Isolation and initial characterization of eIF6 – Eukaryotic initiation factor 6 (eIF6), a monomeric protein of about 26.5 kDa, was originally isolated from the postribosomal supernatant of both wheat germ (Russel and Spremulli 1979) and mammalian cell extracts (Valenzuela et al. 1982) based on an in vitro assay that measured the ability of the protein to bind specifically to the 60S ribosomal subunit and to prevent the association of the 60S subunit to the 40S ribosomal subunit to form the 80S ribosomes. Because, the assembly of the 80S initiation complex during initiation of protein synthesis requires a cellular pool of free 40S and 60S ribosomal subunits, eIF6 was thought to play a direct role in the provision of free ribosomal subunits required for initiation of protein synthesis. The protein was therefore classified as a eukaryotic translation initiation factor (eIF), although its function in translation was poorly characterized. Notably, it was shown that eIF6-bound 60S ribosomal subunit is incapable of joining the 40S (48S) initiation complex to form the 80S initiation complex (Valenzuela et al. 1982). It should be noted that recent cryo-electron microscopic studies reveal that eIF6 and the 40S ribosomal subunit share a common binding region on the 60S subunit and cannot bind simultaneously. Thus the ribosomal subunit anti-association activity of eIF6 is due to its ability to physically block intersubunit bridge formation (Gartmann et al. 2010).
- (b)Functional characterization of eIF6 using molecular genetic analysis in yeast: To facilitate further characterization of eIF6, first a human cDNA (Si et al. 1997) and then the single-copy essential Saccharomyces cerevisiae gene (Si and Maitra 1999; Sanvito et al. 1999) encoding functionally active eIF6, each of 245 amino acids (calculated Mr.,26,558 for human eIF6 and 25,550 for yeast eIF6) were cloned and expressed in Escherichia coli. The two proteins are 72% identical. A molecular genetic analysis in yeast was therefore used by investigators to elucidate the cellular function of eIF6 (Si and Maitra 1999; Sanvito et al. 1999). Using a mutant yeast strain having a conditional eIF6 expression system, these investigators showed that depletion of eIF6 from this mutant strain resulted in inhibition of both cell growth and rate of in vivo protein synthesis. Analysis of the polysome profiles of wild-type and eIF6-depleted cells showed that eIF6-depletion caused a marked reduction in the number of polyribosomes compared with that in wild-type cells (Fig. 1a). However, in contrast to cells depleted of an essential initiation factor, where a reduction in the size of polyribosomes is always accompanied by a marked increase in the number of 80S ribosomes and both free 40S and 60S ribosomal subunits, the decrease in polyribosome content in eIF6-depleted cells is accompanied by a decrease (not increase) in the number of both 80S and 60S ribosomes and concomitant accumulation of half-mer polyribosomes (representing stalled 43S initiation complexes at the 5’-UTR and at the AUG codon of mRNAs awaiting association with 60S ribosomal subunits). Direct determination of total 40S and 60S subunit content of eIF6-depleted cells confirmed that there was a selective reduction of total 60S with respect to total 40S ribosomal subunits, causing a stoichiometric imbalance in the 60S/40S subunit ratio (Fig. 1b) resulting in the formation of half-mer polysomes. These results, along with the observation that lysates of yeast cells lacking eIF6 remained active in translation of mRNA in vitro (Si and Maitra 1999), led to the conclusion that eIF6 does not function as a canonical translation initiation factor for global protein synthesis. Additionally, it was shown that the stability of mature 60S ribosomal particles, synthesized in yeast cells in the presence of eIF6, was not significantly affected following removal of eIF6 from these cells (Basu et al. 2001). Rather evidence clearly indicated that in eIF6-depleted cells, the reduction of 60S ribosomal subunits is due to severe inhibition in the biogenesis of 60S ribosomal subunits. To understand how depletion of eIF6 affects 60S ribosome biogenesis, it is necessary to review very briefly the salient features of ribosome biogenesis.
