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Protein synthesis is an important and fundamental process in the cell to ensure normal growth. Proteins are necessary for different cellular processes including structural integrity of the cell, nuclear transportation, regulation of gene expression and enzymatic catalysis of biochemical reactions.
Translation is the process in which the mRNA is decoded by ribosome to form amino acids and thus proteins. It is a key regulatory step in the process of protein synthesis and cellular growth. The mechanism of translation is divided into different steps: initiation, elongation, and termination; however, the main key regulatory step in the protein synthesis is translation initiation despite the importance of elongation and termination (Preiss and Hentzz 2003). An alternation in any step of the translation process whether at the level of the gene in the mRNA or proteins involved in the translation regulation process is known to cause many diseases like VWM (Vanishing white matter leukoencephalopathy) . Scientists have been focusing on the translation initiation factors, one of the main leading causes of such diseases.
Mechanism and control of translation initiation
Translation initiation in eukaryotes begins after the assembly of 80S ribosome bound to tRNA (Met-tRNAi) and its correct positioning at the initiation codon (AUG) in the mRNA (Pavitt and Proud, 2009; Price and Proud, 1994). This is a multistep regulated process were many initiation eukaryotic factors are involved to guide the assembly of the 80S ribosome and correctly position its P site at the AUG initiation codon of the mRNA (Pavitt, 2005). mRNA models have been used like globin mRNA to understand the different mechanism that lead to translation initiation (Pavitt and Proud, 2009). Initially, Met-tRNAi is selected by eIF2 (eukaryotic initiation factor 2) from a pool of tRNAs and is delivered to the small subunit 40S of the ribosome as part of a ternary complex with eIF2.GTP. The binding of the eIF2/GTP/Met-tRNAi ternary complex and other elongation initiation factors to the small 40S submit form a 43S preinitiation complex (Pavitt 2005; Price and Proud, 1994; Lastra, Rivas and Barria, 2005). This binding is facilitated by eIF1, 1A and 3 (Lastra et.al. 2005). mRNA recruitment, the process of 43S complex binding to mRNA, involves the recognition of m7G cap at the mRNA 5'-terminus by eIF4E (cap binding) subunit of eIF4F (comprising eIF4E, eIF4G, and eIF4A) (Lastra, Rivas and Barria, 2005). Following the attachment of 43S preinitiation complex to the mRNA, the 43S complex will start scanning along the 5' untranslated region (5'UTR) to locate the AUG initiation codon (Pestova, 2001, ADD). Once AUG is encountered by the 43S complex, base pairing occurs between the initiation codon and anticodon in the tRNAi. The eIF5 stimulates the hydrolysis of eIF2.bound.GTP to GDP releasing the inactive form eIF2.GDP which is catalyzed back to eIF2.bound.GTP by eIF2B for another round of translation initiation. (Pavitt, 1998; Pavitt and Proud, 2009; Pavitt 2005; Lastra, Rivas and Barria, 2005). The role of eIF2B is essential to ensure the availability of eIF2.GTP. It is speculated that the initiation factors unbind at this stage from the 48S complex (reference). Furthermore, another initiation factor eIF5B associates with eIF1A to stimulate the joining of 60S ribosome to the small subunit 40 or 43S complex (check) to form the 80S ribosome complex. (Reference)
Figure 1: Mechanism of translation initiation in eukaryotes. eIF2B converts the inactive form eIF2.GDP into the active form eIF2.GTP. The eIF2.GTP bind to Met-tRNA to form a ternary complex which assembles with other elongation factors to form the 43S pre-initiation complex. mRNA recruitment in which the m7G cap at the mRNA 5'-terminus is recognized by eIF4E (cap binding) subunit of eIF4F (comprising eIF4E, eIF4G, and eIF4A). Scanning of the mRNA by 43S complex along the 5' untranslated region (5'UTR) to locate the AUG initiation codon and once located a pairing occurs between the anticodon on tRNA and AUG. Finally, eIF5B associates with eIF1A to stimulate the joining of 60S ribosome to the small subunit 40 or 43S complex (check) to form the 80S ribosome complex.
