Viral and cellular mRNA-specific activators harness PABP and eIF4G to promote translation initiation downstream of cap binding

Abstract

Regulation of mRNA translation is a major control point for gene expression and is critical for life. Of central importance is the complex between cap-bound eukaryotic initiation factor 4E (eIF4E), eIF4G, and poly(A) tail-binding protein (PABP) that circularizes mRNAs, promoting translation and stability. This complex is often targeted to regulate overall translation rates, and also by mRNA-specific translational repressors. However, the mechanisms of mRNA-specific translational activation by RNA-binding proteins remain poorly understood. Here, we address this deficit, focusing on a herpes simplex virus-1 protein, ICP27. We reveal a direct interaction with PABP that is sufficient to promote PABP recruitment and necessary for ICP27-mediated activation. PABP binds several translation factors but is primarily considered to activate translation initiation as part of the PABP-eIF4G-eIF4E complex that stimulates the initial cap-binding step. Importantly, we find that ICP27-PABP forms a complex with, and requires the activity of, eIF4G. Surprisingly, ICP27-PABP-eIF4G complexes act independently of the effects of PABP-eIF4G on cap binding to promote small ribosomal subunit recruitment. Moreover, we find that a cellular mRNA-specific regulator, Deleted in Azoospermia-like (Dazl), also employs the PABP-eIF4G interaction in a similar manner. We propose a mechanism whereby diverse RNA-binding proteins directly recruit PABP, in a non-poly(A) tail-dependent manner, to stimulate the small subunit recruitment step. This strategy may be particularly relevant to biological conditions associated with hypoadenylated mRNAs (e.g., germ cells/neurons) and/or limiting cytoplasmic PABP (e.g., viral infection, cell stress). This mechanism adds significant insight into our knowledge of mRNA-specific translational activation and the function of the PABP-eIF4G complex in translation initiation.D espite the importance of translational control, the mechanisms by which specific subsets of mRNAs are translationally regulated are only well defined in a handful of cases. Nevertheless, it is clear that regulation is most often mediated by factors recruited to the 3′ untranslated region (UTR) of mRNAs and frequently occurs at the level of initiation (1). Initiation is a multistep process involving several mRNA-dependent steps, each of which requires eukaryotic initiation factors (eIFs) (2). Initially, the m 7 GpppX cap is bound by eIF4F, comprising a large scaffold protein, eIF4G, bound to the cap-binding protein, eIF4E, and an RNA helicase, eIF4A. The small (40S) ribosomal subunit, initiator tRNA, and associated initiation factors are then recruited as a 43S preinitiation complex. Recruitment is facilitated by eIF4A-dependent removal of RNA secondary structure and by the interaction of 40S-associated eIF3 with eIF4G. The 43S small ribosomal subunit complex then scans the 5′ UTR to locate a start codon, recognition of which promotes release of initiation factors and joining of the large (60S) ribosomal subunit to form an 80S ribosome. Like the cap,...

Translational Control in Virus-Infected Cells

“…These include an eIF4G-binding protein encoded by HSV-1 (ICP6) that stimulates eIF4E binding to eIF4G, promotes eIF4E phosphorylation, and stimulates viral mRNA translation (Walsh and Mohr 2006). To capitalize on limiting cytoplasmic PABP availability in HSV-1-infected cells, the viral ICP27 RNA-binding protein may stimulate initiation downstream of cap binding by harnessing PABP and eIF4G in a mechanism similar to that used by the cellular regulator Dazl (Deleted in azoospermia-like) (Smith et al 2017). HCMV increases the overall abundance of eIFs and PABP.…”

Epstein-Barr Virus Protein EB2 Stimulates Translation Initiation of mRNAs through Direct Interactions with both Poly(A)-Binding Protein and Eukaryotic Initiation Factor 4G

“…Here, we show that EB2 interacts with the cap-binding complexes (CBC in the nucleus and eIF4F in the cytoplasm) and PABP to stabilize the capped structure of the mRNA. Interestingly, the same type of mechanism has recently been proposed for ICP27 (28), suggesting that these two particular viral proteins target the PABP-eIF4G complex to enhance translation initiation from intronless viral mRNAs. Since EB2 is also able to interact with the nuclear cap-binding complex, we can speculate that the nuclear loading of EB2 onto the target mRNA is required in order to facilitate the subsequent recruitment of PABP onto the cytoplasmic eIF4F cap-binding complex.…”

“…This suggests that PABP could stabilize the interaction between EB2 and eIF4G. In a recent publication, Smith et al have reported an interaction between ICP27, the EB2 homologous protein from HSV-1, and PABP (28). Moreover, they have shown that although eIF4G and ICP27 do not seem to interact directly, eIF4G associates with ICP27 in the presence of PABP.…”

“…UL69 from HCMV stimulates viral mRNA translation through its interactions with the translation initiation factor eukaryotic initiation factor 4A (eIF4A) and the poly(A)binding protein (PABP) (25). Several studies have shown that ICP27 from HSV-1 also enhances translation of unspliced viral mRNAs (26,27), and a recent report showed that ICP27 targets PABP and eIF4G to promote translation initiation (28). Regarding EB2 from EBV, it has been previously demonstrated that besides its role as a nuclear export factor, it also strongly stimulates translation of unspliced mRNAs without affecting overall cellular translation (29).…”

The role of CSDE1 in translational reprogramming and human diseases

“…Although CSDE1 mainly reprograms cap-independent translation initiation, CSDE1 can also stimulate and repress cap-dependent translation initiation. In capdependent translation processes, eukaryotic translation initiation factor 4G (eIF4G) and poly(A)-binding protein 1 (PABP1) interact with each other to promote the circularization of terminal ribosomes from the 3' end to the 5' end [49][50][51]. Through enhancing the eIF4G-PABP1 interaction, CSDE1 increased the interaction between the 5' cap and poly(A) tail to promote capdependent translation.…”