FASTKD5 processes mitochondrial pre-mRNAs at noncanonical cleavage sites

Area of Science:

• Mitochondrial biology
• Gene expression
• Molecular genetics

Background:

• Mitochondrial gene expression involves post-transcriptional processing of polycistronic transcripts.
• Processing of specific noncanonical messenger RNAs (mRNAs) requires the protein FASTKD5.
• Defects in mitochondrial gene expression lead to OXPHOS assembly defects.

Purpose of the Study:

• To investigate the molecular mechanism of FASTKD5 in mitochondrial RNA processing.
• To identify the role of FASTKD5 in the synthesis of mitochondrial-encoded polypeptides.
• To understand the substrate specificity and catalytic activity of FASTKD5.

Main Methods:

• Creation of knockout human cell lines lacking FASTKD5.
• Analysis of OXPHOS assembly and mRNA translation in knockout cells.
• In vitro reconstitution assays with wild-type and mutant FASTKD5.

Main Results:

• Absence of FASTKD5 caused severe OXPHOS defects due to unprocessed mRNAs (CO1, CO3, CYB).
• FASTKD5 exhibits RNA substrate specificity, with a key active site residue crucial for processing all three noncanonical pre-mRNAs.
• Mutating the active site residue did not affect RNA binding but abolished cleavage activity.

Conclusions:

• FASTKD5 is the essential enzyme for the complete post-transcriptional processing of mitochondrial transcripts.
• FASTKD5 is critical for the translation of mitochondrial-encoded proteins and proper OXPHOS function.
• The study elucidates the biochemical mechanism of FASTKD5 in mitochondrial gene expression.