Gel shift experiments with fragments of the Trypanosoma brucei RNA-binding protein RBP10

OBJECTIVE: Trypanosoma brucei is a parasite of mammals and Tsetse flies, and control of mRNA stability is critical for parasite survival in the two different hosts. T. brucei RBP10 is a protein with a single RNA Recognition Motif (RRM) which is expressed only in the mammalian (bloodstream) form. Numerous observations suggest that RBP10 binds to procyclic-specific mRNAs and targets them for destruction, and there is also some evidence for selective binding of RBP10 to RNAs containing the motif UA(U)(6). We here investigated this binding further. RESULTS: We tested in vitro binding of RBP10 to two different probes in solution. One contained two copies of UA(U)(6), and the other two copies of a mutant version, UACUCUCU, which is inactive in regulation. An N-terminal segment of RBP10, including the RRM domain and 90 residues to its C-terminus, could be produced as soluble protein. This could bind both probes in vitro with similar affinities in the low micromolar range, which is not atypical for a single RRM. Soluble RBP10 therefore did not distinguish between UA(U)(6) and UACUCUCU. Since no other sequences were tested, the requirements for RBP10 RNA binding remain to be determined.