EEF1G (Eukaryotic Translation Elongation Factor 1 Gamma) is a protein coding gene that starts at 62,559,601 nt and ends at 62,573,988 nt from qter and with a length of 14388 bp. The current reference sequence is NC_000011.10 and contains 10 exons. It is proximal to the nucleotidyl transferase TUT1 (terminal uridylyl transferase 1) gene and to the AHNAK nucleoprotein gene. Inside the nucleotidic sequence of EEF1G there is also a short non-coding sequence of the microRNA MIR6747 that starts from 62567011 bp and ends at 62567071 bp and is 61 bp long. Around the genomic locus of EEF1G take place different promoter or enhancer transcriptional elements. Two strong elements are closer to the sequence of EEF1G gene and are located at -1.4 kb and at +2.4 kb respectively and have a high influence of different kind of genes in their proximity, such as EEF1G1 and TUT1.

EEF1G mRNA is 1446 bp long with a reference sequence reported in GeneBank as NM_001404.5. The 5UTR sequence is not very long and counts 49 nt. The CDS is extended from 50 to 1363 nt, while the 3UTR starts from 1364 until 1446 nt. EEF1G is expressed ubiquitously in human tissues with a different expression level in relation to the specific tissue type. Minor expression levels are reported for brain, liver, lung, pancreas, salivary glands and testis while on the contrary a significantly higher expression level is found in the ovary (Fagerberg et al., 2014).
Five splice variants for EEF1G were observed (Table.1): the main reference variant is EEF1G-001 and the others are EEF1G-002, EEF1G-003, EEF1G-004 and EEF1G-201 (Figure.1). Only two of which codify for a protein, i.e. EEF1G-001 and EEF1G-201, while the others are non-coding RNA sequences (ncRNAs), classified as processed transcripts, i.e. nucleotide sequences that do not contain an open reading frame (ORF) and alternatively spliced transcripts, i.e. retained intron sequences. Furthermore, there is a potential readthrough with the inclusion of TUT1 gene.

Variant	Name	RefSeq (1)	Transcript ID	Exons	Type	Lenght (bp)	RefSeq (2)	Lenght (aa)
1	EEF1G-001	NM_001404.5	-	10	Protein coding	1446	NP_001395.1	437
2	EEF1G-002 (EEF1G-203)	AK092787.1	ENST00000525340.5	9	Retained intron	2496	-	-
3	EEF1G-003 (EEF1G-204)	-	ENST00000532986.1	5	Processed transcript	578	-	-
4	EEF1G-004 (EEF1G-202)	-	ENST00000524420.5	5	Processed transcript	557	-	-
5	EEF1G-201	AK300203.1	-	10	Protein coding	1631	BAG61974.1	487

Table.1. Splice variants of EEF1G gene (reworked from http://grch37.ensembl.org)

According to Entrez Gene, the analysis of the human genome revealed the presence of nine pseudogenes for EEF1G (Table.2) classified as processed pseudogenes and probably originated by retrotransposition. If these elements have any regulatory roles on the expression of the respective gene as described for others (Hirotsune et al., 2003), is only speculation in the absence of experimental evidence.

Gene	Gene  name	Gene ID	RefSeq	Locus	Location	Start	End	Lenght (nt)
EEF1GP1	EEF1G1 Pseudogene 1	646837	NC_000007.14	Chromosome 7	7q31.33	125033433	125035389	1957
EEF1GP2	EEF1G1 Pseudogene 2	100130260	NC_000005.10	Chromosome 5	5q32	147922182	147923422	1241
EEF1GP3	EEF1G1 Pseudogene 3	651628	NC_000003.12	Chromosome 3	3p22.1	40596122	40597519	1398
EEF1GP4	EEF1G1 Pseudogene 4	100129403	NC_000003.12	Chromosome 3	3q26.1	161324837	161326238	1402
EEF1GP5	EEF1G1 Pseudogene 5	642357	NC_000023.11	Chromosome X	Xq23	115702791	115704195	1405
EEF1GP6	EEF1G1 Pseudogene 6	100421733	NC_000006.12	Chromosome 6	6q16.1	96750800	96751728	929
EEF1GP7	EEF1G1 Pseudogene 7	645311	NC_000001.11	Chromosome 1	1p32.3	52573115	52573818	704
EEF1GP8	EEF1G1 Pseudogene 8	391698	NC_000004.12	Chromosome 4	4q28.2	129903001	129904244	1244
LOC729998	EEF1G1 Pseudogene 9	729998	NC_000007.14	Chromosome 7	7q33	133034515	133035940	1426

Table.2 EEF1G pseudogene (reworked from https://www.ncbi.nlm.nih.gov/gene/1937)

Are described two isoforms for eEF1G and it is shown that this protein has a glutathione S-transferase activity. In addition, eEF1G is a structural constituent of a more greater protein complex that is in relation to the ribosome and plays a role in the elongation step of protein synthesis.

