Neuronal RNA-Binding Protein HuD Interacts with Translation Initiation Factor eIF3

Abstract

Translation initiation is the rate-limiting step of protein synthesis and is the main target of translation regulation. RNA-binding proteins (RBPs) are key mediators of the spatiotemporal control of translation and are critical for cell proliferation, development, and differentiation. We have previously shown that HuD, one of the neuronal RBPs, enhances cap-dependent translation through the direct interaction with eukaryotic initiation factor 4A (eIF4A) and poly(A) tail using a HeLa-derived in vitro translation system. We have also found that translation stimulation of HuD is essential for HuD-induced neurite outgrowth in PC12 cells. However, it remains unclear how HuD is involved in the regulation of translation initiation. Here, we report that HuD binds to eukaryotic initiation factor 3 (eIF3) via the eIF3b subunit, which belongs to the functional core of mammalian eIF3. eIF3 plays an essential role in recruiting the 40S ribosomal subunit onto mRNA in translation initiation. We hypothesize that the interaction between HuD and eIF3 stabilizes the translation initiation complex and increases translation efficiency. We also showed that the linker region of HuD is required for the interaction with eIF3b. Moreover, we found that eIF3b-binding region of HuD is conserved in all Hu proteins (HuB, HuC, HuD, and HuR). These data might also help to explain how Hu proteins stimulate translation in a cap- and poly(A)-dependent way.

INTRODUCTION

Hu proteins, one of the RNA-binding proteins, have four types (HuB, HuC, HuD, and HuR) in mammals; HuR expresses ubiquitously, while HuB, HuC, and HuD express specifically in neural tissues. These Hu proteins have been reported to be involved in the regulation of mRNA stability by binding to AU-rich element found in the 3′ untranslated region (UTR) of target mRNAs.^1,2) We have previously shown that HuD stimulates cap-dependent translation and induces neurite outgrowth in PC12 cells depending on its activity. We also found that HuD interacts with the cap-binding complex, including eukaryotic translation initiation factor (eIF) 4E, eIF4G, eIF4A, and poly(A)-binding protein (PABP). In addition, we revealed that both the poly(A)- and eIF4A-binding ability of HuD is essential for the interaction with the cap-binding complex.³⁾ However, it is still unclear how HuD associated with the cap-binding complex eIF4F via eIF4A stimulates translation.

eIF3 is a large translation factor made of 13 subunits conserved from yeast to humans.^4–7) eIF3 binds the solvent-exposed side of the small ribosomal subunit (40S ribosome), thereby organizing its interaction with other eIFs and Met-tRNAiMet.^8–10) The hence formed 43S pre-initiation complex (PIC) includes eIFs 1, 1A, 2, 3, 5, Met-tRNAiMet and the 40S ribosome.^11,12) The cap-binding complex eIF4F recruits the 43S PIC to the capped 5′-end of the mRNA, thereby forming the 48S PIC competent for the scanning process to locate the start codon. The c, d, and e subunits of eIF3 forms the bridge with the eIF4G subunit of eIF4F,¹³⁾ as demonstrated in a recent cryo-EM study.¹⁴⁾ The beta (Greek)-propeller structure of eIF3b binds the ribosomal protein uS4, linking the eIF3bgi subcomplex that binds the mRNA at the leading edge of the scanning PIC.¹⁴⁾ eIF3 in the 43S PIC directly interacts with eIF4B via the eIF3a subunit. Hence, eIF4B, a coactivator that promotes ATP-dependent RNA helicase activity of eIF4A,¹⁵⁾ contributes to further the linkage between mRNA and 43S PIC.¹⁶⁾ Moreover, a phosphorylation of eIF4B by RSK (ribosomal protein S6 kinase), also known as S6 kinase (S6K), increases the interaction of eIF4B with eIF3.¹⁷⁾ PABP-interacting protein (Paip1) is known to directly bind to the eIF3g subunit of eIF3, stabilizing the translation initiation complex and promoting translation efficiency via its interaction with Paip1.^18,19) Furthermore, a model has also been proposed in which eIF3 is involved in the scanning and recognition of AUG start codon during translation initiation after the recruitment of 43S PICs onto mRNA.^20,21) We therefore hypothesized that HuD stimulates translation through the interaction with eIF3, consolidate circularization of the mRNA via binding to poly(A) tail and eIF4A like as a strategy by Paip1-mediated translation stimulation.

In this study, to elucidate the mechanism of HuD-mediated translation stimulation, the interacting states of HuD-eIF3 were analyzed using HuD deletion mutants. We revealed that HuD binds to eIF3b, one of the subunits of eIF3, and that the linker region between RBD2 and RBD3 of HuD is required for binding to HuD-eIF3b. Furthermore, HuD-eIF3 interaction is conserved among all Hu proteins.

