Eukaryotic initiation factor 6, an evolutionarily conserved regulator of ribosome biogenesis and protein translation

Abstract

We recently identified Receptor for Activated C Kinase 1 (RACK1) as one of the molecular links between abscisic acid (ABA) signaling and its regulation on protein translation. Moreover, we identified Eukaryotic Initiation Factor 6 (eIF6) as an interacting partner of RACK1. Because the interaction between RACK1 and eIF6 in mammalian cells is known to regulate the ribosome assembly step of protein translation initiation, it was hypothesized that the same process of protein translation in Arabidopsis is also regulated by RACK1 and eIF6. In this article, we analyzed the amino acid sequences of eIF6 in different species from different lineages and discovered some intriguing differences in protein phosphorylation sites that may contribute to its action in ribosome assembly and biogenesis. In addition, we discovered that, distinct from non-plant organisms in which eIF6 is encoded by a single gene, all sequenced plant genomes contain two or more copies of eIF6 genes. While one copy of plant eIF6 is expressed ubiquitously and might possess the conserved function in ribosome biogenesis and protein translation, the other copy seems to be only expressed in specific organs and therefore may have gained some new functions. We proposed some important studies that may help us better understand the function of eIF6 in plants.

Eukaryotic Initiation Factor 6 (eIF6) was originally purified from wheat germ¹ and was found to function as a ribosome dissociation factor through binding to the 60S ribosome subunit and preventing its association with the 40S ribosome subunit.² Its homologous proteins were later purified from rabbit reticulocyte lysates,³ calf liver⁴ and human cells.⁴ The action of eIF6 in preventing the ribosome subunits association was later found to involve another two proteins, the activated Protein Kinase C (PKC) and the Receptor for Activated C Kinase 1 (RACK1) in mammalian cells.⁵ PKC is a family of proteins that can be activated by elevated cellular concentration of Ca²⁺ or diacylglycerol and is involved in multiple signal transduction pathways in mammalian cells.⁶ RACK1 was identified as a receptor for activated PKC, anchoring PKC to the subcellular location where its substrate is present.^7,8 In this protein complex, RACK1 serves as a scaffold protein that simultaneously binds to eIF6 and activated PKC to bring these two proteins to close proximity. PKC then phosphorylates eIF6, leading to its dissociation from the 60S ribosome subunit and consequently allowing the association between the 40S and 60S ribosome subunits to assemble a functional 80S subunit to initiate protein translation⁵ (Fig. 1). Genetic studies supported the role of mammalian eIF6 in protein translation initiation as well as in cell growth.⁹ More recent structural studies supported a similar role of eIF6 in regulating 80S ribosome assembly in yeast.^10,11 Yeast eIF6 (Tif6p) was also known to regulate 60S ribosome biogenesis.^12,13 Very recently, eIF6 was identified as a component of a protein complex that interacts with the RNA-induced silencing complex and plays a role in microRNA-directed gene silencing.¹⁴ For a more comprehensive review of eIF6's function in mammalian cells and in yeast, readers should refer to the following review article.¹⁵

Figure 1.

A schematic presentation of the proposed molecular mode of action of eIF6, RACK1, PKC and CK1 in ribosome assembly and protein translation. Nuclear CK1 phosphorylates eIF6 at Serine 174 and Serine 175. This phosphorylation is required for the shuttling of eIF6 from nucleus into cytosol. eIF6 prevents joining of the cytosolic 60S ribosome subunit with the 40S subunit from forming a functional 80S ribosome. RACK1, via binding simultaneously to the eIF6 and the activated PKC, can facilitate the phosphorylation of eIF6 at Serine 235 by PKC. The Serine 235 phosphorylated eIF6 then disassociates from 60S ribosome, thus allowing the assembly of functional 80S ribosome and initiation of protein translation. CK1, Casein Kinase 1; 60S, 60S ribosome subunit; 40S, 40S ribosome subunit; eIF6, Eukaryotic Initiation Factor 6; RACK1, Receptor for Activated C Kinase 1; PKC, Protein Kinase C; pi, phosphate.

