Article

The Transcriptome of the Human Pathogen Trypanosoma brucei at Single-Nucleotide Resolution

School of Public Health, Yale University, New Haven, Connecticut, United States of America.
PLoS Pathogens (Impact Factor: 8.06). 09/2010; 6(9):e1001090. DOI: 10.1371/journal.ppat.1001090
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ABSTRACT The genome of Trypanosoma brucei, the causative agent of African trypanosomiasis, was published five years ago, yet identification of all genes and their transcripts remains to be accomplished. Annotation is challenged by the organization of genes transcribed by RNA polymerase II (Pol II) into long unidirectional gene clusters with no knowledge of how transcription is initiated. Here we report a single-nucleotide resolution genomic map of the T. brucei transcriptome, adding 1,114 new transcripts, including 103 non-coding RNAs, confirming and correcting many of the annotated features and revealing an extensive heterogeneity of 5' and 3' ends. Some of the new transcripts encode polypeptides that are either conserved in T. cruzi and Leishmania major or were previously detected in mass spectrometry analyses. High-throughput RNA sequencing (RNA-Seq) was sensitive enough to detect transcripts at putative Pol II transcription initiation sites. Our results, as well as recent data from the literature, indicate that transcription initiation is not solely restricted to regions at the beginning of gene clusters, but may occur at internal sites. We also provide evidence that transcription at all putative initiation sites in T. brucei is bidirectional, a recently recognized fundamental property of eukaryotic promoters. Our results have implications for gene expression patterns in other important human pathogens with similar genome organization (Trypanosoma cruzi, Leishmania sp.) and revealed heterogeneity in pre-mRNA processing that could potentially contribute to the survival and success of the parasite population in the insect vector and the mammalian host.

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    • "Recent RNA-sequencing studies of T. brucei and L. major have revealed that many genes can harbour multiple alternative processing sites [11] [12]. While the functional significance of these sites has yet to be determined on a genome wide scale, it is likely that some of these alternative sites are important to the regulation and/or function of the final transcript. "
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    ABSTRACT: The Kinetoplastida are a diverse and globally distributed class of free-living and parasitic single-celled eukaryotes that collectively cause a significant burden on human health and welfare. In kinetoplastids individual genes do not have promoters, but rather all genes are arranged downstream of a small number of RNA polymerase II transcription initiation sites are thus transcribed in polycistronic gene clusters. Production of individual mRNAs from this continuous transcript occurs co-transcriptionally by trans-splicing of a ∼39 nucleotide capped RNA and subsequent polyadenylation of the upstream mRNA. SLaP mapper (Spliced-Leader and Polyadenylation mapper) is a fully automated web-service for identification, quantitation and gene-assignment of both spliced-leader and polyadenylation addition sites in Kinetoplastid genomes. SLaP mapper only requires raw read data from paired-end Illumina RNAseq and performs all read processing, mapping, quality control, quantification, and analysis in a fully automated pipeline. To provide usage examples and estimates of the quantity of sequence data required we use RNAseq obtained from two different library preparations from both Trypanosoma brucei and Leishmania mexicana to show the number of expected reads that are obtained from each preparation type. SLaP mapper is an easy to use, platform independent webserver that is freely available for use at http://www.stevekellylab.com/software/slap. Example files are provided on the website.
    Molecular and Biochemical Parasitology 08/2014; 196(2). DOI:10.1016/j.molbiopara.2014.07.012 · 2.24 Impact Factor
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    • "We focus first on transcriptional read-through, a hallmark of defects in transcriptional termination. As in previous studies in L. tarentolae and T. brucei [7] [11] [18], in WT L. major the vast majority of SL-containing reads map to the coding strand, with very few mapping to antisense regions beyond cSSRs (see Fig. 2 C-D, Supplemental Figs. S3, S4). "
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    ABSTRACT: Regions of transcription initiation and termination in kinetoplastid protists lack known eukaryotic promoter and terminator elements, although epigenetic marks such as histone variants and the modified DNA base J have been localized to these regions in Trypanosoma brucei, Trypanosoma cruzi, and/or Leishmania major. Phenotypes of base J mutants vary significantly across trypanosomatids, implying divergence in the epigenetic networks governing transcription during evolution. Here, we demonstrate that the histone variants H2A.Z and H2B.V are essential in L. major using a powerful quantitative plasmid segregation-based test. In contrast, H3.V is not essential for viability or normal growth in Leishmania. Steady-state transcript levels and the efficiency of transcription termination at convergent strand switch regions (SSRs) in H3V-null parasites were comparable to WT parasites. Our genetic tests show a conservation of histone variant phenotypes between L. major and T. brucei, unlike the diversity of phenotypes associated with genetic manipulation of the DNA base J modification.
    Molecular and Biochemical Parasitology 09/2013; DOI:10.1016/j.molbiopara.2013.09.005 · 2.24 Impact Factor
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    • "RNAi directed against the 3′UTR of EF-Tu (EF-Tu-3′UTR) was done using a 465 bp region immediately downstream of the stop codon. The 3′UTR of the EF-Tu mRNA is estimated to be longer than 1 kb (Kolev et al., 2010). For the three remaining RNAi cell lines we used the following inserts: a 455 bp fragment (nucleotides 4–459) of the EF-Ts gene (Tb927.3.3630); a 626 bp fragment (nucleotides 1051–1677) of the EF-G1 gene (Tb927.10.5010) and a 653 bp fragment (nucleotides 3–656) of the RF1 gene (Tb927.3.1070). "
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