Mutually exclusive amino acid residues of L13a are responsible for its ribosomal incorporation and translational silencing leading to resolution of inflammation

Abstract

Eukaryotic ribosomal protein L13a is a member of the conserved universal ribosomal uL13 protein family. Structurally, L13a is distinguished from its prokaryotic counterparts by the presence of a ~55 amino acid-long C-terminal α-helical extension. The importance of these evolved residues in the C-terminal extension for mammalian ribosome biogenesis as well as L13a’s extra-ribosomal function in GAIT (gamma interferon-activated inhibitor of translation) complex-mediated translation silencing during inflammation is not understood. Here, we present biochemical analyses of L13a mutant variants identifying several mutually exclusive amino acid residues in the eukaryote-specific C-terminal extension of human L13a (Tyr149-Val203) important for ribosomal incorporation and translational silencing. Specifically, we show that mutation of Arg169, Lys170 and Lys171 to Ala abrogate GAIT-mediated translational silencing, but not L13a incorporation into ribosomes. Moreover, we show that the C-terminal helix alone can silence translation of GAIT element-containing mRNAs in vitro. We also show through cellular immunofluorescence experiments that nuclear but not nucleolar localization of L13a is resistant to extensive amino acid alterations, suggesting that multiple complex nuclear import signals are present within this protein. These studies provide new insights into L13a structure and its ribosomal and extra-ribosomal functions in model human cells.