Despite considerable progress that has been made in the identification of central components of plant hormone abscisic acid (ABA) signaling, little is known about the molecular mechanism of the long-recognized effect of ABA on protein translation. Our group has been working on the functional analysis of Arabidopsis RACK1 gene family,^16–19 and has identified RACK1 as a negative regulator of ABA responses.¹⁸ Recently, we discovered that RACK1 may play a role in ribosome assembly and 60S ribosome subunit biogenesis and therefore serve as one of the molecular links between ABA signaling and its control on protein translation.²⁰ In addition, we discovered that RACK1 physically interacts with eIF6 in a yeast two-hybrid assay and in a Bi-molecular Fluorescence Complementation assay in an Arabidopsis leaf mesophyll protoplast system. The conserved interaction between RACK1 and eIF6 in plants and in mammals implies an evolutionarily conserved role of eIF6 and RACK1 in ribosome biogenesis, assembly and protein translation.

Conservation of eIF6

eIF6 is highly conserved across eukaryotic organisms including mammals, plants, yeast, worm and green algae (Fig. 2). Homologs of eIF6 were even discovered in Archaea lineage, although no eIF6 was found in the Eubacteria lineage.¹⁵ Interestingly, the phosphorylation site (Serine 235) for PKC of eIF6 protein identified in human cell lines⁵ is not present in non-mammal organisms (Fig. 2), implying that either a different phosphorylation site might be responsible for the regulation of the dissociation of eIF6 from the 60S ribosome, or a different regulatory mechanism is present in other organisms. In addition to its anti-association effect, eIF6 is also known to regulate the 60S ribosome biogenesis.^12,21 Its function in this process relies on the phosphorylation status of Serine 174 and Serine 175. Such phosphorylations also regulate eIF6's nucleus to cytoplasm shuttling.²¹ The kinases that phosphorylate these residues are the Casein Kinase 1 (CK1) family of serine/threonine kinases.¹³ CK1 family is present in Arabidopsis,²² and the proposed phosphorylation sites of eIF6 for CK1 are highly-conserved in eIF6A/At-eIF6;1 (but not in eIF6B/At-eIF6;2) (Fig. 2). In a more recent study, eIF6A/At-eIF6;1 was found to be able to complement yeast eIF6 mutant (tif6).²³ Therefore it is plausible that Arabidopsis eIF6A/At-eIF6;1 and CK1 might also work together to regulate 60S ribosome subunit biogenesis.

Figure 2.

Amino acid alignment of eIF6 in five different species from different lineages. The bar below each amino acid indicates the percentage of conservation at that position across different species. The Casein Kinase 1 phosphorylation sites are marked with “**” and the Protein Kinase C phosphorylation site is marked with “#”. The phylogenetic tree of the six eIF6 homologs in five species was constructed using a web-based phylogeny pipeline (http://www.phylogeny.fr/).²⁴ HseIF6, Homo sapiens, Gene Bank ID: 220675563; CseIF6, Caenorhabditis elegans, Gene Bank ID: 3874970; TIF6, Saccharomyces cerevisiae, Gene Bank ID: 6325273; CreIF6, Chlamydomonas reinhardtii, Gene Bank ID: 158283109; AteIF6A, At3g55620, Arabidopsis thaliana); AteIF6B, At2g39820, Arabidopsis thaliana.

Divergence of eIF6 in Plants

While only a single copy of eIF6 gene is found in non-plant genomes, all the sequenced plant genomes contain two eIF6 genes except the Populus genome which contains three eIF6 genes (Fig. 3). Although both Arabidopsis eIF6A/At-eIF6;1 and eIF6B/At-eIF6;2 were able to interact with RACK1,²⁰ the expression patterns of eIF6A/At-eIF6;1 and eIF6B/At-eIF6;2 across various tissues and organs are very different. The Arabidopsis eIF6A/At-eIF6;1 is expressed ubiquitously (Fig. 4A) whereas eIF6B/At-eIF6;2 is only expressed in pollen (Fig. 4B). Furthermore, a key difference between eIF6A/At-eIF6;1 and eIF6B/At-eIF6;2 was also observed at the amino acid level. eIF6B/At-eIF6;2 lacks a key phosphorylation site (Ser 174) that is highly conserved across various species serving as one of the phosphorylation sites for CK1 (Figs. 2 and 3). Interestingly, a similar difference in expression patterns of eIF6 homologous genes was also present in rice, with one copy of eIF6 gene (Os07g44620) expressed ubiquitously (Fig. 4C) whereas the other copy (Os01g17330) expressed only at inflorescence and very early stage of seed development (Fig. 4D). This phenomenon implies that a duplication of eIF6 gene event may have occurred after the separation of plant and animal lineage during evolution, and that one copy of plant eIF6 gene might have gone through the process of subfunctionalization or neofunctionalization after the separation.

