C8ORF88: A Novel eIF4E-Binding Protein

Abstract

Translation initiation in eukaryotes is regulated at several steps, one of which involves the availability of the cap binding protein to participate in cap-dependent protein synthesis. Binding of eIF4E to translational repressors (eIF4E-binding proteins [4E-BPs]) suppresses translation and is used by cells to link extra- and intracellular cues to protein synthetic rates. The best studied of these interactions involves repression of translation by 4E-BP1 upon inhibition of the PI3K/mTOR signaling pathway. Herein, we characterize a novel 4E-BP, C8ORF88, whose expression is predominantly restricted to early spermatids. C8ORF88:eIF4E interaction is dependent on the canonical eIF4E binding motif (4E-BM) present in other 4E-BPs. Whereas 4E-BP1:eIF4E interaction is dependent on the phosphorylation of 4E-BP1, these sites are not conserved in C8ORF88 indicating a different mode of regulation.

Keywords: C8ORF88; cap-dependent translation; eIF4E; gene expression; mRNA; protein synthesis; translation initiation.

Figure 1

C8ORF88 amino acid sequence alignment with 4E-BP homologs. (a) Conserved sequences in C8ORF88 and 4E-BPs implicated with eIF4E binding activity are identified with boxes and include the canonical 4EBM (C-4EBM) (red), the elbow loop domain (E-loop) (blue) and the non-canonical 4EBM (NC-4EBM) (green). The conserved tyrosine (Y) and leucine (L) of the canonical 4EBM sequence YXXXXLΦ are highlighted in grey. Sequence alignment was performed using the Clustal Omega Multiple Sequence Alignment Tool. Asterisks underneath the sequence denote phosphorylation sites previously identified in 4E-BP1 [15]. Downward arrow denotes C8ORF88 S70, which according to PhosphoSitePlus (https://www.phosphosite.org/proteinAction.action?id=35695900&showAllSites=true, accessed on 15 February 2022) represents a single minor phosphorylation site. (b) Superimposition of 4E-BP1 and C8ORF88. Predicted structure of C8ORF88 flanking 4EBM (https://alphafold.ebi.ac.uk/entry/P0DMB2, accessed on 20 July 2023) was superimposed on 4E-BP1:eIF4E crystal structure (PDB 1WKW) using PyMOL. C8ORF88 is in cyan, 4E-BP1 in green and eIF4E in grey. The canonical 4EBM is colored violet.

Figure 2

Tissue expression pattern of C8ORF88. (a) Bulk tissue C8ORF88 expression from GTEx Portal (https://www.gtexportal.org/home/gene/C8ORF88, accessed on 15 July 2023). (b) Ct values obtained from C8Orf88 and β-actin amplification using RT-qPCR. The dotted line represents the Ct value of the background signal from β-actin’s blank control. The downward arrow identifies highest expression levels of C8Orf88 in testes. (c) Northern blot analysis of C8Orf88 mRNA from mouse tissues. (d) Single cell data showing C8ORF88 expression levels in testis as obtained from The Human Protein Atlas (https://www.proteinatlas.org/ENSG00000253250-C8orf88/single+cell+type, accessed on 15 July 2023).

Figure 3

In vitro association between C8ORF88 and eIF4E. (a) Pulldown of GST-tagged C8ORF88, 4E-BP1 and C8ORF88(Δ6) recombinant proteins in combination with His-4E or His-4E^W73A. (b) Recombinant proteins were generated by in vitro translations in wheat germ extracts in the presence of [³⁵S]-labelled methionine. Reactions were then incubated with m⁷GTP affinity resin. After binding, the resin was washed three times with LCB buffer and twice with 500 µM GTP before eluting with 500 µM m⁷GTP. For each reaction, the input, last GTP wash and m⁷GTP elution were resolved by SDS-PAGE, EN³HANCED, and visualized using autoradiography.

Figure 4

Interaction between C8ORF88 and eIF4E in cell culture. (a) Immunoprecipitation of transfected HEK293T cell lysates with anti-Flag antibody conjugated magnetic beads. Samples were resolved by SDS-PAGE and analyzed by Western blotting. (b) m⁷GTP cap-affinity chromatography of HEK293T cell lysates. The resin was washed three times with LCB buffer and twice with 500 µM GTP before elution in 500 µM m⁷GTP. For each sample the input, last GTP wash and m⁷GTP elution were resolved by SDS-PAGE and analyzed by Western blotting.

Figure 5

Overexpression of C8ORF88 leads to a reduction in global translation rates. (a) HeLa cells transfected to overexpress Flag-C8ORF88, Flag-C8ORF88(Δ6) or empty vector, were treated with [³⁵S]-labelled protein mix for 1 h, 48 h after transfection. Incorporation of [³⁵S]-labelled proteins was assessed via scintillation counting and standardized to the empty vector control. p values were determined using Dunnett’s multiple comparison test. (b) Expression of Flag-tagged proteins confirmed by Western blotting.