Fig. 1: TbRAP1 binds the active VSG RNA in vivo and contains an RNA Recognition Motif (RRM) domain.
From: The RRM-mediated RNA binding activity in T. brucei RAP1 is essential for VSG monoallelic expression
a RNA CLIP experiments were performed in TbRAP1F2H+/- cells that express VSG2. qRT-PCR was performed to estimate the amount of the VSG2 RNA and the TbTERT, SNAP50, and PKAC1 RNAs in the RNA CLIP product. Enrichment of the VSG2, TbTERT, SNAP50, and PKAC1 RNAs (CLIP/Input) was calculated for the CLIP experiment using the HA antibody 12CA5 and that using IgG. Relative enrichment was calculated using the enrichment of IgG CLIP as a reference. Average and standard deviation were calculated from three (SNAP50 & PKAC1), five (TbTERT), and seventeen (VSG2) independent experiments. P values of two-sided unpaired t-tests (compared to VSG2 RNA enrichment) are shown. b RNA CLIP was performed in VSG9-expressing PVS3-2/OD1-1 cells using a TbRAP1 rabbit antibody15 and IgG and the enrichment of the VSG9 RNA in the CLIP product was calculated. Average and standard deviation were calculated from three independent experiments. Error bars represent standard deviation. Source data are provided as a Source Data file. c Domain structure of TbRAP1. Inset, an enlarged diagram of the TbRAP1 MybLike domain (aa 639–761)15, which contains an RRM (aa 653–727) and the DNA Binding (DB) domain (aa 734–761)18. Arrowheads mark the conserved F655 and F694 residues. d Superposition of TbRAP1653-727 (green) with the RRM1 domain of hnRNP A1 (orange) bound with a short RNA oligo (golden) [https://doi.org/10.2210/pdb5MPG/pdb]28. Inset highlights that F655 and F694 in TbRAP1 superimpose well with F17 and F59 in hnRNP A1 that form stacking interactions with the RNA substrate.