Figure 3.

Alignment of eIF6 amino acid sequences in six different sequenced plant genomes. The bar below each amino acid indicates the percentage of conservation at that position across different genomes. The Casein Kinase 1 phosphorylation sites are marked with “**”. AteIF6A, At3g55620, Arabidopsis thaliana; AteIF6B, At2g39820, Arabidopsis thaliana; PteIF6A, Populus trichocarpa, Gene Bank ID: 222864377; PteIF6B, Populus trichocarpa, Gene Bank ID: 222855867); PteIF6C, Populus trichocarpa, Gene Bank ID: 222853012; OseIF6A, Os01g17330, Oryza sativa Japonica Group; OseIF6B, Os07g44620, Oryza sativa Japonica Group; BdeIF6A, Bradi1g19960.1, Brachypodium distachyon; BdeIF6B, Bradi2g10860, Brachypodium distachyon; SbeIF6A, 03g011410, Sorghum bicolor; SbeIF6B, 07g022630, Sorghum bicolor; PpeIF6A, Physcomitrella patens subsp. patens, Gene Bank ID: 162671129; PpeIF6B, Physcomitrella patens subsp. patens, Gene Bank ID: 162664054.

Figure 4.

The relative expression level of (A) Arabidopsis eIF6A, (B) Arabidopsis eIF6B, (C) Rice eIF6 homolog Os07g44620 and (D) Rice eIF6 homolog Os01g17330. Heatmaps were generated using the eFP browser.²⁵

Concluding Remarks and Future Directions

While many studies have been performed to characterize the function of eIF6 in yeasts and mammals, little is known about the function of eIF6 in plants. In order to better understand how ABA signaling regulates protein translation, it would be interesting to further investigate the role of eIF6 in this process. However, because the null allele of eIF6A/At-eIF6;1 is embryo lethal,^20,23 it is necessary to generate weak alleles of eIF6A/At-eIF6;1 (e.g., using RNA interference approach) to examine the role of eIF6 in ribosome subunit biogenesis, assembly and protein translation, and its role in ABA signaling. Another interesting question is whether eIF6A/At-eIF6;1 and eIF6B/At-eIF6;2 have different functions. A genetic complementation approach in which a chimeric fusion of eIF6B/At-eIF6;2 gene with eIF6A/At-eIF6;1 promoter introduced into eIF6A/At-eIF6;1 mutant background can be used to explore the functional equivalency of eIF6A/At-eIF6;1 and eIF6B/At-eIF6;2 at the protein level. Furthermore, it would be interesting to identify the kinases that use eIF6 as substrate. The plant genomes do not contain genes encoding apparent PKCs, and the proposed PKC phosphorylation site of eIF6 proteins seems to be mammal-specific. Therefore the interaction between RACK1 and eIF6 and their potential roles in the regulation of protein translation might involve different mechanisms or different protein kinases. In contrast, the phosphorylation sites of eIF6 at Serine 174 by CK1 are highly conserved in plants, although this phosphorylation site of eIF6 is very likely responsible for its role in 60S ribosome biogenesis. Our recent study pointed to a possibility that eIF6, by interacting with RACK1 and possibly other partners, might regulate protein translation.²⁰ In addition, similar to RACK1, eIF6 might also be a part of the molecular link between ABA signaling and its downstream events in regulating protein translation. Therefore, understanding the function of plant eIF6 in ribosome subunit biogenesis and assembly, and especially its potential role in ABA-regulated protein translation, may help advance our understanding of ABA signaling network.

Abbreviations

40S

40S ribosome subunit

60S

60S ribosome subunit

ABA

abscisic acid

CK1

casein kinase 1

eIF6

eukaryotic initiation factor 6

PKC

protein kinase C

RACK1

receptor for activated C kinase 1

Addendum to: Guo J, Wang S, Valerius O, Hall H, Zeng Q, Li JF, et al. Involvement of Arabidopsis RACK1 in protein translation and its regulation by abscisic acid. Plant Physiol. 2011;155:370–383. doi: 10.1104/pp.110.16066.