eIF6 is Essential for the Biogenesis of 60S Ribosomal Subunits
Ribosome biogenesis (reviewed in Kressler et al. 2010; Venema and Tollervey 1999) is a highly conserved process from yeast to mammals and occurs primarily in the nucleolus, where four ribosomal rRNAs (25S in yeast or 28S in mammals), 18S, 5.8S, and 5S are formed, modified co-transcriptionally by pseudouridylation and methylation, and processed during their assembly with 78 (in yeast) and 79 (in mammals) ribosomal proteins into mature 40S and 60S ribosomal subunits. In yeast, where the process has been best characterized, the 18S rRNA of the 40S subunit and the 25S (28S in mammals) and 5.8S rRNAs of the 60S subunit are transcribed in the nucleolus from the rDNA transcription unit by RNA polymerase I as a single large precursor RNA known as the 35S pre-RNA in yeast and 45S pre-rRNA in mammals. The fourth rRNA 5S, also a constituent of the 60S subunit, is transcribed independently from 5S DNA by RNA polymerase III. Immediately following the synthesis of the 35S pre-rRNA, many ribosomal proteins (mostly of the 40S subunits) as well as a large number of transacting nonribosomal proteins and small nucleolar RNPs (SnoRNPs) associate with the 35S pre-rRNA to form the 90S ribonucleoprotein (RNP) particle. Association of the transacting nonribosomal proteins as well as snoRNAs with the preribosomal RNP particles are required for accurate pre-RNA processing, pre-rRNA modification, and ribosome assembly. The 35S pre-rRNA present in the 90S RNP then undergoes a sequence of ordered exonucleolytic trimming and endonucleolytic cleavage reactions (see Fig. 1c) giving rise to several intermediate RNP particles of decreasing size in the nucleus and eventually to a mature 40S ribosomal subunit containing 18S rRNA and a mature 60S ribosomal subunit containing 5.8S and 25S rRNAs. As shown in Fig. 1c, the very first endonucleolytic cleavage at the A2 site results in the formation of pre-40S (43S) and pre-60S (66S) particles. Each precursor particle then follows an independent pathway for subsequent biogenesis in the nucleolus and nucleoplasm, nuclear export and final maturation in the cytoplasm. It should be noted that not all transacting assembly proteins associate simultaneously with the 90S or intermediate pre-40S and pre-60S RNP particles. Rather, these proteins associate with preribosomal RNP particles at different steps of the processing reactions. As the biogenesis and maturation of the pre-60S and pre-40S particles proceed first in nucleolus and then in the nucleoplasm, most of the bound transacting proteins (not the ribosomal proteins) are sequentially released along the biogenesis pathway and recycled in the nucleolus for new rounds of biogenesis. The pre-40S and pre-60S particles that emerge from the nuclear pore complex to the cytoplasm contain only a few bound transacting proteins (reviewed in Kressler et al. 2010; Panse and Johnson 2010). These bound proteins are released in sequence from the preribosomal particles in the cytoplasm to form translation-competent mature ribosomal subunits.
eIF6 is one of the essential 60S ribosomal assembly proteins that associates with the pre-60S ribosomal particles in the nucleolus and is required for the subsequent maturation of the 60S ribosomal particles. In eIF6-depleted cells, while the 20S precursor RNA present in the pre-40S particles is processed to 18S rRNA quite efficiently, most of the 27S pre-rRNA present in the pre-60S particles is degraded without forming 25S and 5.8S rRNAs (Basu et al. 2001). Specifically, depletion of eIF6 blocks the processing of the intermediate 27SB pre-rRNA to 7S and 25.5S pre-rRNAs that are the precursors of 5.8S and 25S mature rRNAs, respectively (Basu et al. 2001 and Fig. 1c). Thus, eIF6 is necessary for the formation of 60S ribosomal subunits because it is necessary for the formation of 25S and 5.8S rRNAs, constituents of the 60S ribosomal subunit.
eIF6 (Tif6p) is Phosphorylated In Vitro and In Vivo in Mammalian and Yeast Cells
In earlier studies (Basu et al. 2003; Ray et al. 2008), it was observed that in both mammalian and yeast cells, eIF6 (Tif6p) is phosphorylated at Ser-174 (major site) and Ser-175 (minor site) by the nuclear isoform of CK1 (CK1α or β in mammals and Hrr25p in yeast). In yeast cells, these are the only sites that are phosphorylated in vivo. Mutation of Ser-174 alone to alanine abolishes phosphorylation of yeast eIF6 by >75% while mutation of both Ser-174 and Ser-175 to alanine or depletion of Hrr25 from yeast cells causes total abolition of eIF6 phosphorylation. More importantly, failure to phosphorylate eIF6 in vivo either by depletion of Hrr25p from yeast cells or alanine replacement of Ser-174 and Ser-175 of Tif6p inhibited efficient processing of preribosomal RNA to form the mature 25S and 5.8S rRNAs and thus 60S ribosome biogenesis in yeast (Ray et al. 2008). Conversely, mutation of Ser-174 alone to alanine abolishes yeast cell growth and viability. Taken together, these results suggest that phosphorylation of Tif6p at Ser-174 and Ser-175 plays an important regulatory role in the function of Tif6p. However, the molecular basis of phosphorylation of eIF6 (Tif6p), is not apparent from these studies.
