Drosophila Me31B a novel type of eIF4E interacting protein in P-bodies

VILARDO E; HERNÁNDEZ G; RIVERA POMAR R; LAYANA C

Congreso; SAIB-SAMIGE 2020; 2020

Eukaryotic translation initiation factor 4E (eIF4E) is a key factor involved in different aspects of mRNA metabolism. Drosophila melanogaster genome encodes eight eIF4E isoforms, and the canonical isoform eIF4E-1 is a ubiquitous protein that plays a key role in mRNA translation. eIF4E-3 is specifically expressed in testis and controls translation during spermatogenesis. In eukaryotic cells, translational control and mRNA decay is highly regulated in different cytoplasmic ribonucleoprotein foci, which include the processing bodies (PBs). Previous results of our laboratory showed that Drosophila eIF4E-1 and eIF4E-3 occur in PBs where might play a role in mRNA storage and translational repression. We also demonstrated that the DEAD-box RNA helicase Me31B, a component of PBs, physically interacts with eIF4E-1 and eIF4E-3 both in the yeast two-hybrid system and FRET in Drosophila S2 cells. Moreover, truncated Me31B proteins indicate that the binding sites of Me31B for eIF4E-1 and eIF4E-3 are located in different domains. Here we wanted to identify the two eIF4E-binding sites (4E-BSs) present in Me31B: at the carboxy-terminal, residues Y401-L407 are essential for interaction with eIF4E-1 whereas residues F63-L70 (at the amino-terminal) are critical for interaction with eIF4E-3. A comparison of the 4E-BSs occurring in other eIF4E-interacting proteins suggested two putative 4E-BSs within the regions identified of Me31B. To test the functionality of the putative 4E-BSs, we performed site-directed mutagenesis to replace residues F63 to Ala and L70 to Arg in the eIF4E-3 interacting region and Y401 to Ala and L407 to Arg in the eIF4E-1 interacting site. The substitution of both aromatic residues Y401 and F63 to a non-aromatic residue prevented the interaction of Me31B with eIF4E-1 and eIF4E-3, respectively. Therefore, we conclude that Me31B interacts with eIF4E-1 and eIF4E-3 through independent binding sites specific for each isoform. Thus, Me31B represents a novel type of eIF4E-interacting protein. Our observations suggest that Me31B might recognize different eIF4E isoforms in different tissues, which could be the key to silencing specific messengers. They provide further evidence that alternative forms of eIF4E and their interactions with different partners add complexity to the control of gene expression in eukaryotes.