Strand-specific RNA-Seq reveals widespread and developmentally regulated transcription of natural antisense transcripts in Plasmodium falciparum

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.

Landscape and Dynamics of Transcription Initiation in the Malaria Parasite Plasmodium falciparum

“…These numbers are in agreement with previous microarrays and RNA-Seq studies and confirm that the parasite transcriptional cascade is recapitulated at the TSS level. Furthermore, comparison of our data with a recent RNA-Seq analysis (Siegel et al, 2014) showed that the detected transcriptional initiation events correlated well with gene expression levels (Figure S4C). This suggests that the majority of these events are associated with the production of full-length transcripts.…”

“…Many of these non-coding transcription units correspond to transcription initiation in an antisense orientation to coding genes. More generally, this configuration was widespread across the genome, regardless of the coding potential of the sense/antisense pairs, as previously observed (López-Barragán et al, 2011; Militello, 2005; Siegel et al, 2014). Such arrangements that presumably yield overlapping transcripts may carry a regulatory role, as shown by the expression of certain sense-antisense transcript pairs in a mutually exclusive manner.…”

“…For instance, the mRNA fragmentation and the reverse transcription reaction were performed using conditions previously established for RNA-seq and DNA microarrays in P. falciparum (Bozdech et al, 2003b; Hoeijmakers et al, 2013a). Additionally, the second strand synthesis as well as the amplification of the cDNA libraries were performed using the KAPA DNA polymerase enzyme that has a very low AT-bias and is now commonly used when preparing P. falciparum sequencing libraries (Oyola et al, 2012; Siegel et al, 2014). About 600,000 unique 5′ long tags (> 90 nucleotides) were recovered on average per independent library (Figure S1C, Table S1) and collapsed on their first base to define the position of the transcription start sites (TSSs) at single-nucleotide resolution.…”

“…More particularly, the processes underlying the regulation of transcription initiation, such as the recruitment of RNA polymerase II (RNA PolII) or transcription start site (TSS) selection, still remain to be deciphered. Generally, characterizing the transcriptome architecture of P. falciparum has been technically challenging, given the low-complexity of its genome and the potential for reverse-transcription-derived artifacts (Siegel et al, 2014). Nevertheless, several single gene studies have attempted to identify transcription initiation sites (Horrocks et al, 2009), while the FULL-Malaria project mapped TSS positions for ∼25% of P. falciparum genes by cloning and subsequently sequencing full-length cDNA molecules (Watanabe et al, 2002).…”

A Plasmodium yoelii Mei2-Like RNA Binding Protein Is Essential for Completion of Liver Stage Schizogony

“…Conversely, transcriptome sequencing data sets suggest that the majority of the genome is pervasively transcribed throughout blood stage development (3). Few Plasmodium transcription factors have been identified, with the exception of the 27 members of the Apicomplexan Apetala 2 (ApiAP2) family of transcription factors (4), which are homologous to plant AP2/ethylene response factor DNA-binding proteins, the second largest family of transcription factors in the model plant Arabidopsis thaliana (5).…”