Analysis of eEF1Bγ interactome in the nuclear fraction of A549 human lung adenocarcinoma cells

L. M. Kapustian, I. L. Lysetsky, T. V. Bondarchuk © 2019 L. M. Kapustian et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDC 577.217.535 + 577.322.23


Introduction
The high molecular weight complex of translation elongation factors, which is called eEF1B, provides efficient GDP/GTP exchange in the molecule of another translation factor, eEF1A [1]. eEF1A*GTP binds aminoacyl-tRNA of any specificity and brings it to the pre-A site of the 80S ribosome [2,3]. Hydrolysis of GTP finalizes the step of codonanticodon recognition permitting an aminoacyl-tRNA to be fully established in the A site.
After some pause, length of which may depend on tRNA specificity [4], eEF1A*GDP leaves the ribosome. In principle, the exchange of GDP for GTP in eEF1A can occur spontaneously as the cellular concentration of GTP is much higher than that of GDP. Nevertheless, the eEF1B complex accelerates the nucleotide exchange process leading to the formation of a new eEF1A*GTP*aminoacyl-tRNA complex [5]. ISSN 1993-6842 (on- eEF1B comprises eEF1Bα, eEF1Bβ and eEF1Bγ subunits. eEF1Bγ is the only subunit of eEF1B which does not catalyze nucleotide exchange in eEF1A. Instead, it is believed to serve as "glue" holding all subunits of eEF1B together [6]. However, the existence of free eEF1Bγ was detected in human cancer tissues. Moreover, cancer cells showed a sign of nuclear localization of eEF1Bγ [7,8]. As there is a general belief that protein synthesis occurs exclusively in cytoplasm [9], the cyto-nucleo transfer of eEF1Bγ implies some non-translational role of the subunit.

Structure and Function of Biopolymers
Here, in an attempt to clarify novel functions of the eEF1Bγ subunit in cancer cells, we carried out bioinformatic and experimental analysis of the protein partners of this subunit in the nuclear fraction of human lung cancer cells A549 and envisaged non-translational processes and networks involving eEF1Bγ.
As a result, we propose the splicing of pre-mRNA and regulation of mRNA stability as two main processes in which nuclear eEF1Bγ can be involved.

Preparation of nuclear fraction of human lung cells
Human lung cancer cells A549 were grown up to 7.5×10 6 cells/ml and harvested with Trypsin-EDTA. The nuclear fraction was isolated as in [10]. Briefly, cells were lysed in 1.5 volume of the buffer containing 10 mM HEPES pH7,9; 1,5 mM MgCl 2 ; 10 mM KCl; 0,5 % NP-40; 0,2 mM PMSF; 0,5 mM DTT for 20 min on ice. The centrifugation was performed (400g, 10 min) and the precipitate was resuspended in 4.5 volumes of the buffer (10 mM HEPES, 0.25 mM sucrose, 1.5 mM MgCl 2 , 10 mM KCl, 0.1 % NP-40, 0.5 mM DTT, 0.2 mM protease inhibitor PMSF) on ice for 10 min to provide the protein extraction. The sucrose cushion (2M) centrifugation was performed at 400g for 10 min. Nuclei were re-suspended in the lysis buffer and centrifuged at 1500g for 10 min. The procedure was double-repeated. The nuclear pellet was re-suspended in a half of initial volume of the nucleus lysis buffer (20 mM HEPES pH 7.9, 25 % glycerol, 0.42 M NaCl, 1.5 mM MgCl 2 , 0.2 mM DTT, 0.2 mM EDTA, 0.2 mM PMSF), incubated on ice for 30 min and centrifuged at 16000g for 30 min. The supernatant was used as a protein the nuclear fraction. The absence of the cytoplasmic fraction admixture was controlled by Western blot with anti-Tubulin antibodies (Abnova, Taiwan) [8].

Co-immunoprecipitation
The nuclear extract was pre-cleared with Protein G Sepharose (Sigma, USA) for 1 hour at 4 °C. Anti-eEF1Bγ antibodies (Abnova, Taiwan) (1.5 μg of antibodies per 1 mg of total protein) were added to pre-cleared lysate for overnight incubation at 4 °C. After addition of the Protein G Sepharose slurry the incubation persisted for 2 hours at 4 °C with continuous shaking. The eluted proteins were loaded on 12 % PAGE [11]. The protein bands of interest were cut and processed for massspectrometry analysis (LC-MS/MS).