The eukaryotic elongation factor 1-gamma (alias eEF1G, eEF1γ, heEF1γ, eEF1Bγ) is a subunit of the macromolecular eukaryotic elongation factor-1 complex (alias eEF1, also called eEF1H), a high-molecular-weight form made up of an aggregation of different protein subunits: eEF1A (alias eEF1α), eEF1B (alias eEF1 β, eEF1Bα, eEF1B2), eEF1G, eEF1D (alias eEF1δ, eEF1Bδ) and valyl t-RNA synthetase (valRS). eEF1H protein complex plays central roles in peptide elongation during eukaryotic protein biosynthesis, in particular for the delivery of aminoacyl-tRNAs to ribosome mediated by the hydrolysis of GTP. In fact, during the translation elongation step, the inactive GDP-bound form of eEF1A (eEF1A-GDP) is converted to its active GTP-bound form (eEF1A-GTP) by eEF1BGD complex-mediated GTP hydrolysis so it acts as a guanine nucleotide exchange factor (GEF), regenerating eEF1A-GTP for the successive elongation cycle. The physiological role of eEF1G in the translation context is still not well defined, however eEF1G seems to be necessary for this nucleotide exchange. Studies did not confirm its direct involvement in this process but it is supposed that it may stimulate the activity of eEF1B and guarantee stability to entire eEF1H complex (Ejiri, 2002; Mansilla et al., 2002). In addition, studies revealed that eEF1G sequence does not contain any consensus sequence for ATP or GTP binding (Maessen et al., 1987).
There are known two isoforms produced by alternative splicing: the isoform 1 (RefSeq NP_001395.1; UniParc, P26641-1), that has been chosen as the canonical sequence, is formed by 437 residues and has an overall molecular weight of 50.12 kDa, while the isoform 2 (RefSeq BAG61974.1; UniParc, P26641-2) is 487 amino acids long with 56.15 kDa of molecular weight. The sequence of this isoform differs from the canonical sequence for the substitution of the first four amino acids (MAAG) by the insertion of other fifty residues (MAERWVAPAVLRRARFASTFFLSPQIYAHKDGDLRSAFFILSFKRGEFIPFLNW) with the creation of an alternative amino acid sequence (Ota et al., 2004). No experimental data or other studies were performed for this isoform, so its biological role is totally unknown.
eEF1G shown hydrophobic properties, has a relatively high isoelectric point (pI ≈ 7) (van Damme et al., 1991) and the analysis of its primary and secondary structures revealed some interesting characteristics. In fact, it is a multi-domain protein which consists of three main domains: from the amino to carboxyl half terminal there are a glutathione S-transferase (GST)-like N-terminus domain (NT-eEF1G), a glutathione S-transferase (GST)-like C-terminus domain (CT-eEF1G) and a conserved C-terminal domain (CD-eEF1G) (Achilonu et al.,2014). NT-eEF1G and CT-eEF1G domains show a homology to the theta class of glutathione S-transferases (GSTs) (Koonin et al., 1994; Janssen and Möller, 1988).
The NT-eEF1G domain of eEF1G, by using secondary structure prediction algorithms, seems to have a predominant α-helix secondary structure (Achilonu et al.,2014) and it was demonstrated that interacts directly with the N-terminal domain of eEF1B (van Damme et al., 1991) and also with the N-terminal domain of eEF1D in independent manner (Cao et al., 2014; Mansilla et al., 2002; Janssen et al., 1994), although different interactional models were proposed (Le Sourd et al., 2006; Jiang et al.,2005; Sheu and Traugh, 1999; Minella et al., 1998). It does not seem to have a direct interaction with other eEF1H elements. In addition, the presence of an enzymatically active GST element could be involved in detoxification of oxygen radicals and the over-expression of eEF1G in cancer cells could influence their response to oxidative stress and their aggressiveness (Koonin et al., 1994).
The calculated secondary structure of the CT-eEF1G domain of eEF1G shows both α-helix and β-strand secondary structure elements (Achilonu et al.,2014) and does not interact with eEF1B but instead is the candidate domain to has transient interactions with other proteins or cell structures.
The CD-eEF1G domain of eEF1G seems to be mostly in α-helix secondary structure with few β-strands (Achilonu et al.,2014) and currently it does not show particular elements or interactions.
It is interesting that eEF1G shows two internal repeats of eight amino acids (VFGEXNXS) at positions 35 - 42 and 355 - 362 respectively, that are located in its amino-terminal and carboxy-terminal halves. The roles of these two octapeptides are still unknown even if they could cover the function of binding-sites (van Damme et al., 1991; Maessen et al., 1987).
eEF1G seems to have in human cells a dimeric nature, forming homodimers, while in rabbit shows a trimeric nature and in yeast was observed that it acts as a monomer (Achilonu et al.,2014; Koonin et al., 1994). Furthermore, it has hydrophobic properties that enable it to attach to membranes (Mansilla et al., 2002).
eEF1G interacts mainly with EEF1B2 and EEF1D, even if other interactions are documented in protein databases and in literature, i.e. with the histidyl-tRNA synthetase ( HARS), leucyl-tRNA synthetase ( LARS), cysteine-tRNA synthetase ( CARS), leucine zipper putative tumor suppressor 1 ( LZTS1), enoyl-CoA hydratase 1( ECH1), plasminogen receptor ( PLGRKT), small ubiquitin-related modifier 2 ( SUMO2), ATP-binding cassette sub-family C member 9 ( ABCC9), tripartite motif containing 55 ( TRIM55), E3 ubiquitin-protein ligase ( TRIM63), interleukin enhancer binding factor 2 ( ILF2), vascular cell adhesion protein 1 ( VCAM1), eukaryotic translation elongation factor 1 delta pseudogene 3 ( EEF1DP3), RNA-binding protein 6 ( RBM6) (HuRI database - http://interactome.baderlab.org/), ATP-dependent DNA helicase Q5 ( RECQL5) and fasciculation and elongation protein zeta 1 ( FEZ1)(Ishii et al., 2001) although the nature of these interactions are poorly understood.
Post-translational modifications. Some post-translational modifications are observed, such as phosphorylation, acetylation and S-nitrosylation.
1) Phosphorylation: it was demonstrated that eEF1G is a target of the kinases, in particular the cell cycle protein kinase CDK1 /cyclin B (Le Sourd et al., 2006; Mansilla et al., 2002). There are at least four positions for phosphorylation: two on threonine residues (T43, T230) and two on serine residues (S286, S406) (Olsen et al., 2010; http://hprd.org). It is assumed that these phosphorylations play a regulatory role, but their exact functional significance is poorly understood.
2) Acetylation: there are three positions for acetylation on lysine residues (K132, K147, K434) (Choudhary et al., 2009; http://hprd.org).
3) S-nitrosylation: there is one most probable position for S-Nitrosylation on a cysteine residue (C194) (Han and Chen, 2008; http://hprd.org).