MATERIALS AND METHODS

Plasmids

Plasmids encoding T7-tagged mouse HuD (D14-385) proteins, T7-tagged mHuD mutants (HuDmt, D14-302, D216-385), T7-tagged HuD, T7-tagged HuB, T7-tagged HuC, T7-tagged HuR, T7-tagged green fluorescent protein (GFP), FLAG-tagged HuD were described previously (Kasashima et al., Saito et al.). To obtain the expression plasmids encoding FLAG-tagged mHuD mutants (D236-385, D250-385, and D277-385), the coding region of each cDNA fragment was inserted into the HindIII-SalI site of pFLAG-CMV-2 plasmid (Sigma-Aldrich, St. Louis, MO, U.S.A.). To obtain the expression plasmids encoding T7-tagged mHuD mutant (D14-215), the coding region of cDNA fragment was inserted into the BamHI site of the pEF-BOS-T7 plasmid (Kasashima et al.). Plasmids encoding HuD deletion mutants were constructed by PCR-based mutagenesis of the plasmid encoding the FLAG-tagged or T7-tagged wild-type HuD.

Cell Culture and Transfection

HeLa cells were cultured in Dulbecco’s modified Eagle medium (Wako Pure Chemical Corporation, Osaka, Japan) supplemented with 10% fetal bovine serum. Transient transfection of cells was carried out using PEI MAX (Cosmo Bio, Tokyo, Japan).

Immunoprecipitation Assay

Hela cells that have been cotransfected with the constructs coding for FLAG-GFP or FLAG-eIF3b and T7-HuD, T7-HuD mutants, T7-HuB, T7-HuC, T7-HuR, and HeLa cells that have been transfected with constructs coding for FLAG-HuD, D236-385, D250-385 or D277-385 were lysed in TNE buffer (20 mM Tris–HCl pH 7.5, 2 mM ethylenediaminetetraacetic acid (EDTA), 150 mM NaCl, 1% NP-40, 1 mM phenylmethylsulfonyl fluoride, 10 mg/mL aprotinin, and 10 mg/mL leupeptin). In nuclease-treated cell extracts, Benzonase nuclease (Sigma-Aldrich) was added to the final concentration of 10 µg/mL and incubated at 4 °C for 1 h. Anti-FLAG M2 monoclonal antibody (Sigma-Aldrich) was added to the extracts with protein G-Sepharose beads. Bound proteins were washed with TNE buffer 1 mL ×5 times, followed by elution with sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) loading buffer. These eluates were subjected to SDS-PAGE and immunoblotting (IB) using anti-T7 monoclonal antibody and anti-FLAG polyclonal antibody.

RESULTS

The Interaction of HuD with eIF3

In this study, we first aimed to investigate the involvement of HuD in the 48S PIC, which is the rate-limiting step for translation initiation, and thus examined its interaction with eIF3, a core factor. eIF3, composed of 13 subunits, is known to be unable to form without the eIF3a or eIF3b subunit.²²⁾ Therefore, we performed immunoprecipitation (IP) analyses to confirm the interaction of HuD with endogenous eIF3b using nuclease-treated or -untreated HeLa cell extracts overexpressed FLAG-tagged HuD or GFP as a negative control. We analyzed IP products by immunoblotting, and we detected that FLAG-HuD co-purify with endogenous eIF3b but not with GFP (Fig. 1A). Notably, HuD-eIF3b interaction remains under the nuclease-treatment condition. Furthermore, we performed IP-assays using HuDmt, which contains amino acid substitutions in all RNA-binding domains and consequently lacks any RNA-binding activity,^3,23) and found that HuDmt also co-purifies with endogenous eIF3b (Fig. 1B). Thus, these data imply that the interaction of HuD with eIF3 is independent of RNA binding.

Fig. 1. RNA-Independent Interaction between HuD and eIF3b

(A) Specific co-immunoprecipitation of eIF3b with HuD. HeLa cells were transfected with FLAG-GFP or FLAG-HuD-coding plasmids. Cytoplasmic extracts from transfected cells treated Nuclease (right panel) or not (left panel) were immunoprecipitated with anti-FLAG antibody. IP-products were monitored by immunoblotting (IB) with indicated antibodies. (B) RNA-binding affinity of HuD is not required for binding to eIF3b. HeLa cells were transfected with T7-GFP, T7-HuD, or T7-HuDmt-coding plasmids. Cytoplasmic extracts from transfected cells were immunoprecipitated with anti-T7 antibody. IP-products were monitored by CBB stain and immunoblotting (IB) with indicated antibodies.

