An unconventional RNA-binding protein defines the mRNA-fate of the major variant surface antigen in Trypanosoma brucei

ELIEZER CRUZ; M. DOS NASCIMENTO ; EGLER, F.; ARNOLD, K. ; CLAYTON, C.; ESTEBAN ERBEN

Colonia del Sacramento

Congreso; 1er Encuentro Binacional de Clubes del ARN de Argentina y Uruguay: Prof. Otto Pritsch; 2022

Club del ARN del Uruguay

Trypanosoma brucei is the causative agent of human sleeping sickness. The parasites’ variant surface glycoprotein (VSG) enables them to evade adaptive immunity via antigenic variation. VSG comprises almost 10% of total mRNA and the high stability of VSG mRNA is essential for trypanosome survival. To determine how VSG mRNA stability is maintained, we used mRNA affinity purification to identify all its associated proteins. CFB2 (Cyclin F-box protein 2), an unconventional RBP, was specifically enriched with VSG mRNA. Further investigation demonstrated that, via an unknown RNA binding domain, CFB2 recognizes a conserved 16mer element that is found in the 3´-UTRs of all VSG mRNAs. Moreover, we could demonstrate the mechanism by which CFB2 acts: recruitment of a stabilizing complex that includes MKT1, PBP1, PABP2, and the cap-binding translation initiation complex EIF4E6/G5. CFB2 has a putative RNA-binding domain (RRM) surrounded by an N-terminal cyclin-F-box domain and a C-terminal MKT1-interaction linear motif. F-box proteins are found in SCF complexes (SKP1-Cullin-F-Box), which are E3 ligase components of the ubiquitination machinery. Our results suggested that the interaction of CFB2 with SKP1 promotes CFB2 degradation and that such interaction is required for CFB2 activity. CFB2 variants containing mutated or deleted F-Box domains are unable to promote reporter expression. Intriguingly, a CFB2 variant lacking conserved residues in the RBD domain is still active. To provide new insights into the mechanism by which CFB2 binds to VSG mRNA while being actively degraded, we adopted a random mutagenesis approach driven by error prone PCR to generate a library of CFB2 mutants. The library contains a ‘faithful’ C terminus and a mutagenized central region spanning the F-Box and RRM domains. Once transfected into cells expressing a reporter encoding a lethal gene under the control of the VSG 3′-untranslated region, plasmid inserts will be amplified from survival mutants unable to increase reporter expression and sequenced. Our approach not only will provide novel insights into the singular CFB2 RNA-binding activity but also could shed light into how SCF-complex formation and nucleic acid binding activity are coordinated.