eIF2B (Eukaryotic Initiation Factor 2B)
1.2.1 Structure and Role of eIF2B
eIF2B is a multimeric complex protein composed of five subunits α, β, γ, ? and Æ that are encoded by GCN3/EIF2B1, GCD7/EIF2B2, GCD1/EIF2B3, GCD2/EIF2B4, and GCD6/EIF2B5 respectively, in yeast/humans (Benjamin and Proud, 1997; Van Der Knaap et.al. 2002; Price et.al. 1996; Pavitt and Proud, 2009). eIF2Bα is essential for the full activity of eIF2B in mammals unlike in yeast in which it is required for the GCN4 translation and amino acid starvation [Richardson et.al. 2004; William et.al. 2001; Craddock et.al. 1996, Liu et.al. 2011).The five subunits form two different subcomplexes: regulatory and catalytic. eIF2B α, eIF2B β and eIF2Bgamma subunits have high sequence similarity and represent the regulatory subcomplex that is controlled by the phosphorylated serine Ser51 in the N-terminal of the α subunit of eIF2 (eIF2[αP]); in addition, the regulatory subcomplex is demonstrated to have a higher affinity to phosphorylated eIF2 than to non-phosphorylated (Pavitt and Proud, 2009; Rowlands, 1998; Wek et.al. 2006) Studies have shown that a phosphorylated α subunit of eIF2 indirectly reduces the TC complex level by acting as a competitive inhibitor of eIF2B, and thus hindering the translation initiation leading to a slow growth of yeast by inactivating its GDP exchange activity (Pavitt, 1998; Rowlands et.al. 1998; Wek et.al. 2006). In cases such as amino acid starvation and stress, protein kinase R (PKR), HCR, and GCN2 phosphorylate eIF2 which in turn, inhibit eIF2B activity by binding to its α (Pavitt, 1998). A small amount of phosphorylated eIF2 is needed to exert a large inhibitory effect on eIF2B since in normal cases eIF2 is present in larger amounts than eIF2B (Boeson et.al. 2004). On the other hand, a single missense mutation in any gene encoding the three subunits of eIF2B can counteract the inhibitory function of the phosphorylated eIF2 and disrupt the regulatory complex (Pavitt, 1998; Reid 2012; Richardson et.al. 2004]. Alternatively, eIF2BFourth and eIF2BÆ subunits show extensive sequence similarity among each other and form the catalytic subcomplex of eIF2B that functions as a GEF (Guanine nucleotide exchange factor) (Price et.al. 1996; Pavitt, 1998; Boeson et.al. 2004). Furthermore, these subunits are highly conserved from rat, yeast, and Drosophila. This subcomplex binds both the eIF2 and eIF2(αP) without being affected by the phosphorylated eIF2α (Pavitt, 1998; Boeson et.al. 2004)
In the translation initiation process, two main regulatory steps exist to control the protein synthesis: the binding of Met-tRNAi to the P site of 40S ribosomal subunit that is induced by eIF2 bound to GTP and the ribosome recruitment of mRNA (Ashe et.al. 2001). The catalytic subcomplex of eIF2B plays an integral role in the regulation of translation initiation thus protein synthesis since it is necessary to promote the conversion of the inactive form eIF2.GDP into the active form eIF2.GTP by promoting the release of GDP to be replaced by GTP to enable ternary complex formation (Pavitt and Proud, 2009: Pavitt 2005; Ashe et.al. 2001).
eF2BÆ is an essential gene of eIF2B (Wang et.al. 2012). It is important to note that the eIF2BÆ alone can exert the catalytic activity in vitro; however, the rate of nucleotide exchange increases when the complete eIF2B complex is formed (Leng et.al. 2011). eIF2BÆ has three major regions: the NT, the I-patch, and the Cat (Catalytic domain) (Gomez and Pavitt, 2000; Wang et.al. 2012).
