Search for: All records

Abstract Ribonuclease (RNase) MRP is a conserved RNA-based enzyme best known for its essential role in the maturation of ribosomal RNA (rRNA) in eukaryotes. However, the composition and RNA substrate specificity of this multisubunit ribonucleoprotein complex in higher eukaryotes remain a mystery. Here, we identify NEPRO and C18ORF21 (which we renamed RMP64 and RMP24, respectively) as constitutive subunits of metazoan RNase MRP. These proteins are unique to RNase MRP and absent from the closely related RNase P, which processes transfer RNA (tRNA) precursors and tRNA-like substrates. We find that RMP64 and RMP24 are integral subunits of RNase MRP, stabilize its catalytic RNA, and are required for rRNA maturation and cell proliferation. Leveraging these discoveries, we identify a broad suite of in vivo RNA-binding targets of each enzyme, including potential cleavage sites at nucleotide resolution. Our findings identify the first metazoan RNase MRP-specific protein subunits and define the RNA-targeting repertoire of this essential enzyme in mammalian cells. 

Abstract In oxygen (O₂)‐controlled cell culture, an indispensable tool in biological research, it is presumed that the incubator setpoint equals the O₂tension experienced by cells (i.e., pericellular O₂). However, it is discovered that physioxic (5% O₂) and hypoxic (1% O₂) setpoints regularly induce anoxic (0% O₂) pericellular tensions in both adherent and suspension cell cultures. Electron transport chain inhibition ablates this effect, indicating that cellular O₂consumption is the driving factor. RNA‐seq analysis revealed that primary human hepatocytes cultured in physioxia experience ischemia‐reperfusion injury due to cellular O₂consumption. A reaction‐diffusion model is developed to predict pericellular O₂tension a priori, demonstrating that the effect of cellular O₂consumption has the greatest impact in smaller volume culture vessels. By controlling pericellular O₂tension in cell culture, it is found that hypoxia vs. anoxia induce distinct breast cancer transcriptomic and translational responses, including modulation of the hypoxia‐inducible factor (HIF) pathway and metabolic reprogramming. Collectively, these findings indicate that breast cancer cells respond non‐monotonically to low O₂, suggesting that anoxic cell culture is not suitable for modeling hypoxia. Furthermore, it is shown that controlling atmospheric O₂tension in cell culture incubators is insufficient to regulate O₂in cell culture, thus introducing the concept of pericellular O₂‐controlled cell culture.