Mass-spectrometry LC-MS/MS
The nuclear extract incubated with plain G-Sepharose was used as a control of nonspecific binding. The electrophoretic bands that were not present in the control or were much more extensive than in the control were cut and processed for mass spectrometry analysis at the Mass Spectrometry Laboratory of the Institute of Biochemistry and Biophysics (Warsaw, Poland) as described before [12].

Bioinformatic analysis
Cytoscape 3.2.0 Program [13] interaction database BIOGRID was supplemented with newly identified nuclear protein partners of eEF1Bγ and analyzed by MCODE plugin which finds highly interconnected regions (clusters) in any network loaded into Cytoscape. These clusters have been shown to represent protein complexes and/or parts of pathways [14]. For the sake of clarity, the known protein partners of eEF1Bγ: eEF1A1, eEF1A2 and UBC (polyubiquitin-C) were excluded from the database [12]. Also, we limited analysis by the first (direct) partners of the eEF1Bγ partners. MCODE analysis was performed on the hybrid supercomputer "SCIT-4" of the Glushkov Institute of Cybernetics (GIC) of National Academy of Sciences of Ukraine (http://icybcluster.org.ua).
Analysis of the nuclear protein partners which are co-fractionated with eEF1Bγ was done using the Mapofthecell program (http:// www.mapofthecell.org). All protein partners of the nucleus-localized eEF1Bγ identified by co-precipitation studies were tested for a possibility of their co-fractionation with eEF1Bγ as described in [10] for eEF1Bβ.

