Characterization of the human FASTKD4 post-transcriptional regulator

Mitochondria are multifunctional organelles responsible for vital biological processes such as energy production and programmed cell death. Mitochondrial plasticity and homeostasis are largely orchestrated by nucleic acid binding proteins tasked with regulating the mitochondrial genome, transcriptome, and proteome. Tight control of mitochondrial RNA processing events is especially important since aberrant mitochondrial RNA causes a variety of pathologies including cancer. The FASTK protein family are an emerging class of mitochondrial post-transcriptional regulators linked to diverse mitochondrial RNA processing events including RNA maturation, modification, and turnover. The FASTKD4 member is functionally linked to RNA maturation at non-canonical junctions, an important RNA processing event that liberates select RNA transcripts from its polycistronic RNA precursor. Despite its central role in RNA maturation and mitochondrial activity, FASTKD4 function and regulation remains unclear. This knowledge gap is largely due to the complete absence of biophysical and biochemical studies describing the molecular properties of FASTKD4. To address this hurdle, we reconstituted human FASTKD4 and launched a comprehensive structure-function program to identify its intrinsic molecular properties. We performed size exclusion chromatography to determine FASTKD4 is monomeric in solution. Using a quantitative fluorescence-based assay, we establish that FASTKD4 has a strong preference for single stranded RNA and correlated binding affinity with substrate length. Together, these data begin to elucidate the molecular principles driving FASTKD4's function in mitochondrial RNA maturation.