Poly(A) binding KPAF4/5 complex stabilizes kinetoplast mRNAs in Trypanosoma brucei

Area of Science:

• Molecular Biology
• Parasitology
• RNA Biology

Background:

• Mitochondrial pre-mRNAs in Trypanosoma brucei require extensive processing, including 3' modifications and editing.
• Pentatricopeptide repeat (PPR) Kinetoplast Polyadenylation Factors (KPAFs) regulate these 3' modifications.
• Previous work established roles for KPAF3, KPAF4, and the KPAF1/2 dimer in transcript stabilization and polyadenylation.

Purpose of the Study:

• To elucidate the role of KPAF4 in complex with KPAF5 during mitochondrial mRNA processing.
• To investigate the function of the KPAF4/5 heterodimer in poly(A) tail recognition and mRNA stabilization.
• To explore the interaction between 5' and 3' end processing complexes in mitochondrial mRNA quality control.

Main Methods:

• Co-immunoprecipitation to identify KPAF complexes.
• RNA binding assays to assess poly(A) tail recognition.
• In vitro assays to study mRNA processing and stabilization.
• Crosslinking and immunoprecipitation (CLIP) to map RNA-protein interactions.

Main Results:

• KPAF4 forms a heterodimer with KPAF5, a protein lacking known RNA-binding motifs.
• The KPAF4/5 heterodimer specifically binds to poly(A) tails, stabilizing them during RNA editing.
• This interaction prevents premature translational activation of partially edited transcripts.
• Evidence suggests an RNA editing substrate binding complex bridges the 5' PPsome and 3' polyadenylation complexes, potentially mediating mRNA circularization.

Conclusions:

• The KPAF4/5 heterodimer acts as a crucial poly(A) binding element within the mitochondrial polyadenylation complex.
• This complex plays a vital role in stabilizing mitochondrial mRNAs during the editing process.
• Interactions between 5' and 3' processing complexes may facilitate mRNA circularization, contributing to mitochondrial mRNA stability and quality control.