Nuclear Export of eIF6 (Tif6p) Bound to the pre-60S Ribosomal Particles and Its Subsequent Release
Although the nascent pre-60S particles are mostly assembled in the nucleolus and to a lesser extent in the nucleoplasm, they are exported out of the nucleus to the cytoplasm where final maturation of the pre-60S ribosomal particles occurs to form the mature translationally competent 60S ribosomal subunits. Most of the transacting protein factors that associate with the preribosomal particles during their nucleolar assembly are released in the nucleus prior to the export of the pre-60S particles in the cytoplasm. However, a small number of protein factors including eIF6 (Tif6p) remain bound as the pre-60S particles exit the nucleus and enter the cytoplasm. The cytoplasmic ribosomal maturation pathway involves sequential and ordered release of these bound protein factors by the action of specific energy-consuming cytoplasmic ATPases or GTPases, each of which associates with the preribosomal particles to affect the release of a specific bound factor. In addition, some critical ribosomal proteins are also added at this stage to the 60S particles to make functional 60S ribosomes. The released factors are recycled back to the nucleus for another round of pre-60S ribosome assembly and export. In the ordered release of the bound factors, Tif6p and the nuclear export adapter Nmd3 are the last proteins to be released. Following their release, the pre-60S particles become mature 60 ribosomal subunits and competent to participate in translation.
Mechanism of Release of eIF6 (Tif6p) from the pre-60S Particles
In earlier studies it was reported that in mammalian cells, release of eIF6 from the pre-60S particles is triggered by phosphorylation of eIF6 at Ser-235 by protein kinase C (PKC) and RACK1 (receptor for activated kinase C) (Ceci et al. 2003). However, molecular genetic analysis in yeast cells provided compelling evidence that two cytoplasmic proteins – the GTPase elongation factor-like 1 (Efl1p) (Becam et al. 2001; Senger et al. 2001) and Sdo1, the yeast ortholog of highly conserved mammalian Shwachman–Bodian–Diamond Syndrome (SBDS) protein that is mutated in the inherited bone marrow failure and predisposition to leukemia disorder – genetically interact with the pre-60S particles containing bound eIF6 in the cytoplasm, and act cooperatively to facilitate the release of eIF6 from the pre-60S particles (Menne et al. 2007).
In yeast cells, deletion of either EFL1 or SDO1 confers a very slow growth phenotype. It was also observed that while in wild-type yeast cells, eIF6 (Tif6p) localized predominantly to the nucleolus, there was a large accumulation of Tif6p bound to the pre-60S particles in the cytoplasm of efl1Δ and sdo1Δ cells. Importantly, multiple gain of function TIF6 alleles (having a missense mutation in TIF6) that rescued the growth defect of either sdo1Δ or efl1Δ cells also restored both the nuclear export defect and nucleolar localization of Tif6p. Biochemical analysis showed that in contrast to wild-type Tif6p, which binds to 60S ribosomal subunits with a relatively high affinity, mutant eIF6 in the suppressor strains has a much reduced affinity for 60S subunits and can thus bypass the requirement of Sdo1 and Efl1p for eIF6 release (Menne et al. 2007).
Direct biochemical evidence in support of the requirement of SDO1 and EFL1 in the release of eIF6 from the pre-60S particles came from the recent elegant work of Finch et al. (2011) using a reconstituted in vitro system. These investigators isolated stalled pre-60S ribosomal particles containing bound eIF6 from sbds-deleted mouse liver. Incubation of these eIF6-bound pre-60S particles with purified recombinant human SBDS and EFL1 proteins resulted in the release of eIF6 from the pre-60S particles. This reaction requires GTP binding to EFL1 and subsequent energy of hydrolysis of GTP coupled to eIF6 release. EFL1 alone has a low intrinsic GTPase activity which is markedly stimulated by the addition of purified mature mammalian 60S ribosomal subunits (Senger et al. 2001). Addition of SBDS stimulated further 60S-dependent GTP hydrolysis by EFL1 (Finch et al. 2011). However, despite its 60S ribosome-dependent GTPase activity, EFL1 alone in the absence of SBDS is unable to promote the release of eIF6 from the 60S-found. eIF6 complex although GTP hydrolysis still occurs under these conditions. Further mechanistic studies showed that the essential role of SBDS is to tightly couple the activation of EFL1-mediated GTP hydrolysis on the 60S ribosome to eIF6 release. A conserved lysine residue at position 151 of SBDS, which is mutated in SDS disorder, is required for cooperativity with EFL1 and is essential for this coupling process.
