LARP4 interacts with poly(A)-binding protein (PABP) to protect messenger RNAs (mRNAs) from deadenylation and decay, and recent data indicate it can direct the translation of functionally related mRNA subsets. LARP4 was known to bind RACK1, a ribosome-associated protein, although the specific regions involved and relevance had been undetermined. Here, through a combination of in-cell and in vitro methodologies, we identified positions 615-625 in conserved region-2 (CR2) of LARP4 (and 646-656 in LARP4B) as directly binding RACK1. Consistent with these results, AlphaFold2-Multimer predicted high-confidence interaction of CR2 with RACK1 propellers 5 and 6. CR2 mutations strongly decreased LARP4 association with cel... More
LARP4 interacts with poly(A)-binding protein (PABP) to protect messenger RNAs (mRNAs) from deadenylation and decay, and recent data indicate it can direct the translation of functionally related mRNA subsets. LARP4 was known to bind RACK1, a ribosome-associated protein, although the specific regions involved and relevance had been undetermined. Here, through a combination of in-cell and in vitro methodologies, we identified positions 615-625 in conserved region-2 (CR2) of LARP4 (and 646-656 in LARP4B) as directly binding RACK1. Consistent with these results, AlphaFold2-Multimer predicted high-confidence interaction of CR2 with RACK1 propellers 5 and 6. CR2 mutations strongly decreased LARP4 association with cellular RACK1 and ribosomes by multiple assays, whereas PABP association was less affected, consistent with independent interactions. The CR2 mutations decreased LARP4's ability to stabilize a β-globin mRNA reporter containing an AU-rich element (ARE) to higher degree than β-globin and GFP (green fluorescent protein) mRNAs lacking the ARE. We show LARP4 robustly increases translation of β-glo-ARE mRNA, whereas the LARP4 CR2 mutant is impaired. Analysis of nanoLuc-ARE mRNA for production of luciferase activity confirmed LARP4 promotes translation efficiency, while CR2 mutations are disabling. Thus, LARP4 CR2-mediated interaction with RACK1 can promote translational efficiency of some mRNAs.
