A Genome-Wide Tethering Screen Reveals Novel Potential Post-Transcriptional Regulators in Trypanosoma brucei

Esteban D. Erben, Abeer A. Fadda, Smiths Lueong, Jörg D. Hoheisel, Christine Clayton

Research output: Contribution to journalArticle

42 Citations (Scopus)

Abstract

In trypanosomatids, gene expression is regulated mainly by post-transcriptional mechanisms, which affect mRNA processing, translation and degradation. Currently, our understanding of factors that regulate either mRNA stability or translation is rather limited. We know that often, the regulators are proteins that bind to the 3′-untranslated region; they presumably interact with ribonucleases and translation factors. However, very few such proteins have been characterized in any detail. Here we describe a genome-wide screen to find proteins implicated in post-transcriptional regulation in Trypanosoma brucei. We made a library of random genomic fragments in a plasmid that was designed for expression of proteins fused to an RNA-binding domain, the lambda-N peptide. This was transfected into cells expressing mRNAs encoding a positive or negative selectable marker, and bearing the "boxB" lambda-N recognition element in the 3′-untranslated region. The screen identified about 300 proteins that could be implicated in post-transcriptional mRNA regulation. These included known regulators, degradative enzymes and translation factors, many canonical RNA-binding proteins, and proteins that act via multi-protein complexes. However there were also nearly 150 potential regulators with no previously annotated function, or functions unrelated to mRNA metabolism. Almost 50 novel regulators were shown to bind RNA using a targeted proteome array. The screen also provided fine structure mapping of the hit candidates' functional domains. Our findings not only confirm the key role that RNA-binding proteins play in the regulation of gene expression in trypanosomatids, but also suggest new roles for previously uncharacterized proteins.

Original languageEnglish
Article numbere1004178
JournalPLoS Pathogens
Volume10
Issue number6
DOIs
Publication statusPublished - 2014
Externally publishedYes

Fingerprint

Trypanosoma brucei brucei
Genome
Proteins
RNA-Binding Proteins
RNA Stability
Protein Biosynthesis
3' Untranslated Regions
Messenger RNA
Genomic Library
Gene Expression Regulation
Proteome
Ribonucleases
Plasmids
RNA
Gene Expression
Peptides
Enzymes

ASJC Scopus subject areas

  • Microbiology
  • Parasitology
  • Virology
  • Immunology
  • Genetics
  • Molecular Biology
  • Medicine(all)

Cite this

A Genome-Wide Tethering Screen Reveals Novel Potential Post-Transcriptional Regulators in Trypanosoma brucei. / Erben, Esteban D.; Fadda, Abeer A.; Lueong, Smiths; Hoheisel, Jörg D.; Clayton, Christine.

In: PLoS Pathogens, Vol. 10, No. 6, e1004178, 2014.

Research output: Contribution to journalArticle

Erben, Esteban D. ; Fadda, Abeer A. ; Lueong, Smiths ; Hoheisel, Jörg D. ; Clayton, Christine. / A Genome-Wide Tethering Screen Reveals Novel Potential Post-Transcriptional Regulators in Trypanosoma brucei. In: PLoS Pathogens. 2014 ; Vol. 10, No. 6.
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