MSI1 Knockout Cell Line (HEK293)

Musashi RNA-binding proteins are important post-transcriptional regulators of stem cell homeostasis and are known to be involved in viral infections. However, their role in SARS-CoV-2 infection remains largely unknown. Using computational studies, in vivo RNA immunoprecipitation, and biochemical assays, here, we establish that Musashi 1 (Msi1) interacts with viral genomic RNA through direct binding to the SARS-CoV-2 3'UTR. Importantly, binding of Msi1 to the viral 3'UTR results in translational repression that could be mediated by inhibition of poly(A) binding protein. Conversely, Msi1 knockout promotes robust viral replication and increased viral protein expression. Using 2D cell cultures, stem cells, and 3D organoids, we show that depletion of Msi1 in intestinal cells augments infection. This finding explains why the human intestine serves as a reservoir for SARS-CoV-2, in which differentiated enterocytes with negligible Msi1 levels are particularly affected. Contrarily, stem cells, which are enriched for Msi1 expression, are known to be less permissive to SARS-CoV-2 infection despite expressing the entry receptors. Our findings show how translational repression of SARS-CoV-2 by stem cell RNA-binding proteins, such as Msi1, could help evade infection.

Thyroid hormone (TH) is a critical regulator of cellular function and cell fate. The circulating TH level is relatively stable, while tissue TH action fluctuates according to cell type-specific mechanisms. Here, we focused on identifying mechanisms that regulate TH action through the type 2 deiodinase (D2) in glial cells. Dio2 mRNA has an unusually long 3'UTR where we identified multiple putative MSI1 binding sites for Musashi-1 (MSI1), a highly conserved RNA-binding cell cycle regulator. Binding to these sites was confirmed through electrophoretic mobility shift assay. In H4 glioma cells, shRNA-mediated MSI1 knockdown increased endogenous D2 activity, whereas MSI1 overexpression in HEK293T cells decreased D2 expression. This latter effect could be prevented by the deletion of a 3.6 kb region of the 3'UTR of Dio2 mRNA containing MSI1 binding sites. MSI1 immunoreactivity was observed in 2 mouse Dio2-expressing cell types, that is, cortical astrocytes and hypothalamic tanycytes, establishing the anatomical basis for a potential in vivo interaction of Dio2 mRNA and MSl1. Indeed, increased D2 expression was observed in the cortex of mice lacking MSI1 protein. Furthermore, MSI1 knockdown-induced D2 expression slowed down cell proliferation by 56% in primary cultures of mouse cortical astrocytes, establishing the functionality of the MSI1-D2-T3 pathway. In summary, Dio2 mRNA is a target of MSI1 and the MSI1-D2-T3 pathway is a novel regulatory mechanism of astrocyte proliferation with the potential to regulate the pathogenesis of human glioblastoma.