Native mitochondrial RNA-binding complexes in kinetoplastid RNA editing differ in guide RNA composition.

Area of Science:

• * Molecular Biology
• * Genetics
• * Biochemistry

Background:

• * Kinetoplastid mitochondrial mRNAs undergo extensive U-insertion/deletion RNA editing.
• * This editing process is guided by guide RNAs (gRNAs) and involves RNA editing core complexes (RECC).
• * The mitochondrial RNA-binding complex 1 (MRB1), also known as gRNA-binding complex (GRBC), plays a role in RNA editing through mechanisms yet to be fully elucidated.

Purpose of the Study:

• * To investigate the composition and function of native subcomplexes of the MRB1 complex.
• * To understand how MRB1 interacts with other mitochondrial factors involved in RNA editing.
• * To explore the role of MRB1 in substrate specificity and developmental stage-specificity of RNA editing.

Main Methods:

• * Next-generation sequencing of native MRB1 subcomplexes immunoselected using antibodies against RNA helicase 2 (REH2) or MRB3010.
• * RNA photo-crosslinking to identify associated proteins.
• * Analysis of gRNA and mRNA association within purified MRB1 complexes.

Main Results:

• * MRB1 subcomplexes contain either REH2 or MRB3010, along with shared core proteins like GAP1.
• * The MRB3010-containing complex is enriched for initiating gRNAs, suggesting a role in early editing steps.
• * MRB1 particles copurify with both edited and pre-edited mRNAs, indicating binding to editing substrates.

Conclusions:

• * Multiple MRB1 subcomplexes with distinct compositions likely act as scaffolds for assembling the RNA editing holoenzyme.
• * The MRB3010-subcomplex may facilitate early RNA editing stages by recruiting specific gRNAs.
• * Fast evolution of mRNA 3' ends and strain-specific editing impact mitochondrial physiology.