Table_1_Effects of Hydrogen Peroxide Stress on the Nucleolar Redox Environment and Pre-rRNA Maturation.DOCX

Identifying biologically relevant molecular targets of oxidative stress may provide new insights into disease mechanisms and accelerate development of novel biomarkers. Ribosome biogenesis is a fundamental prerequisite for cellular protein synthesis, but how oxidative stress affects ribosome biogenesis has not been clearly established. To monitor and control the redox environment of ribosome biogenesis, we targeted a redox-sensitive roGFP reporter and catalase, a highly efficient H₂O₂ scavenger, to the nucleolus, the primary site for transcription and processing of rRNA in eukaryotic cells. Imaging of mouse 3T3 cells exposed to non-cytotoxic H₂O₂ concentrations revealed increased oxidation of the nucleolar environment accompanied by a detectable increase in the oxidative damage marker 8-oxo-G in nucleolar RNA. Analysis of pre-rRNA processing showed a complex pattern of alterations in pre-rRNA maturation in the presence of H₂O₂, including inhibition of the transcription and processing of the primary 47S transcript, accumulation of 18S precursors, and inefficient 3′-end processing of 5.8S rRNA. This work introduces new tools for studies of the redox biology of the mammalian nucleolus and identifies pre-rRNA maturation steps sensitive to H₂O₂ stress.