EEF1G mRNA is expressed widely as previous reported while the presence of eEF1G protein in human tissues shows unexpected differences i.e. higher levels of protein are observed in cerebellum, hippocampus, esophagus, stomach, small intestine and pancreas while its low expression levels are reported in oral mucosa, bronchus, lung, parathyroid glands, adrenal glands, smooth muscle, prostate and urinary bladder. No protein presence is found in bone marrow, heart muscle, kidney, liver and skeletal muscle (Fagerberg et al., 2014). Furthermore was revealed the presence of the protein both in the human physiological secretions (cerumen, saliva, milk, urine, seminal plasma) and in pathological intercellular fluids (ascites) (https://www.proteomicsdb.org).
The expression pattern in cell lines tested for its presence is similar and without significantly differences except in one study that revealed a higher expression in human embryonic kidney HEK293 cell line and in human liver hepatocellular carcinoma HepG2 cell line (Cao et al, 2014).

eEF1G is located mostly in the cytoplasm where forming a gradient from the nucleus to the periphery of the cell, but some studies find it also in cellular nucleus, nucleolus, mitochondria and in relation with endoplasmic reticulum and plasma membrane (Cho et al., 2003; Minella et al., 1996; Sanders et al., 1996). It was reported also its localization in extracellular exosomes (Principe et al., 2013).