Results and Discussion
Having established a procedure for the cytonucleo fractionation of human lung carcinoma A549 cells [10] we isolated the nuclear fraction and conducted co-immunoprecipitation of the eEF1Bγ protein partners using anti-eEF1Bγ antibodies. Subsequent LC-MS/MS identification of precipitated proteins has delivered 234 interacting partners of eEF1Bγ.
These partners were used for further analysis by the Cytoscape program as described before [15]. Based on published data the Cytoscape protein interaction network of eEF1Bγ encompassed 11 proteins (including eEF1Bγ) [15].
A complementary approach to identify the cellular processes potentially involving eEF1Bγ is to combine our eEF1Bγ co-immunoprecipitation data with the proteomic data derived from precise co-fractionation of the cellular proteins with eEF1Bγ, with subsequent analysis of the functions of the proteins common for both data sets. Precise co-fractionation is considered an alternative way to estimate a possibility of protein-protein interaction in cell [17]. Recently we used this approach to identify the proteins co-fractionated with the eEF1Bβ subunit of eEF1B in human cancer cells [10]. For this aim the Mapofthecell database of the subcellular localization of the proteins in cancer human cells was employed [17].
Here we utilized the same approach to find out the proteins of the nuclear fraction of human cancer cells, co-fractionated with the eEF1Bγ subunit. To do this we used the whole list of 234 protein partners of eEF1Bγ identified by Co-IP and MS in the nuclear fraction to check a possibility of their co-fractionation with eEF1Bγ according to the Mapofthecell database. Indeed, nine protein partners were found to colocalize with eEF1Bγ during precise co-fractionation ( Fig. 2). All these proteins showed mixed cyto-nuclear localization according to the Subcellular localization database COMPARTMENTS (https://compartments.jensenlab.org) which is consistent with their pres-ence in the nuclear fraction of A549 cells. Interestingly, two groups of the eEF1Bγ partners separated by fractionation were observed on the map (Fig. 2). ELAVL1, TIA1, CD2AP, PCBP2 and DAZAP1 represented Group 1, while SERBP1, YTHDC2, YTHDF2 and GIGYF2 represented Group 2. Peculiarly, all but one proteins possessed RNA-binding proper ties.
Interestingly, these proteins showed quite different abundancy in human cancer cells [17]. The copy number of the eEF1Bγ estimated as number of molecules per cell (11 587 782) was comparable with the amount of the PCBP2, SERBP1 and FLAVL1 proteins. The copy number of YTHDF2 and DAZAP1 was 15-fold, CD2AP and TIA1 about 40-fold, GIGYF2-64-fold and YTHDC2-141-fold lower that the amount of the eEF1Bγ molecules per cell. The characteristics of the eEF1Bγ partners are given below.
PCBP2 is linked to the cancer development, in particular, it is overexpressed in pancreatic ductal adenocarcinoma [26], glioma [27] [37]. Also, CD2AP exemplifies pro-viral activity, for instance it binds unstructured subunits of Chikungunya virus replicase [38] and stimulates chronic hepatitis C virus propagation and steatosis by disrupting insulin signaling [39]. TIA1 (T cell intracellular antigen-1) is prion-related RNA-binding protein [40]. During arsenic stress inducing global shortening of 3'UTRs, TIA1 preferentially interacts with shorter 3'UTR sequences through U-rich motifs, correlating with stress granule association and mRNA decay of long 3'UTR isoforms [41]. TIA1 protein essentially contributes to the fidelity of mRNA maturation, translation, and RNA-stress-sensing pathways in human cells [42]. TIA1 is a key component of stress granules which is regulated by Zn2+ ions [43]. DNA damage due to mitogens activation promotes mRNA relocation and translation in part due to dissociation of Tia1 from its mRNA targets [44].
ELAVL1 (ELAV-like protein 1) which is also called HuR (human antigen R), is an RNA-binding protein involved in differentiation and stress response that acts primarily by stabilizing mRNA targets [56][57][58]. It binds 3'-untranslated region of BECN1/Beclin1 mRNA regulating ferroptosis , recently recognized form of controlled cell death that is characterized by lipid peroxidation, in liver fibrosis [59]. Its binding to PARG mRNA positively affects DNA repair and increases resistance to PARP inhibitors [60]. HuR/ ELAVL1 binding to SCN5A mRNA increases its stability, with subsequent reduction of arrhythmic risk in heart failure [61]. HuR/ ELAVL1 is involved in telomerase function, as it associates with TERC and promotes the assembly of the TERC/TERT complex by facilitating TERC C106 methylation [62]. It participates in spermatogenesis [63].
HuR/ELAVL1 demonstrates pro-viral activity [71]. This protein is linked to Parkinson's disease [72]. Disruption of ELAVL1/HuR nuclear export is consistent with the effects of inborn errors of vitamin B12 (cobalamin) metabolisms on brain development, neuroplasticity and myelin formation [73].
