Comprehensive Identification of mRNA-Binding Proteins of Leishmania donovani by Interactome Capture

PLoS One. 2017 Jan 30;12(1):e0170068. doi: 10.1371/journal.pone.0170068. eCollection 2017.

Abstract

Leishmania are unicellular eukaryotes responsible for leishmaniasis in humans. Like other trypanosomatids, leishmania regulate protein coding gene expression almost exclusively at the post-transcriptional level with the help of RNA binding proteins (RBPs). Due to the presence of polycystronic transcription units, leishmania do not regulate RNA polymerase II-dependent transcription initiation. Recent evidence suggests that the main control points in gene expression are mRNA degradation and translation. Protein-RNA interactions are involved in every aspect of RNA biology, such as mRNA splicing, polyadenylation, localization, degradation, and translation. A detailed picture of these interactions would likely prove to be highly informative in understanding leishmania biology and virulence. We developed a strategy involving covalent UV cross-linking of RBPs to mRNA in vivo, followed by interactome capture using oligo(dT) magnetic beads to define comprehensively the mRNA interactome of growing L. donovani amastigotes. The protein mass spectrometry analysis of captured proteins identified 79 mRNA interacting proteins which withstood very stringent washing conditions. Strikingly, we found that 49 of these mRNA interacting proteins had no orthologs or homologs in the human genome. Consequently, these may represent high quality candidates for selective drug targeting leading to novel therapeutics. These results show that this unbiased, systematic strategy has the promise to be applicable to study the mRNA interactome during various biological settings such as metabolic changes, stress (low pH environment, oxidative stress and nutrient deprivation) or drug treatment.

MeSH terms

  • Gene Ontology
  • Humans
  • Leishmania donovani / metabolism*
  • Mass Spectrometry
  • Protein Domains
  • Protein Interaction Mapping / methods*
  • Proteome / metabolism
  • Protozoan Proteins / chemistry
  • Protozoan Proteins / metabolism*
  • RNA, Messenger / genetics
  • RNA, Messenger / metabolism
  • RNA-Binding Proteins / chemistry
  • RNA-Binding Proteins / metabolism*

Substances

  • Proteome
  • Protozoan Proteins
  • RNA, Messenger
  • RNA-Binding Proteins

Grant support

This work was funded by Grants MOP – 125879 to NER and 77688 to LJF from the Canadian Institutes for Health Research. The funding agencies had no role in design, collection, analysis, or interpretation of data; neither were they involved in writing the manuscript; or in the decision to submit the manuscript for publication.