Abstract
BackgroundAdvances in high-throughput sequencing have led to the discovery of widespread transcription of natural antisense transcripts (NATs) in a large number of organisms, where these transcripts have been shown to play important roles in the regulation of gene expression. Likewise, the existence of NATs has been observed in Plasmodium but our understanding towards their genome-wide distribution remains incomplete due to the limited depth and uncertainties in the level of strand specificity of previous datasets.ResultsTo gain insights into the genome-wide distribution of NATs in P. falciparum, we performed RNA-ligation based strand-specific RNA sequencing at unprecedented depth. Our data indicate that 78.3% of the genome is transcribed during blood-stage development. Moreover, our analysis reveals significant levels of antisense transcription from at least 24% of protein-coding genes and that while expression levels of NATs change during the intraerythrocytic developmental cycle (IDC), they do not correlate with the corresponding mRNA levels. Interestingly, antisense transcription is not evenly distributed across coding regions (CDSs) but strongly clustered towards the 3′-end of CDSs. Furthermore, for a significant subset of NATs, transcript levels correlate with mRNA levels of neighboring genes.Finally, we were able to identify the polyadenylation sites (PASs) for a subset of NATs, demonstrating that at least some NATs are polyadenylated. We also mapped the PASs of 3443 coding genes, yielding an average 3′ untranslated region length of 523 bp.ConclusionsOur strand-specific analysis of the P. falciparum transcriptome expands and strengthens the existing body of evidence that antisense transcription is a substantial phenomenon in P. falciparum. For a subset of neighboring genes we find that sense and antisense transcript levels are intricately linked while other NATs appear to be regulated independently of mRNA transcription. Our deep strand-specific dataset will provide a valuable resource for the precise determination of expression levels as it separates sense from antisense transcript levels, which we find to often significantly differ. In addition, the extensive novel data on 3′ UTR length will allow others to perform searches for regulatory motifs in the UTRs and help understand post-translational regulation in P. falciparum.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-150) contains supplementary material, which is available to authorized users.
Highlights
Advances in high-throughput sequencing have led to the discovery of widespread transcription of natural antisense transcripts (NATs) in a large number of organisms, where these transcripts have been shown to play important roles in the regulation of gene expression
A thorough comparison of different strand-specific RNA-Seq methods indicated that the least amount of ‘false’ antisense RNA was generated when libraries were prepared by using the ‘RNA-ligation method’ involving the sequential ligation of 3′-preadenylated and 5′-adapters to RNA followed by reverse transcription using a primer complementary to the 3′-adapter [17] (Figure 1A)
To determine if P. falciparum contains NATs whose expression is regulated independently from that of adjacent genes, we investigated the correlation between the changes in mRNA levels of the downstream genes and the changes in antisense transcript levels of the corresponding upstream genes during the intraerythrocytic developmental cycle (IDC)
Summary
Advances in high-throughput sequencing have led to the discovery of widespread transcription of natural antisense transcripts (NATs) in a large number of organisms, where these transcripts have been shown to play important roles in the regulation of gene expression. The publication of the P. falciparum genome sequence in 2002 [1] was followed by transcriptome analyses using microarrays [2,3] and, more recently, high-throughput sequencing of cDNA (RNA-Seq) [4,5,6,7]. These analyses allowed determination of transcript levels for a large number of genes, helped to refine the original gene model and revealed a tight regulation of gene expression throughout the intraerythrocytic developmental cycle (IDC) of P. falciparum. Information regarding 5′ and 3′ untranslated regions (UTRs) and the degree of antisense transcription is still missing for most genes
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