Taken together, these observations of Menne et al. (2007) and Finch et al. (2011) provide compelling evidence that the mechanism of eIF6 release from the pre-60S particles in the cytoplasm during the final maturation of 60S ribosomal subunits is highly conserved between yeast and mammals and involves cooperative interaction of SBDS and EFL1 in mediating the GTP hydrolysis-dependent release of eIF6 from the pre-60S particles. In contrast to the report of Ceci et al. (2003), there was no evidence for the requirement of phosphorylation of eIF6 at Ser-235 for its release.
Recycling of eIF6: Opposing Action of Casein Kinase 1 and Calcineurin Phosphatase in Nucleo-Cytoplasmic Shuttling of eIF6
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The presence of a biologically important CK1 phosphorylation site and a calcineurin docking motif in nucleated species gave rise to the hypothesis that the nuclear entry and export of eIF6 might be regulated by CK1 phosphorylation and dephosphorylation by calcineurin as was described for NFAT family of transcription factors in vertebrates and a stress-responsive transcription factor, Crz1p, in yeast (reviewed in Biswas et al. (2011)).
eIF6, a highly conserved protein from yeast to mammals, binds to the 60S ribosomal subunit and functions as a ribosomal subunit anti-association factor by preventing premature association with the 40S ribosomal subunit. Molecular genetic analysis in yeast has provided compelling evidence that eIF6 is not a canonical translation initiation factor. Rather, it is essential for 60S ribosome biogenesis and assembly. Specifically, eIF6 associates with the pre-60S particles in the nucleolus along with many other transacting protein factors and is essential for 60S ribosome assembly and pre-rRNA processing to form the mature rRNAs, constituents of the 60S ribosomal particle. Association of eIF6 with the pre-60S particles is also required for the export of the assembled pre-60S particles from the nucleus to the cytoplasm where the release of eIF6 occurs. Nuclear export of the pre-60S particles containing bound eIF6 requires phosphorylation of eIF6 at Ser-174 and Ser-175 by the nuclear isoform of CK1. In the cytoplasm, during the final maturation of the pre-60S particles, two cytoplasmic proteins SBDS/Sdo1 and EFL1/Efl1p interact with the pre-60S particles and catalyze the release of eIF6 coupled to hydrolysis of GTP by EFL1. The released eIF6 that is presumably in the phosphorylated form then interacts with Ca2+/calinodulin-dependent protein phosphatase calcineurin and the dephosphorylated form of eIF6 is imported to the nucleus to participate in another round of 60S ribosome assembly and biogenesis. These observations suggest eIF6 plays an important regulatory role in 60S ribosome biogenesis in eukaryotic cells.
Since the original isolation of eIF6 as a ribosomal subunit anti-association factor, it is now clear that this activity of eIF6 is not utilized to generate free 40S and 60S ribosomal subunits required for initiation of protein synthesis. Rather, association of eIF6 with the pre-60S particles in the nucleolus prevents their premature interaction with the pre-40S particles both in the nucleus and cytoplasm. eIF6 is released only when all other bound factors dissociate from the pre-60 particle leading to the formation of mature 60S ribosomal subunit. Secondly, the essential requirement of SBDS (along with the GTPase EFL1) in the release of eIF6 from the pre-60S particles underscores the importance of this protein in 60S ribosome biogenesis. Mutation of the SBDS gene has been shown to be associated in the inherited leukemia predisposition disorder Shwachman–Diamond syndrome (SDS). Some of the disease-causing mutant proteins are unable to catalyze the release of eIF6 from the pre-60S particles causing defects in 60S ribosome maturation and biogenesis. Thus, SDS has been defined as a “ribosomopathy” by Finch et al. (2011). Finally, the requirement of Ca2+-activated calcineurin phosphatase in the nuclear import of eIF6 could provide a means by which changes in intracellular Ca2+ levels could modulate nuclear import of eIF6, and consequently, nucleolar 60S ribosome biogenesis. Clearly, a detailed molecular investigation of these steps is necessary to understand various quality control mechanisms in the pathway of ribosome biogenesis.
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