Termination codon readthrough of NNAT mRNA regulates calcium-mediated neuronal differentiation.

Termination codon readthrough (TCR) is a process in which ribosomes continue to translate an mRNA beyond a stop codon generating a C-terminally extended protein isoform. Here, we demonstrate TCR in mammalian NNAT mRNA, which encodes NNAT, a proteolipid important for neuronal differentiation. This is a programmed event driven by cis-acting RNA sequences present immediately upstream and downstream of the canonical stop codon and is negatively regulated by NONO, an RNA-binding protein known to promote neuronal differentiation. Unlike the canonical isoform NNAT, we determined that the TCR product (NNATx) does not show detectable interaction with the sarco/endoplasmic reticulum Ca2+-ATPase isoform 2 Ca2+ pump, cannot increase cytoplasmic Ca2+ levels, and therefore does not enhance neuronal differentiation in Neuro-2a cells. Additionally, an antisense oligonucleotide that targets a region downstream of the canonical stop codon reduced TCR of NNAT and enhanced the differentiation of Neuro-2a cells to cholinergic neurons. Furthermore, NNATx-deficient Neuro-2a cells, generated using CRISPR-Cas9, showed increased cytoplasmic Ca2+ levels and enhanced neuronal differentiation. Overall, these results demonstrate regulation of neuronal differentiation by TCR of NNAT. Importantly, this process can be modulated using a synthetic antisense oligonucleotide.

Drug targeting Nsp1-ribosomal complex shows antiviral activity against SARS-CoV-2.

The SARS-CoV-2 non-structural protein 1 (Nsp1) contains an N-terminal domain and C-terminal helices connected by a short linker region. The C-terminal helices of Nsp1 (Nsp1-C-ter) from SARS-CoV-2 bind in the mRNA entry channel of the 40S ribosomal subunit and blocks mRNA entry, thereby shutting down host protein synthesis. Nsp1 suppresses host immune function and is vital for viral replication. Hence, Nsp1 appears to be an attractive target for therapeutics. In this study, we have in silico screened Food and Drug Administration (FDA)-approved drugs against Nsp1-C-ter. Among the top hits obtained, montelukast sodium hydrate binds to Nsp1 with a binding affinity (KD) of 10.8 ± 0.2 µM in vitro. It forms a stable complex with Nsp1-C-ter in simulation runs with -95.8 ± 13.3 kJ/mol binding energy. Montelukast sodium hydrate also rescues the inhibitory effect of Nsp1 in host protein synthesis, as demonstrated by the expression of firefly luciferase reporter gene in cells. Importantly, it shows antiviral activity against SARS-CoV-2 with reduced viral replication in HEK cells expressing ACE2 and Vero-E6 cells. We, therefore, propose montelukast sodium hydrate can be used as a lead molecule to design potent inhibitors to help combat SARS-CoV-2 infection.

Evidence for low-level translation in human erythrocytes.

It is generally believed that human mature erythrocytes do not possess functional ribosomes and therefore cannot synthesize proteins. However, the absence of translation is not consistent with the long lifespan of mature erythrocytes. They stay viable and functional for about 115 d in the circulatory system. Here, using a highly pure preparation of human mature erythrocytes, we demonstrate the presence of translation by polysome profiling, [35S]methionine labeling, and RiboPuromycylation. [35S]methionine labeling revealed that the translation in mature erythrocytes is about 10% of that observed in reticulocytes. We could observe polysomes by transmission electron microscopy in these cells. RNA-seq and quantitative real-time PCR performed on polysome fractions of these cells revealed that HBA (α-globin) and HBB (ß-globin) transcripts are translated. Using a luciferase-based reporter assay and mutational studies, we show that the sequence of the 5' untranslated region is crucial for the translation of these transcripts. Furthermore, mature erythrocytes showed reduced expression of globin proteins (α- and ß-) when treated with translation inhibitors. Overall, we provide multiple lines of evidence for translation of globin mRNAs in human mature erythrocytes.