Sequestration of eIF4A by angiomotin: A novel mechanism to restrict global protein synthesis in trophoblast cells

Abstract

Enrichment of angiomotin (AMOT) in the ectoplacental cone of E7.5 murine placenta prompted our investigation on the role of AMOT in trophoblast differentiation. We show here that AMOT levels increased in mouse placenta during gestation and also upon induction of differentiation in trophoblast stem cell ex vivo. Proteomic data unravelling AMOT-interactome in trophoblast cells indicated a majority of AMOT interactors to be involved in protein translation. In-depth analysis of AMOT-interactome led to identification of eukaryotic translation initiation factor 4A (eIF4A) as the most plausible AMOT interactor. Loss of function of AMOT enhanced, whereas, gain in function resulted in decline of global protein synthesis in trophoblast cells. Bioinformatics analysis evaluating the potential energy of AMOT-eIF4A binding suggested a strong AMOT-eIF4A interaction using a distinct groove encompassing amino acid residue positions 238 to 255 of AMOT. Co-immunoprecipitation of AMOT with eIF4A reaffirmed AMOT-eIF4A association in trophoblast cells. Deletion of 238 to 255 amino acids of AMOT resulted in abrogation of AMOT-eIF4A interaction. In addition, 238 to 255 amino acid deletion of AMOT was ineffective in eliciting AMOT's function in reducing global protein synthesis. Interestingly, AMOT-dependent sequestration of eIF4A dampened its loading to the m7-GTP cap and hindered its interaction with eIF4G. Furthermore, enhanced AMOT expression in placenta was associated with intrauterine growth restriction in both rats and humans. These results not only highlight a hitherto unknown novel function of AMOT in trophoblast cells but also have broad biological implications as AMOT might be an inbuilt switch to check protein synthesis in developmentally indispensable trophoblast cells.

Molecular regulation of trophoblast stem cell self-renewal and giant cell differentiation by the Hippo components YAP and LATS1

“…Thus, unveiling the kinases responsible for phosphorylating YAP at ser127 in differentiated trophoblast cells or the phosphatases which maintains the pool of unphosphorylated YAP in TSCs require further investigation. However, additional molecular mechanisms of YAP regulation by specific cytoplasmic mediators might exist in differentiated trophoblast cells [ 38 , 59 ]. Therefore, uncovering these non-canonical pathways surrounding YAP which might be functionally segregated from the canonical Hippo signaling pathway needs further study.…”

“…Human term placental samples were collected from healthy pregnant females and mothers with IUGR babies as described previously [ 38 ]. All experimental protocols that used human tissue were approved by Institutional Ethics Committee for Human Research, Calcutta National Medical College, India, in accordance with the relevant guidelines and regulations set forward by Indian Council of Medical Research ( http://icmr.nic.in/human_ethics.htm ).…”

“…Murine blastocyst-derived TSCs (TS 3.5 ) were a kind gift from Professor Janet Rossant, The Hospital for Sick Children (SickKids), Toronto, Canada, cultured as previously reported [ 11 , 38 – 40 ] and detailed in Additional file 1 .…”

“…Western blotting was performed as described previously [ 38 – 42 ]. The procedure and image acquisition have been described in detail in Additional file 1 .…”

“…For immunoprecipitation, 250 μg cell lysate was incubated overnight at 4ºC with the desired capture antibody and immunoprecipitation was performed as described previously [ 38 ]. Details pertaining to immunoprecipitation are described in Additional file 1 .…”