4 kb) are indicated with a purple arrow. (G) Plotting the normalized distribution of antisense RNAs relative to annotated gene bodies revealed a 3’ tail-to-tail bias