Certain NT domain residues and I-patch regions are highly conserved throughout eukaryotes and function in the interaction between subunits in the eIF2B complex, in particular between the catalytic and the other subunits. In brief, the eIF2BÆ has three major different regions that are responsible for its GEF activity, binding to eIF2, and interaction with other subunits to form the eIF2B complex. As mentioned previously, the two subunits Æ and γ show high sequence similarity; accordingly, it was shown that the fragment domain comprising the 44-165 and 4-140 residues of the eIF2BÆ and eIFBγ in human, respectively to be homologous to the NT (Nucleotidyl transferases) and referred to as the NT domain (Wang et.al. 2012). The residues to the N terminal of NT domain are highly conserved between both subunits and between the subunit and other proteins. The residues at the N terminal of the eIF2BÆ are required for the interaction with the other subunits (Wang et.al. 2012). A residue L68 exists in the NT domain that is shown to be mutated in the Vanishing white matter patients (Wang et.al. 2012). The residues from 67-70 are highly conserved in the human eIF2BÆ. L67 and L68 have been shown to be important in the complete formation of the eIF2B complex and form a full activity active eIF2B (Wang et.al. 2012). Wang et.al. (2012) mutated the L67 and L68 to alanine and found that each of them mutated alone has induced a 30% decrease in the eIF2B activity; in addition, L67 has interfered with the interaction between the eIF2BÆ and the regulatory unlike the L68A. Any loss of association between the regulatory and catalytic subcomplex will lead to a decreased eIF2B activity because to have a full active eIF2B, a full complex is required. Mutation of both L67 and L68 caused a large decrease in the Eif2B activity due to the loss of association between the regulatory and catalytic subcomplex (Wang et.al. 2012).
In conclusion, The N terminal of the eIF2B plays a role in the interaction between the epsilon subunit and other subunits to form eF2BÆ complex. This interaction enhances the binding to eIF2 and GEF activity (Gomez and Pavitt, 2000).
In vitro, eIF2BÆ subunit can exert a catalytic function without the need for the other subunits (Boeson et.al. 2004; Gomez et.al. 2002). Genetic analysis in vitro and pull down assays have shown that within the C terminus (518-712/527-726 residues) of the yeast/human eIF2BÆ respectively, encompasses the catalytic activity and the binding to the eIF2 (Boeson et.al. 2004; Wang et.al. 2012; Gomez and Pavitt, 2000; Gomez et.al. 2002; Asano et.al. 1999). In addition, it is the Æ subunit that has the minimal catalytic domain of eIF2B (Boeson et.al. 2004). It has been shown, that different residues are responsible for the different functions, GEF and binding to eIF2B (Wang et.al. 2012). Two functional regions exist in this catalytic domain: the first region comprises 115 amino acid residues and second 65 amino acid residues, in yeast shown to be (518-583), the main catalytic region of the enzyme (Boesen et.al. 2004; Leng et.al. 2011). This second region is well conserved in all eIF2BÆ. Studies have shown that deletions of residues 518-580 result in loss of GEF activity without affecting the binding to eIF2 (Boeson et.al. 2004).
