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

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.

Drug Target Identification Using a Trypanosome Overexpression Library

“…In the course of another project, we created a library of trypanosomes, each of which is capable of overexpressing a different protein fragment in a tetracycline-inducible fashion (15). The overexpression library was constructed using randomly sheared DNA (sizes of 0.7 to 3 kbp) ligated into a plasmid suitable for tetracycline-inducible expression (Fig.…”

“…Authentic trypanosome proteins will be expressed as lambda-N fusion peptides if the insert sequence contains a trypanosome open reading frame (ORF) that is in frame and in the correct orientation. High-throughput sequencing results suggested that approximately one in 25 plasmids fulfilled this criterion, meaning that the library has at least 10-fold coverage of Trypanosoma brucei ORFs (15).…”

“…We transfected the plasmid library DNA into bloodstreamform T. brucei 2T1 cells, taking advantage of endonuclease-facilitated recombination (16), to obtain over 3 million clones (15). Since this is a random shotgun library, most plasmids do not encode full-length trypanosome proteins, and the presence of the N-terminal peptide precludes targeting of proteins to the secretory pathway or mitochondria.…”

Regulatory mechanisms of Leishmania Aquaglyceroporin AQP1

“…,2011) . In trypanosomatids, although indirect results suggest that some proteins cause degradation of target transcripts, this has never been shown directly (Erben et al, 2014).…”

“…Perhaps the best characterized family of instability determinants is the A+U rich elements (ARE) in the 3'-UTR, which are known to regulate a variety of transcripts via mRNA degradation and/or translational repression (Knapinska et al, 2011). Some known AUBPs (A + U binding proteins) are AUF1/hnRNP (heterogeneous nuclear ribonucleoprotein), TTP, BRF1 (Butyrate response factor), KSRP (KH-type splicing regulatory protein), TIA-1 (Tcell internal antigen 1), and TIAR (TIA-1 related protein) (Barreau et al, 2005 although indirect results suggest that some proteins cause degradation of target transcripts, this has never been shown directly (Erben et al, 2014). Since AQP1 mRNA in the VL species is more unstable compared to the Cutaneous leishmaniasis species, and this phenomenon is driven by their respective 3'-UTRs, we analyzed the predicted 3'-UTR RNA structures of the most resistant (L. infantum) and the most sensitive (L.…”