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. Much less is known about the regulatory roles of mRNA binding by ribosomal proteins to specific mRNA translation and ribosome specialization. Here we used CRISPR-Cas9, to mutate the RPS26 C-terminal tail (RPS26dC) predicted to bind AUG upstream nucleotides at the exit channel. RPS26 binding to positions -10 to -16 of short 5'UTR mRNAs exerts positive and negative effects on translation directed by Kozak and 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 AMPK is activated in RPS26dC cells, mirroring energy-deprived 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. Overall design: We used CRISPR-Cas9 mediated mutagenesis in mammalian cells to delete endogenous RPS26 C terminus (RPS26dC cells). For global translation analysis, the collected samples were merged to create the Light (2-5 ribosomes) and Heavy (6+ ribosomes) translated fractions. Equivalent RNA concentrations of Input, Light and Heavy translated ribosome were taken for the library preparation for MARS-Seq.