eEF1G shows canonical functions and multiple non-canonical roles (moonlighting roles) inside the cell.
Canonical function: eEF1G binds to eEF1B and eEF1D and is supposed that its primary role may be to ensure the proper scaffolding and stability of its binding partners in the eEF1BDG macromolecular complex and then it could anchors the entire EF1H complex to the endoplasmic reticulum together with the ribosome (Mansilla et al., 2002; Janssen et al., 1994). However, the complete significance of the role of human eEF1G remains unknown and needs to be more studied.
Non-canonical roles: eEF1G has shown to interact with cytoskeleton, RNA polymerase II, TNF receptor-associated protein 1 ( TRAP1) and membrane-bound receptors. In addition, it was observed that it has mRNA binding properties and it is a positive regulator of NF-kB signaling pathway.
1) eEF1G and cytoskeleton: it was discovered that eEF1G is a structural component of the cytoskeleton (Coumans et al., 2014), in fact it can bind both keratin intermediate filaments (Kim et al., 2017) and the tubulin (Janssen and Möller, 1988). This suggests that it may have an influence on cytoskeletal architecture, cell morphology and motility, but these implications and the roles of these bindings are still need to clarify.
2) eEF1G and interaction with RNA polymerase II: it physically interacts with RNA polymerase II (pol II) core subunit 3 ( POLR2C), both in isolation and in the context of the holo-enzyme (Corbi et al., 2010).
3) interaction between eEF1G and TNF receptor-associated protein 1 (TRAP1): TRAP1 is the main mitochondrial member of the heat shock protein (HSP) 90 family and it was revealed that there is an interaction between this protein and some members of eEF1H complex, including eEF1G. The role of the interaction between eEF1G and TRAP1 is related to the translational control (Matassa et al., 2013) and maybe also in the protection to oxidative stress (Pisani et al., 2016).
4) eEF1G and membrane-bound receptors (dopamine D3 receptor): it was observed that there is an interaction between eEF1G and dopamine D3 receptor ( DRD3) and that they have a co-localization on the plasma membrane. This interaction involve also eEF1B subunit after its protein kinase-mediated phosphorylation on its serine residues. eEF1G acts as a bridge for the relation between eEF1B and DRD3 and these three factors together forming a new macromolecular complex on the plasma membrane that obviously play some roles even if its functional meaning is still understood (Cho et al., 2003).
5) mRNA binding properties: the presence of eEF1G was detected on the genomic locus corresponding to the promoter region of human vimentin gene VIM and this permits to suppose that eEF1G regulates vimentin gene by contacting both pol II and the vimentin promoter region. In addition was shown that it can bind to 3UTR of vimentin mRNA and so it can shuttling/nursing the vimentin mRNA from its gene locus to its appropriate cellular compartment for translation. In fact, depletion of eEF1G causes the incorrect compartmentalizing of the vimentin protein and seriously compromise cellular shape and mitochondria localization (Corbi et al., 2010). Furthermore, was shown that eEF1G can bind also AATF (Che-1) and transcription and their promoter regions (Pisani et al., 2016).
6) regulation of NF-kB signaling pathway: eEF1G can binds to the CARD domain of mitochondrial antiviral-signaling protein ( MAVS) and thus significantly promotes the activities of transcription factor NF-kB functioning as its positive regulator. The discover offers a new regulating mechanism of the antiviral responses that promotes the downstream pro-inflammatory cytokines CXCL8 (interleukin-8 (IL8)) and interleukin-6 ( IL6) (Liu et al., 2014).

eEF1G is highly and abundant conserved in many species, with sometimes the lack of NT-eEF1G domain. The homology for eEF1G protein between species is reported in Table.3

Organism (1)	Organism (2)	Symbol	Similarity (%)
Human	H.sapiens	eEF1G	100
Chimpanzee	P.troglodytes	eEF1G	100
Gorilla	G.gorilla gorilla	eEF1G	99
Cat	Felis catus	eEF1G	99
Mouse	M.musculus	eEF1G	98
Rat	R.norvegicus	eEF1G	98
Zebrafish	D.rerio	eEF1G	75
Drosophila	D.melanogaster	Ef1gamma	58
Caenorhabditis	C.elegans	F17C11.9	52
Yeast	S.cerevisiae	TEF4	38

Table.3 eEF1G homology (reworked from https://cgap.nci.nih.gov;

The great number of mutations in the genomic sequence or in the amino acid sequence for EEF1G was discovered in cancer cells that are obviously genetically more unstable respect normal cells. The genomic alterations observed include the formation of novel fusion genes as EBF3/EEF1G, EEF1G/ALK, EEF1G/ENG, EEF1G/GFAP, EEF1G/MTA2, EEF1G/MYH9, EEF1G/NXF1, EEF1G/OOEP [t(6;11)(q13;q12) EEF1G/OOEP], EEF1G/PPP6R3 [t(11;11)(q12;q13) EEF1G/PPP6R3], EEF1G/TOX2, EEF1G/UBXN1, ETFB/EEF1G, HNRNPUL2/EEF1G, IGHG1/EEF1G, IGHM/EEF1G and NCEH1/EEF1G (Klijn et al., 2015; http://atlasgeneticsoncology.org/Bands/11q12.html; http://quiver.archerdx.com), however there are no experimental data yet to understand the repercussions on cellular behavior and so the implications in cancer of these fusion genes are unclear. There is one chromosomic translocations with production of a novel fusion gene that is more investigated and it is t(2;11)(p23;q12.3) EEF1G/ALK.