The cell type-specific ER membrane protein UGS148 is not essential in mice.

Genomes of higher eukaryotes encode many uncharacterized proteins, and the functions of these proteins cannot be predicted from the primary sequences due to a lack of conserved functional domains. In this study, we focused on a poorly characterized protein UGS148 that is highly expressed in a specialized cell type called tanycytes that line the ventral wall of the third ventricle in the hypothalamus. Immunostaining of UGS148 revealed the fine morphology of tanycytes with highly branched apical ER membranes. Immunoprecipitation revealed that UGS148 associated with mitochondrial ATPase at least in vitro, and ER and mitochondrial signals occasionally overlapped in tanycytes. Mutant mice lacking UGS148 did not exhibit overt phenotypes, suggesting that UGS148 was not essential in mice reared under normal laboratory conditions. We also found that RNA probes that were predicted to uniquely detect UGS148 mRNA cross-reacted with uncharacterized RNAs, highlighting the importance of experimental validation of the specificity of probes during the hybridization-based study of RNA localization.

Establishment of a Rapid Detection System for ISG20-Dependent SARS-CoV-2 Subreplicon RNA Degradation Induced by Interferon-α.

Inhaled nebulized interferon (IFN)-α and IFN-ß have been shown to be effective in the management of coronavirus disease 2019 (COVID-19). We aimed to construct a virus-free rapid detection system for high-throughput screening of IFN-like compounds that induce viral RNA degradation and suppress the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We prepared a SARS-CoV-2 subreplicon RNA expression vector which contained the SARS-CoV-2 5'-UTR, the partial sequence of ORF1a, luciferase, nucleocapsid, ORF10, and 3'-UTR under the control of the cytomegalovirus promoter. The expression vector was transfected into Calu-3 cells and treated with IFN-α and the IFNAR2 agonist CDM-3008 (RO8191) for 3 days. SARS-CoV-2 subreplicon RNA degradation was subsequently evaluated based on luciferase levels. IFN-α and CDM-3008 suppressed SARS-CoV-2 subreplicon RNA in a dose-dependent manner, with IC50 values of 193 IU/mL and 2.54 µM, respectively. HeLa cells stably expressing SARS-CoV-2 subreplicon RNA were prepared and treated with the IFN-α and pan-JAK inhibitor Pyridone 6 or siRNA-targeting ISG20. IFN-α activity was canceled with Pyridone 6. The knockdown of ISG20 partially canceled IFN-α activity. Collectively, we constructed a virus-free rapid detection system to measure SARS-CoV-2 RNA suppression. Our data suggest that the SARS-CoV-2 subreplicon RNA was degraded by IFN-α-induced ISG20 exonuclease activity.