The Linker Region of HuD Is Essential for Interaction with eIF3b

Hu protein has three RNA-binding domains (RBDs) (RBD1, RBD2, and RBD3). RBD1 and RBD2 bind to AU-rich elements (ARE), regulatory sequences on mRNA involved in rapid degradation in eukaryotes.^24,25) RBD3 is a domain that binds specificity to poly(A) tail.^26,27) There is a linker region between RBD2 and RBD3, which is crucial to the interacting factor for HuD. We have shown that the linker region of HuD includes the eIF4A-binding site, and the eIF4A-binding mutant of HuD cannot associate with the cap-binding complex nor stimulate cap-dependent translation.³⁾ In addition, we also reported that HuD interacts specifically and directly with active Akt1 through its linker region to induce neurite outgrowth.²⁸⁾ Thus, the linker region is considered to be crucial for the interaction of HuD with other factors, and we wished to test whether HuD interacts with eIF3b via the linker region. To address this question, we mapped linker region of HuD to identify the critical interaction domain by immunoprecipitation assays using HeLa cell lysates expressing T7-tagged HuD deletion mutants or GFP in nuclease-treated condition (Fig. 2A). As expected, removal of the linker region abolishes the ability of HuD to interact with eIF3b (Fig. 2B). Thus, the linker is also important to interact with eIF3b.

Fig. 2. Linker Region of HuD Is Necessary for the Interaction between HuD and eIF3b

(A)Schematic representation of HuD wild-type (D14-385) and HuD deletion mutants (D14-215, D14-302 and D216-385). (B) Linker region between RBD2 and RBD3 of HuD is required for the interaction with eIF3b. HeLa cells were cotransfected with FLAG-GFP or FLAG-eIF3b and T7-HuD or T7-HuD deletion mutants (D14-215, D14-302, D216-385)-coding plasmids. Cytoplasmic extracts from transfected cells were immunoprecipitated with anti-FLAG antibody in the presence of Nuclease. IP-products were monitored by immunoblotting (IB) with indicated antibodies.

All Hu Proteins Can Interact with eIF3b

Although the overall amino acid sequence of the Hu family proteins is highly conserved, the linker region differs among Hu proteins (Fig. 3A). Thus, we next asked whether all Hu proteins can interact with eIF3b. To address this question, we further mapped the key interaction domain embedded within the linker region. To that end, we performed immunoprecipitation assays using nuclease-treated HeLa cell lysates expressing FLAG-tagged HuD deletion mutants or GFP (Fig. 3B) and examined co-immunoprecipitation of endogenous eIF3b with FLAG-tagged HuD or GFP. We find that the HuD mutant D236–385 containing a truncated linker region (236–302) and RBD3 can still interact with eIF3b, although the affinity is weakened. In contrast, removal of the amino acids between aa 236 and 249 (D250–385 and D277–385) abolish the ability of HuD to interact with eIF3b (Fig. 3B). Thus, the amino acids between aa 236 and 249 in the linker region of HuD is required for HuD’s interaction with eIF3b. Since the amino acid sequence in this region is highly conserved among the four Hu proteins, we tested whether the other three Hu proteins bind eIF3b. To address this question, we performed immunoprecipitation assays using nuclease-treated HeLa cell lysates expressing T7-tagged Hu proteins (HuB, HuC, HuD, and HuR) and FLAG-tagged eIF3b or GFP. As expected, we find that all Hu proteins can interact with eIF3b (Fig. 3C). This result implies that eIF3-binding of Hu proteins is important for the formation of the complex controlling translation efficiency and mRNA stability.

Fig. 3. All Hu Proteins Can Interact with eIF3b

(A) Amino acid alignment of the linker region of all Hu proteins. Identical residues are shown in gray. (B) The amino acids sequence between aa 236 and 249 of HuD is important for the interaction between HuD and eIF3b. HeLa cells were transfected with FLAG-GFP, FLAG-HuD or FLAG-HuD deletion mutants (D236-385, D250-385, D277-385)-coding plasmids. (C) The eIF3b-interaction is conserved among all Hu proteins. HeLa cells were transfected with FLAG-GFP or FLAG-eIF3b and T7-HuB, T7-HuC, T7-HuD or T7-HuR-coding plasmids. (B, C) Cytoplasmic extracts from transfected cells were immunoprecipitated with anti-FLAG antibody in the presence of Nuclease. IP-products were monitored by immunoblotting (IB) with indicated antibodies.

