Identification of the mRNAs associated with the protein complex PuREBP1/PuREBP2 (Tb927.4.4550/Tb927.3.3060) in Trypanosoma brucei [RIP-seq]

We have previously characterized a short RNA stem-loop cis-element within the 3'-UTR of the NT8 nucleobase transporter mRNA (PuRE, Purine Responsive Element) that is necessary and sufficient to confer a strong repression of gene expression in response to purines in the human parasite Trypanosoma brucei (PMID: 24813448). In this study, we have identified a protein complex composed of two RNA-binding proteins (Tb927.4.4550 and Tb927.3.3060, named here as PuREBP1 and PuREBP2 respectively) that binds to the PuRE in vitro and to NT8 mRNA in vivo. Depletion of PuREBP1 by RNAi results in the upregulation of just NT8 and the mRNAs encoding the amino acid transporter AATP6 paralogues. Moreover, we found that the PuREBP1/2 complex is associated with only a handful of mRNAs, and that it is responsible for the observed purine-dependent regulation of NT8 expression. Just a handful of mRNAs are associated with the PuREBP1/2 complex: NT8, AATP6, mCUd and other mRNAs encoding proteins of unknown function, mainly membrane proteins Overall design: Tandem-affinity purification (TAP) was used to identify mRNAs associated with the complex. Input RNA vs affinity-purified RNA were compared. As a control, TAP was performed with a functionally-inactive PuREBP1/2 complex