Identification of EPRS domains required for formation of the GAIT mRNP: Key roles of WHEP‐TRS repeats

Glutamyl‐prolyl‐tRNA synthetase (EPRS) is an aminoacyl‐tRNA synthetase (AARS) that catalyzes ligation of Glu and Pro to cognate tRNAs. EPRS contains two catalytic domains separated by a linker of 3 tandem WHEP‐TRS helix‐turn‐helix domains. Single WHEP‐TRS domains are found in four other chordate AARS, but not in any other proteins. WHEP‐TRS domains do not contribute significantly to aminoacylation, suggesting a role in non‐canonical AARS activities. The domains bind weakly to diverse RNAs, but their specific function is unknown. We have reported that EPRS is a component of the GAIT (IFN‐ G amma‐ A ctivated I nhibitor of T ranslation) translation silencing complex. Following U937 cell activation by IFN‐gamma, EPRS is phosphorylated and released from the tRNA multisynthetase complex. Released EPRS joins NSAP1 and two other proteins to form the GAIT complex, which binds the 3′‐UTR GAIT element of ceruloplasmin mRNA and silences its translation. Here we elucidate the EPRS domains responsible for GAIT mRNP formation. Recombinant EPRS linker containing 3 WHEP‐TRS repeats binds the GAIT element specifically and with high affinity (Kd ~5 nM) as shown by BIAcore spectroscopy. Deletion analysis showed that the GAIT RNA element binds WHEP‐TRS repeats 1 and 2. In contrast, NSAP1 binds WHEP‐TRS repeats 2 and 3. Differential binding of the repeats to protein and RNA was unexpected given their high sequence similarity. Our results are the first to show a specific function for a WHEP‐TRS domain. They suggest that EPRS WHEP‐TRS repeats function in formation of the GAIT mRNP and in inflammation‐responsive, transcript‐selective translation silencing. Supported by NIH P01 HL29582.