DAZAP1 (DAZ-associated protein 1) is an RNA-binding protein involved in mammalian development and spermatogenesis [74]. Knockdown or over-expression of DAZAP1 causes a cell proliferation defect while phosphorylation of its C-terminal domain which is sufficient to activate splicing is essential for the nuclear/cytoplasmic translocation of DAZAP1 [75]. DAZAP1 affects splicing of pre-mRNA [76]. It can regulate translation of mRNA as well [77]. SERBP1 (Serpine1 mRNA Binding Protein 1) binds different protein partners which participate in different cellular processes. For instance, it interacts with signaling protein RACK1 involved to signal transduction, mRNA splicing and translation and the cytoskeleton [78]. It interacts with SPIN1, a maternal protein containing Tudor-like domains, which is involved in regulating maternal transcripts to control meiotic resumption by controlling mRNA stability and/or translation [79]. One of the inhibitory mechanisms in this case is the occupation of the ribosomal mRNA entrance channel [80]. It binds dimers of activation-induced cytidine deaminase (AID) which may contribute to DNA-cleavage and recombination [81]. SERBP1 also affects DNA repair [82]. It is involved in transcriptional complex [83]. During stress SERBP1 is distributed simultaneously to cytoplasmic stress granules and nucleoli, two ribonucleoproteinenriched subcellular compartments [84]. Cytonuclear distribution can be regulated by methylation of its arginine residues [85].
SERBP1 was markedly upregulated in prostate cancer tissues and was significantly associated with tissue metastasis and Gleason score. The loss of miR-26a-5p promotes proliferation, migration, and invasion through targeting SERBP1 [86].
GIGYF2 (GRB10 Interacting GYF Protein 2) is a specific RNA-binding protein linked to repression of translation. It shows at least two distinct mechanisms of repression: one depends on 4EHP binding and mainly affects translation; the other is 4EHP-independent and involves the CCR4/NOT complex and its deadenylation activity [87]. Protein GIGYF2 is also a regulator of miRNA-mediated translation repression [88]. GIGYF2 is an autophagy regulator controlling neuron and muscle homeostasis [89] which is possibly involved in the regulation of signaling at endosomes [90]. It is also involved in mammalian development [91].
GIGYF2 mutations may be associated with increased risk of Parkinson's disease [92,93] and macrocephaly [94]. Increased expression of GIGYF2 might contribute to the development of diabetes-associated cognitive disorder via negatively regulating IGF1R signaling pathway [95].
YTHDC2 (YTH Domain Containing 2) protein is a N6-methyladenosine (m6A) reader that specifically recognizes and binds modified nucleotides in RNA. YTHDC2 enhances the translation efficiency of its targets and also decreases their mRNA abundance [96].
Recently it was shown that YTHDC2 interacts with the small ribosomal subunit in close proximity to the mRNA entry/exit sites and controls specific mRNAs by recruitment of the RNA degradation machinery to regulate the stability of m6A-containing mRNAs and by utilizing its distinct RNA-binding domains to bridge interactions between m6A-containing mRNAs and the ribosomes to facilitate their efficient translation [97]. Regulation of gene expression by YTHDC2 is considered an evolutionarily ancient strategy for controlling the germline transition into meiosis [98,99]. YTHDC2 is found to regulate mammalian spermatogenesis [96]. YTHDC2 promotes cancer metastasis [100].
YTHDF2 (YTH N6-Methyladenosine RNA Binding Protein 2) is another m6A reader which reduces the stability of target transcripts [101]. Due to this, YTHDF2 plays a role in maternal-to-zygotic transition during the early life of embryos [102,103]. YTHDF2 may recognize and bind the m6 A site of FAM134B that plays a pivotal role in lipid homeostasis to reduce its mRNA lifetime and reduce its protein abundance [104]. It binds to the peroxisome proliferator-activated receptor α to mediate its mRNA stability to regulate lipid metabolism [105]. It recognized and decayed methylated mRNAs of Cyclin-A2 and kinase CDK2, thereby prolonging cell cycle progression and suppressing adipogenesis [106]. YTHDF2 plays an important role in regulating hematopoietic stem cells ex vivo expansion by regulating the stability of multiple mRNAs critical for HSC self-renewal of these cells [107]. Ythdf2 modulates neural development by promoting m6A-dependent degradation of neural development-related mRNA target [108]. YTHDF2 is mainly present in the cytosol, however, nearly all YTHDF2 translocated from the cytosol into the nucleus after heat shock [109].
YTHDF2 is a negative regulator of interferon response as it facilitates the fast turnover of interferon mRNAs and consequently helps viral propagation [110]. YTHDF2 plays positive roles in viral gene expression and HIV-1 particle assembly, suggesting that HIV-1 interacts with mRNA decay components to successfully accomplish viral replication [111,112]. Overexpression of YTHDF2 induces more rapid viral replication, and larger viral plaques, in SV40 infected BSC40 cells [113].
YTHDF2 directly binds the m6A modification site of EGFR 3'-UTR to promote the degradation of EGFR mRNA in HCC cells acting as a tumor suppressor to repress cancer cell proliferation and growth [114,115].
The information is summarized in Table 1. Supplementation of the bioinformatic Cytoscape approach with the experimental pull-down data permitted to pinpoint a number of different cellular processes potentially involving eEF1Bγ in nucleus. Among those could be the main molecular biological processes: replication/reparation, transcription and translation. According to Cytoscape, eEF1Bγ may take part in apoptosis, cell regulation and cytoskeleton-membrane interaction, including exosomal trafficking. Use of the Mapofthecell database relying on precise cellular sub-fractionation, provided a basis for narrowing the wide functional variety of potential functions of eEF1Bγ expected from a number of its cellular partners in nuclear fraction. Consequently, we analyzed the functions of the experimentally proved eEF1Bγ partners which were predicted by both Cytoscape and Mapofthecell approaches. We reasoned that the coincidence of the functions of the eEF1Bγ partners identified by different approaches increases the probability of eEF1Bγ involvement in fulfillment of this function.
