GSE193394
The RRM-mediated RNA binding activity in T. brucei RAP1 is essential for VSG monoallelic expression
Trypanosoma brucei is a protozoan parasite that causes human African trypanosomiasis. Its major surface antigen VSG is expressed from subtelomeric loci in a strictly monoallelic manner. We previously showed that the telomere protein TbRAP1 binds dsDNA through its 737RKRRR741 patch to silence VSGs globally. How TbRAP1 permits transcription of the single active VSG is unknown. Through NMR structural analysis, we unexpectedly identify an RNA Recognition Motif (RRM) in TbRAP1, which is unprecedented for RAP1 homologs. Assisted by the 737RKRRR741 patch, TbRAP1 RRM recognizes consensus sequences of VSG 3'UTRs in vitro and binds the active VSG RNA in vivo. Mutating conserved RRM residues abolishes the RNA binding activity, significantly decreases the active VSG RNA level, and derepresses silent VSGs. The competition between TbRAP1's RNA and dsDNA binding activities suggests a novel mechanism of monoallelic expression, in which the active VSG's abundant RNA antagonizes TbRAP1's silencing effect, thereby sustaining its full-level transcription. Overall design: High-throughput paired-end RNA sequencing on the Illumina platform was performed. Mutant T. brucei strain has one WT TbRAP1 allele flanked by loxP repeats (floxed) and a FLAG-HA-HA and an SV40 NLS- tagged TbRAP1-2FA&5A or a FLAG-HA-HA tagged TbRAP1-2FQ allele. Control T. brucei strain has one floxed and a WT TbRAP1 allele. Both mutant and control cells were induced for Cre induction for 30 hrs (to remove the floxed TbRAP1 allele) before total RNA was isolated. Three independent induction were performed for both mutants. RNA samples were sent to Novogene for subsequent experiments. Poly(A) RNA was purified for generating cDNA library and paired-end sequencing was done using Illumina. Gene expression profiles were compared between the mutant and control cells by differential gene expression analysis.