A brief explanation is presented regarding the mutated gene (gcd6-F250L) that has been part of this study. gcd6-F250L is a missense mutation in the N terminal half of the eIF2BÆ (Gomez and Pavitt, 2000). The gcd6-F250L mutant complex is shown to impair the GEF activity of the complex eIF2B and thus translation initiation resulting in the slow growth phenotype without affecting the eIF2BÆ subunit alone (Gomez and Pavitt, 2000). As mentioned earlier, the catalytic activity is higher in vitro once the eIF2B complex is formed than that of eIF2BÆ alone (Gomez and Pavitt, 2000; Wang et.al. 2012). The gcd6-F250L mutant complex impairs the catalytic activity of the five subunit eIF2B complex, in particular eliminates the enhancement feature obtained once the five subunit of eIF2B is formed and not the eIF2BÆ alone in vitro (Gomez and Pavitt, 2000). It was questioned, whether the effect of gcd6-F250L is due to a decrease in the binding affinity to eIF2 or not; however, studies have shown that the gcd6-F250L mutant complex retained its binding affinity to eIF2 without enhancing the GEF activity once the five subunit complex of eIF2B is formed (Gomez and Pavitt, 2000). This implies that gcd6-F250L mutant does not exert its effect by decreasing the binding affinity to eIF2 rather by affecting residues that further enhance the GEF function of eIF2B. eIF2B five subunit complex enhance the binding affinity to eIF2 and the GEF activity which is reduced in case of gcd6-F250L mutant complex. Also, it was speculated that the GEF activity of eIf2B is enhanced due to increase in binding affinity of eIF2 to eIF2B five subunit complexes (Gomez and Pavitt, 2000); however, as mentioned earlier, it was shown that gcd6-F250L mutant complex impaired the increase in rate of nucleotide exchange without impairing the eIF2 binding affinity indicating that the binding affinity of eIF2 to eIF2B is not related to the GEF activity. In conclusion, gcd6-F250L impairs translation initiation by decreasing the eIF2B rate of exchange activity causing a slow growth phenotype.
1.2.3 VWH/CACH and eIF2B
Vanishing white matter leukoencephalopathy (VWM) or Childhood ataxia with central nervous system hypomyelination (CACH) is a hereditary disorder caused by missense and premature nonsense mutations in any genes of the eIF2B subunit (Boesen et.al. 2004; Pavitt and Proud, 2009; Richardson et.al. 2004: Van der Knaap et.al. 2010). The disorder is an autosomal recessive in which two allelic copies of the mutated gene of eIF2B should be found (Pavitt and Proud, 2009; Wang et.al. 2012). In addition, it is one of the fatal brain disorders in which the symptoms progress with age. Scientists in the Laboratory of Professor Marjo van der Knapp (Free University Amsterdam, Amsterdam, The Netherlands) first identified that mutations in the eIF2B encoding eIF2BÆ in patients with VWM/CACH caused the disease; however, later on, they also discovered that mutations in the genes of any eIF2B subunit could lead to this disease (Pavitt and Proud, 2009). The most common ones lie in the eIF2BÆ. Accordingly, two mutations W628R and E650K in human eIF2BÆ correspond to yeast Trp618 and Met640, respectively have been shown to cause the disease when mutated to Arg and Lys by interfering with the structural integrity of the catalytic domain of the C terminus rather than interfering directly in catalysis of eIF2BÆ (Boesen et.al. 2004). Initial studies demonstrated that these mutations cause a decrease in eIF2B activity resulting in VWM phenotype (Fogli et.al. 2004; Horzinski et.al. 2009); however, it was demonstrated by Wang et. al. (2012) that the majority of the VWM mutations hardly exert an effect on eIF2B activity.
1.2.5 Aims of the Project
The main aim of the project was to try and identify the suppressor gene that suppresses the reduced activity of eIF2B caused by gcd6-F250L, that is basically an unknown mutated gene named by Graham Pavitt as sif1-1 gene. The main focus was on the gcd6-F250L mutated complex of eIF2BÆ. The objective was to select mutated candidate genes from the whole genome of the GP3763 mutated strain after being sequenced. Future aim, in case the gene had been identified was to determine the function of the suppressor gene.
Materials and Methods
2.1 Whole Genome Sequencing
Whole genome of strains GP3755, GP3763 and GP3764 were sequenced at the sequencing facility, Faculty of Life Sciences, University of Manchester.
2.2. Growth conditions for Saccharomyces cerevisiae Strains in a solid media
The four strains used in this study are listed in table 1. A sample of each strain was streaked on the complete media (YPD, 1% [w/v] bactoyeast extract, 2% [w/v] bactopeptone, 2% [w/v] glucose and +/- 2% agar) and incubated overnight at 30°C.