Translation regulation of specific mRNAs by RPS26 C-terminal RNA-binding tail integrates energy metabolism and AMPK-mTOR signaling.

Increasing evidence suggests that ribosome composition and modifications contribute to translation control. Whether direct mRNA binding by ribosomal proteins regulates the translation of specific mRNA and contributes to ribosome specialization has been poorly investigated. Here, we used CRISPR-Cas9 to mutate the RPS26 C-terminus (RPS26dC) predicted to bind AUG upstream nucleotides at the exit channel. RPS26 binding to positions -10 to -16 of short 5' untranslated region (5'UTR) mRNAs exerts positive and negative effects on translation directed by Kozak and Translation Initiator of Short 5'UTR (TISU), respectively. Consistent with that, shortening the 5'UTR from 16 to 10 nt diminished Kozak and enhanced TISU-driven translation. As TISU is resistant and Kozak is sensitive to energy stress, we examined stress responses and found that the RPS26dC mutation confers resistance to glucose starvation and mTOR inhibition. Furthermore, the basal mTOR activity is reduced while AMP-activated protein kinase is activated in RPS26dC cells, mirroring energy-deprived wild-type (WT) cells. Likewise, the translatome of RPS26dC cells is correlated to glucose-starved WT cells. Our findings uncover the central roles of RPS26 C-terminal RNA binding in energy metabolism, in the translation of mRNAs bearing specific features and in the translation tolerance of TISU genes to energy stress.

Cap-independent translation and a precisely located RNA sequence enable SARS-CoV-2 to control host translation and escape anti-viral response.

Translation of SARS-CoV-2-encoded mRNAs by the host ribosomes is essential for its propagation. Following infection, the early expressed viral protein NSP1 binds the ribosome, represses translation, and induces mRNA degradation, while the host elicits an anti-viral response. The mechanisms enabling viral mRNAs to escape this multifaceted repression remain obscure. Here we show that expression of NSP1 leads to destabilization of multi-exon cellular mRNAs, while intron-less transcripts, such as viral mRNAs and anti-viral interferon genes, remain relatively stable. We identified a conserved and precisely located cap-proximal RNA element devoid of guanosines that confers resistance to NSP1-mediated translation inhibition. Importantly, the primary sequence rather than the secondary structure is critical for protection. We further show that the genomic 5'UTR of SARS-CoV-2 drives cap-independent translation and promotes expression of NSP1 in an eIF4E-independent and Torin1-resistant manner. Upon expression, NSP1 further enhances cap-independent translation. However, the sub-genomic 5'UTRs are highly sensitive to eIF4E availability, rendering viral propagation partially sensitive to Torin1. We conclude that the combined NSP1-mediated degradation of spliced mRNAs and translation inhibition of single-exon genes, along with the unique features present in the viral 5'UTRs, ensure robust expression of viral mRNAs. These features can be exploited as potential therapeutic targets.

Assessment of Sacsin Turnover in Patients With ARSACS: Implications for Molecular Diagnosis and Pathogenesis.

BACKGROUND AND OBJECTIVES: Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is caused by variations in SACS gene encoding sacsin, a huge multimodular protein of unknown function. More than 200 SACS variations have been described worldwide to date. Because ARSACS presents phenotypic variability, previous empirical studies attempted to correlate the nature and position of SACS variations with the age at onset or with disease severity, although not considering the effect of the various variations on protein stability. In this work, we studied genotype-phenotype correlation in ARSACS at a functional level. METHODS: We analyzed a large set of skin fibroblasts derived from patients with ARSACS, including both new and already published cases, carrying variations of different types affecting diverse domains of the protein. RESULTS: We found that sacsin is almost absent in patients with ARSACS, regardless of the nature of the variation. As expected, we did not detect sacsin in patients with truncating variations. We found it strikingly reduced or absent also in compound heterozygotes carrying diverse missense variations. In this case, we excluded SACS mRNA decay, defective translation, or faster posttranslational degradation as possible causes of protein reduction. Conversely, our results demonstrate that nascent mutant sacsin protein undergoes cotranslational ubiquitination and degradation. DISCUSSION: Our results provide a mechanistic explanation for the lack of genotype-phenotype correlation in ARSACS. We also propose a new and unambiguous criterion for ARSACS diagnosis that is based on the evaluation of sacsin level. Last, we identified preemptive degradation of a mutant protein as a novel cause of a human disease.