DISCUSSION

eIF3, which recruits 43S PIC onto mRNA, plays a central role in translation initiation, consolidating the closed-loop structure of the mRNA and promoting translation efficiency. Recently, there emerged evidence that the interaction of eIF3 with various RBPs that recognize cis-elements of mRNA determines the preferences of the mRNAs to be translated.^29,30) For instance, YTHDF3 recognizes N⁶-methyladenosine (m⁶A) modification, one of the most common and conserved internal chemical modifications in eukaryotes. YTHDF3 has been shown to recruit eIF3a subunits to m⁶A-rich 5′ UTR of RNAs and stimulates translation initiation of target mRNAs.²⁹⁾ Moreover, it has been recently shown that eIF3a regulates checkpoint kinase 1 (Chk1) and regulatory-associated protein of mammalian target of rapamycin (mTOR) (Raptor) synthesis by binding to the cis-regulatory elements of their mRNA through the interaction with the RNA-binding protein HuR.^30,31)

In this study, we show that HuD interacts with eIF3 via the eIF3b subunit (Fig. 1). We previously reported that HuD stimulates cap-dependent translation, and this stimulatory effect depends on the interaction of HuD with eIF4A and a poly(A) tail.³⁾ We also found that HuD impede microRNA-mediated translational repression in an eIF4A-binding-dependent manner.³²⁾ These findings clearly indicate that HuD associates with the translation initiation complex, although the molecular mechanism underlying how HuD positively regulates translation is not yet clear.

Key for this enhancer function of HuD is the linker region. We previously found that HuD interacts specifically and directly with eIF4A and active Akt1 via the linker region.^3,28) Here, we found that the interaction domain of eIF3b is also embedded with the linker region (Fig. 2B). The linker region is not only essential for HuD-mediated translation stimulation but also contributes to its neurite-inducing activity. Moreover, both the eIF4A-binding and Akt-binding abilities of HuD contribute to its neurite-induction activity. Notably, active Akt1 interacts specifically with the neuronal Hu proteins (HuB, HuC, and HuD) but not with HuR.²⁸⁾ Moreover, HuR cannot bind to eIF4A in immunoprecipitation assay using cell extracts (A.F. and T.F., unpublished data). In contrast, eIF3b can interact with all Hu proteins (Fig. 3C). In this study, we identified the amino acids between aa 236 and 249 in the linker region of HuD as the HuD-eIF3b interaction domain (Fig. 3B). Although the linker region of Hu proteins differs between the neuronal Hu proteins and HuR, the overall amino acid sequence of this region is well conserved (Fig. 3A). We previously identified the amino acid sequence from 277 to 302 in the linker region of HuD as the HuD-Akt interaction domain.²⁸⁾ Contrarily to HuR, eIF3b cannot interact with the HuD mutant D277–385 containing a truncated linker region (277–302) and RBD3. The amino acid sequence in this region is conserved in neural Hu but not in HuR, suggesting that a different set of binding partners in the linker region may confer a functional difference on Hu proteins. Indeed, it is well known that neuronal Hu proteins, but not HuR, can induce neuronal differentiation. Therefore, one might speculate that neural Hu proteins and HuR should form messenger ribonucleoprotein particles (mRNPs) with target mRNAs and common or different binding partners to regulate gene expression.

HuD has been studied as a key factor of post-transcriptional gene regulation in neurons. It has been shown that HuD plays pivotal roles in neuronal development, plasticity, survival, function, and neuronal diseases.^33–35)

Recently, the importance of HuD has been revealed not only in neurons, but also in cancer cells and various endocrine cells.^36–39) It has been reported that high HuD mRNA levels were observed in neuroblastoma cells.⁴⁰⁾ In contrast, reduced HuD mRNA and protein levels were observed in the pancreas of type 2 diabetes model mice.⁴¹⁾ It remains to be fully elucidated how HuD functions are involved in these diseases. Therefore, elucidating the molecular mechanisms of regulating gene expression by HuD is crucial for the treatment of these diseases.

Further analyses of molecular and functional interactions between HuD and eIF3 will provide us with a clear clue to the understanding of how the cooperative interaction of HuD and eIF3 leads to the translation stimulation.

Acknowledgments

We would like to thank William Figoni for proofreading the manuscript. This work was supported by Grants from Grant-in-Aid for Scientific Research (B) (22H02549) (to T.F.). This work was also supported in part by the Naito Foundation (to A.F.) and a Grant from Nagase & Co., Ltd. (to T.F.).

Author Contributions

HN, TT, AF, and TF conceived and designed the experiments; HN, TT, AF, and YF performed the experiments; HN, TT, AF, and TF analyzed the results and wrote the manuscript.

Conflict of Interest

The authors declare no conflict of interest.

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