Peculiarly, two functions based on the mRNA-binding properties of the proteins were markedly represented in both datasets. The first one is the splicing of mRNA presented by HNRNPH1, HNRNPD, SNRNP20, MED23, SF3B1 (Cytoscape) and PCBP2, DAZAP1 (MapoftheCell). Fig. 3 shows a possibility that HNRNPD, DAZAP1 and eEF1Bγ on one side and HNRNPH1, SNRNP20, MED23, SF3B1 and PCBP2 on another side can be involved in different entities in the cell so, the interaction of eEF1Bγ with the second formation can be dynamic rather than stable. Thus, nucleuslocalized eEF1Bγ can be involved in splicing events. Importantly, the experimental proteomic data have suggested that eEF1Bγ could be a member of pre-mRNA 3' processing complex [116]. The second function is the regulation of stability of mRNA which involves HNRNPD, HNRNPH1 (Cytoscape) and GIGYF2, YTHDC2, YTHDF2, TIA1, ELAVL1 (MapoftheCell). Mapofthecell of these partners shows that HNRNPH1does not seem to belong to the stable complex ( Fig. 4) which cannot exclude, however, the existence of the dynamic interaction between HNRNPH1 and other members of the entity. One may suggest that eEF1Bγ can be bound to different mRNAs in nucleus contributing to their stability and transport. Notably, eEF1Bγ has been described to bind the 3' UTRs of vimentin [117] and some other mRNA [16].
Another function of eEF1Bγ partners identified by both approaches is related to cytoskeleton-membrane link and cellular trafficking. The group comprises EZR and HNRNPH1 (Cytoscape) and CD2AP (MapoftheCell). It is widely accepted that ezrin participates in anchoring membrane proteins to the cortical actin network [118]. Nuclear localization of ezrin was also reported, however, the role of nuclear ezrin is not yet deciphered [119]. HNRNPH1 and CD2AP participate in exosome trafficking [33,120]. It is worthy to mention that a role of eEF1Bγ in organelle transport has been shown [16,121]. eEF1Bγ was also co-immunoprecipitated with an essential component of ER-Golgi transport vesicles [122]. Also, it is known that eEF1Bγ interacts with cytoskeleton [123] and membranes [124]. All these facts permit to suggest that eEF1Bγ, along with its partners, may contribute to the cytoskeleton-membrane interaction and perform a transport role.
Based on these data one may suggest that eEF1Bγ is also associated with cancer and may play a central hub role to link together various cancer-related processes. There are a few experimental facts indicating such a possibility [7,8,133]. One may suggest that translation function of eEF1Bγ is linked to its involvement to eEF1B complex, while the induced by cancer appearance of free eEF1Bγ has regulatory consequences relying on its ability to influence pre-mRNA splicing and mRNAs stability. Subsequently, eEF1Bγ could be a novel perspective target for molecular therapy of cancer.
Another function of eEF1Bγ and its partners can be associated with viral propagation. Interestingly, 55 % of the eEF1Bγ partners picked up by Mapofthecell and 37 % of the eEF1Bγ partners picked up by Cytoscape showed pro-or anti-viral activity. Peculiarly, eEF1Bγ by itself is involved in viral infection and propagation [134][135][136].
Interestingly, no indication of the nuclear eEF1Bγ partners link to retinoblastoma was found, contrary to what was observed for cytoplasmic eEF1Bγ [15]. In contrast, a number  of eEF1Bγ partners in the nuclear fraction were shown to be related to neurodegenerative disorders (Alzheimer's disease, Parkinson's disease, epilepsy, intellectual disability) so, possible involvement of nuclear eEF1Bγ in genesis of these diseases should be elucidated in future studies.
Conclusions 234 proteins were identified by co-immunoprecipitation and LC-MS-MS as interacting with eEF1Bγ in the nuclear fraction of lung cancer cells A549. Possible functional networks involving eEF1Bγ and its partners were built with the use of the Cytoscape 3.2.0 program. The networks were related to the DNA replication/reparation, transcription, translation, cell regulation and cytoskeleton-membrane interaction, mRNA splicing and intracellular transportation processes. Additional analysis with Mapofthecell engine based on precise protein co-fractionation, permitted to pinpoint two main processes in which nuclear eEF1Bγ may be involved. They are splicing of mRNA and regulation of mRNA stability. According to our data, eEF1Bγ may also take part in cytoskeleton-membrane linking and cellular trafficking.

Acknowledgments
This work was partially financed by the Interdisciplinary Program of Scientific research of NAS of Ukraine "Molecular and cell biotechnologies for medicine, industry and agriculture" and Collaborative Program of NAS of Ukraine and PAN 2018-2020. This work was supported by the state program "Support of the development of priority directions of scientific research" (КПКВК 6541230).
We thank Prof. Anna El'skaya for her valuable contribution to the preparation of the manuscript. We thank Prof. M. Dadlez for an expert help in performing MS analysis. The contribution of V. Zakon and Dr. I. Groisman to the MS data analysis is appreciated. We are grateful to M. M. Ilchenko for helpful advice.