Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Transcriptome of Trypanosome brucei after PUF2 Knock-down - polysome fractions

ABSTRACT: Polysome fractions of RNAi PUF2 (Tb927.10.12660) 13 hours RNAi induction (TET)

INSTRUMENT(S): Illumina HiSeq 2000

ORGANISM(S): Trypanosoma brucei

SUBMITTER: Christine Clayton

PROVIDER: E-MTAB-5613 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Depletion of the Trypanosome Pumilio domain protein PUF2 or of some other essential proteins causes transcriptome changes related to coding region length.

Jha Bhaskar Anand BA Fadda Abeer A Merce Clementine C Mugo Elisha E Droll Dorothea D Clayton Christine C

Eukaryotic cell 20140328 5

Pumilio domain RNA-binding proteins are known mainly as posttranscriptional repressors of gene expression that reduce mRNA translation and stability. Trypanosoma brucei has 11 PUF proteins. We show here that PUF2 is in the cytosol, with roughly the same number of molecules per cell as there are mRNAs. Although PUF2 exhibits a low level of in vivo RNA binding, it is not associated with polysomes. PUF2 also decreased reporter mRNA levels in a tethering assay, consistent with a repressive role. Dep ...[more]

PMID: 24681684

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Project description:We used all five current human African trypanosomiasis drugs for genome-scale RNA interference (RNAi) target sequencing (RIT-seq) screens in Trypanosoma brucei, revealing the transporters, organelles, enzymes and metabolic pathways that function to facilitate anti-trypanosomal drug action. RIT-seq profiling links more than fifty additional genes to drug action. This data has been described in the following article [doi:10.1038/nature10771] and its further analysis can be freely submitted for publication. For information on the proper use of data shared by the Wellcome Trust Sanger Institute (including information on acknowledgement), please see http://www.sanger.ac.uk/datasharing/. Protocol - Assembly of the bloodstream-form T. brucei RNAi library and the RNAi target sequencing (RIT-seq) approach were reported previously (Alsford, S. et al. High-throughput phenotyping using parallel sequencing of RNA interference targets in the African trypanosome. Genome Res 21, 915-924, 264 doi:gr.115089.110 [pii] 265 10.1101/gr.115089.110 (2011). Briefly, a tetracycline-inducible RNAi plasmid library, containing randomly sheared genomic fragments (mean ~600 bp) under the control of head-to-head T7 promoters, was targeted to a single genomic locus that had been validated for robust expression. For this study, the library was grown under inducing conditions with drug selection and genomic DNA was isolated from surviving populations. For chemical RIT-seq profiling, adapter-ligated sequencing libraries were prepared from each genomic DNA sample and used to amplify DNA fragments containing RNAi cassette-insert junctions in semi-specific PCR reactions; one primer was specific for the RNAi vector and the other for the Illumina adapter. Size-selected DNA was sequenced with 76 cycle paired-end runs on an Illumina GAII. Illumina sequencing reads containing a nine-base RNAi vector junction sequence were then mapped to the T. brucei reference genome. Where loss of function increases drug tolerance, RNAi-target sequence representation is increased relative to the otherwise susceptible population, revealing hot spots.

Dataset Information

Transcriptome of Trypanosome brucei after PUF2 Knock-down - polysome fractions

Publications

Depletion of the Trypanosome Pumilio domain protein PUF2 or of some other essential proteins causes transcriptome changes related to coding region length.

Similar Datasets

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