TY - JOUR A1 - Siegel, T. Nicolai A1 - Hon, Chung-Chau A1 - Zhang, Qinfeng A1 - Lopez-Rubio, Jose-Juan A1 - Scheidig-Benatar, Christine A1 - Martins, Rafeal M. A1 - Sismeiro, Odile A1 - Coppée, Jean-Yves T1 - Strand-specific RNA-Seq reveals widespread and developmentally regulated transcription of natural antisense transcripts in Plasmodium falciparum JF - BMC Genomics N2 - Background 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. 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. Results To 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. Conclusions Our 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. KW - directional RNA-Seq KW - ncRNA KW - natural antisense transcripts KW - 3′ UTR KW - polyadenylation sites KW - genes KW - antisense RNA KW - plasmodium falciparum Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-119892 VL - 15 ER - TY - JOUR A1 - Reynolds, David A1 - Cliffe, Laura A1 - Förstner, Konrad U. A1 - Hon, Chung-Chau A1 - Siegel, T. Nicolai A1 - Sabatini, Robert T1 - Regulation of transcription termination by glucosylated hydroxymethyluracil, base J, in Leishmania major and Trypanosoma brucei JF - Nucleic Acids Research N2 - Base J, beta-d-glucosyl-hydroxymethyluracil, is an epigenetic modification of thymine in the nuclear DNA of flagellated protozoa of the order Kinetoplastida. J is enriched at sites involved in RNA polymerase ( RNAP) II initiation and termination. Reduction of J in Leishmania tarentolae via growth in BrdU resulted in cell death and indicated a role of J in the regulation of RNAP II termination. To further explore J function in RNAP II termination among kinetoplastids and avoid indirect effects associated with BrdU toxicity and genetic deletions, we inhibited J synthesis in Leishmania major and Trypanosoma brucei using DMOG. Reduction of J in L. major resulted in genome-wide defects in transcription termination at the end of polycistronic gene clusters and the generation of antisense RNAs, without cell death. In contrast, loss of J in T. brucei did not lead to genome-wide termination defects; however, the loss of J at specific sites within polycistronic gene clusters led to altered transcription termination and increased expression of downstream genes. Thus, J regulation of RNAP II transcription termination genome-wide is restricted to Leishmania spp., while in T. brucei it regulates termination and gene expression at specific sites within polycistronic gene clusters. KW - RNA-polymerase-II KW - variant surface glycoprotein KW - SWI2/SNF2-like protein KW - messenger RNA KW - polycistronic transcription KW - DNA glycosation KW - hela cells KW - gene expression KW - genome KW - 5-bromodeoxyuridine Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-117863 VL - 42 IS - 15 ER - TY - JOUR A1 - Siegel, T. Nicolai A1 - Vasquez, Juan-José A1 - Hon, Chung-Chau A1 - Vanselow, Jens T. A1 - Schlosser, Andreas T1 - Comparative ribosome profiling reveals extensive translational complexity in different Trypanosoma brucei life cycle stages N2 - While gene expression is a fundamental and tightly controlled cellular process that is regulated at multiple steps, the exact contribution of each step remains unknown in any organism. The absence of transcription initiation regulation for RNA polymerase II in the protozoan parasite Trypanosoma brucei greatly simplifies the task of elucidating the contribution of translation to global gene expression. Therefore, we have sequenced ribosome-protected mRNA fragments in T. brucei, permitting the genome-wide analysis of RNA translation and translational efficiency. We find that the latter varies greatly between life cycle stages of the parasite and ∼100-fold between genes, thus contributing to gene expression to a similar extent as RNA stability. The ability to map ribosome positions at sub-codon resolution revealed extensive translation from upstream open reading frames located within 5' UTRs and enabled the identification of hundreds of previously un-annotated putative coding sequences (CDSs). Evaluation of existing proteomics and genome-wide RNAi data confirmed the translation of previously un-annotated CDSs and suggested an important role for >200 of those CDSs in parasite survival, especially in the form that is infective to mammals. Overall our data show that translational control plays a prevalent and important role in different parasite life cycle stages of T. brucei. KW - Ribosom KW - Profilierung KW - Trypanosoma brucei KW - Entwicklung KW - Lebenszyklus Y1 - 2014 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-112657 ER -