Loss of SNORA73 reprograms cellular metabolism and protects against steatohepatitis

Dyslipidemia and resulting lipotoxicity are pathologic signatures of metabolic syndrome and type 2 diabetes. Excess lipid causes cell dysfunction and induces cell death through pleiotropic mechanisms that link to oxidative stress. However, pathways that regulate the response to metabolic stress are not well understood. Herein, we show that disruption of the box H/ACA SNORA73 small nucleolar RNAs encoded within the small nucleolar RNA hosting gene 3 (Snhg3) causes resistance to lipid-induced cell death and general oxidative stress in cultured cells. This protection from metabolic stress is associated with broad reprogramming of oxidative metabolism that is dependent on the mammalian target of rapamycin signaling axis. Furthermore, we show that knockdown of SNORA73 in vivo protects against hepatic steatosis and lipid-induced oxidative stress and inflammation. Our findings demonstrate a role for SNORA73 in the regulation of metabolism and lipotoxicity. Overall design: All experimental procedures were approved by the Washington University and Joslin Diabetes Center Animal Studies Committees and conducted in accordance with the Public Health Service Policy for the Humane Care and Use of Laboratory Animals. Male C57BL/6J mice were obtained from the Jackson Laboratory (JAX 000664). For in vivo knockdown experiments, locked nucleic acids (LNA, Qiagen) targeting SNORA73 or GFP (control) were injected intraperitoneally into 8-week-old mice at a dose of 3 mg/kg every other day for 3 injections. Animals were euthanized 2 days after the final injection for analyses. Input RNA for transcriptome studies was recovered from liver tissue lysed in polysome buffer using Trizol LS and RNeasy mini kit (Qiagen) including on column DNase digestion. For translatome studies, RNA was recovered from corresponding polysome fractions by Trizol-LS extraction, followed by chloroform-isopropanol-ethanol precipitation, heparinase treatment (New England Biolabs, 15 minutes, 37°C), and RNA cleanup step using RNA Clean & Concentrator (Zymo). Quality of RNA samples was assessed by Agilent Bioanalyzer. Sequencing libraries were prepared using Kapa mRNA Hyper Prep (Roche) according to the manufacturer's protocol. Briefly, total RNA samples (100-1000 ng) were polyA-selected and reverse transcribed into double-stranded cDNA. High-throughput sequencing was performed using a NovaSeq 6000 Instrument (Illumina) at The Molecular Biology Core Facilities (MBCF) at Dana-Farber Cancer institute (DFCI). RNA-seq raw reads were 100bp reversely stranded paired-end reads.