The RNA helicase,eIF4A‐1, is required for ovule development and cell size homeostasis in Arabidopsis

eIF4A is a highly conserved RNA‐stimulated ATPase and helicase involved in the initiation of mRNA translation. The Arabidopsis genome encodes two isoforms, one of which (eIF4A‐1) is required for the coordination between cell cycle progression and cell size. A T‐DNA mutant eif4a1 line, with reduced eIF4A protein levels, displays slow growth, reduced lateral root formation, delayed flowering and abnormal ovule development. Loss of eIF4A‐1 reduces the proportion of mitotic cells in the root meristem and perturbs the relationship between cell size and cell cycle progression. Several cell cycle reporter proteins, particularly those expressed at G2/M, have reduced expression in eif4a1 mutant meristems. Single eif4a1 mutants are semisterile and show aberrant ovule growth, whereas double eif4a1 eif4a2 homozygous mutants could not be recovered, indicating that eIF4A function is essential for plant growth and development.     

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

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.Key words: CK1, eIF6, PKC, protein translation, RACK1, ribosome assembly, ribosome biogenesisEukaryotic 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.¹⁵Open in a separate windowFigure 1A 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.     

Opposing action of casein kinase 1 and calcineurin in nucleo-cytoplasmic shuttling of mammalian translation initiation factor eIF6

Eukaryotic initiation factor 6 (eIF6), a highly conserved protein from yeast to mammals, is essential for 60 S ribosome biogenesis and assembly. Both yeast and mammalian eIF6 are phosphorylated at Ser-174 and Ser-175 by the nuclear isoform of casein kinase 1 (CK1). The molecular basis of eIF6 phosphorylation, however, remains elusive. In the present work, we show that subcellular distribution of eIF6 in the nuclei and the cytoplasm of mammalian cells is mediated by dephosphorylation and phosphorylation, respectively. This nucleo-cytoplasmic shuttling is dependent on the phosphorylation status at Ser-174 and Ser-175 of eIF6. We demonstrate that Ca(2+)-activated calcineurin phosphatase binds to and promotes nuclear localization of eIF6. Increase in intracellular concentration of Ca(2+) leads to rapid translocation of eIF6 from the cytoplasm to the nucleus, an event that is blocked by specific calcineurin inhibitors cyclosporin A or FK520. Nuclear export of eIF6 is regulated by phosphorylation at Ser-174 and Ser-175 by the nuclear isoform of CK1. Mutation of eIF6 at the phosphorylatable Ser-174 and Ser-175 to alanine or treatment of cells with the CK1 inhibitor, D4476 inhibits nuclear export of eIF6 and results in nuclear accumulation of eIF6. Together, these results establish eIF6 as a substrate for calcineurin and suggest a novel paradigm for calcineurin function in 60 S ribosome biogenesis via regulating the nuclear accumulation of eIF6.     

BCCIP is required for nucleolar recruitment of eIF6 and 12S pre-rRNA production during 60S ribosome biogenesis

Ribosome biogenesis is a fundamental process required for cell proliferation. Although evolutionally conserved, the mammalian ribosome assembly system is more complex than in yeasts. BCCIP was originally identified as a BRCA2 and p21 interacting protein. A partial loss of BCCIP function was sufficient to trigger genomic instability and tumorigenesis. However, a complete deletion of BCCIP arrested cell growth and was lethal in mice. Here, we report that a fraction of mammalian BCCIP localizes in the nucleolus and regulates 60S ribosome biogenesis. Both abrogation of BCCIP nucleolar localization and impaired BCCIP–eIF6 interaction can compromise eIF6 recruitment to the nucleolus and 60S ribosome biogenesis. BCCIP is vital for a pre-rRNA processing step that produces 12S pre-rRNA, a precursor to the 5.8S rRNA. However, a heterozygous Bccip loss was insufficient to impair 60S biogenesis in mouse embryo fibroblasts, but a profound reduction of BCCIP was required to abrogate its function in 60S biogenesis. These results suggest that BCCIP is a critical factor for mammalian pre-rRNA processing and 60S generation and offer an explanation as to why a subtle dysfunction of BCCIP can be tumorigenic but a complete depletion of BCCIP is lethal.     

Control of Paip1-Eukayrotic Translation Initiation Factor 3 Interaction by Amino Acids through S6 Kinase

The simultaneous interaction of poly(A)-binding protein (PABP) with eukaryotic translation initiation factor 4G (eIF4G) and the mRNA 3′ poly(A) tail promotes translation initiation. We previously showed that the interaction of PABP-interacting protein 1 (Paip1) with PABP and eukaryotic translation initiation factor 3 (eIF3; via the eIF3g subunit) further stimulates translation. Here, we demonstrate that the interaction of eIF3 with Paip1 is regulated by amino acids through the mTORC1 signaling pathway. The Paip1-eIF3 interaction is impaired by the mTORC1 inhibitors, rapamycin and PP242. We show that ribosomal protein S6 kinases 1 and 2 (S6K1/2) promote the interaction of eIF3 with Paip1. The enhancement of Paip1-eIF3 interaction by amino acids is abrogated by an S6K inhibitor or shRNA against S6K1/2. S6K1 interacts with eIF3f and, in vitro, phosphorylates eIF3. Finally, we show that S6K inhibition leads to a reduction in translation by Paip1. We propose that S6K1/2 phosphorylate eIF3 to stimulate Paip1-eIF3 interaction and consequent translation initiation. Taken together, these data demonstrate that eIF3 is a new translation target of the mTOR/S6K pathway.     

The nucleolar protein SAHY1 is involved in pre-rRNA processing and normal plant growth

Although the nucleolus is involved in ribosome biogenesis, the functions of numerous nucleolus-localized proteins remain unclear. In this study, we genetically isolated Arabidopsis thaliana salt hypersensitive mutant 1 (sahy1), which exhibits slow growth, short roots, pointed leaves, and sterility. SAHY1 encodes an uncharacterized protein that is predominantly expressed in root tips, early developing seeds, and mature pollen grains and is mainly restricted to the nucleolus. Dysfunction of SAHY1 primarily causes the accumulation of 32S, 18S-A₃, and 27SB pre-rRNA intermediates. Coimmunoprecipitation experiments further revealed the interaction of SAHY1 with ribosome proteins and ribosome biogenesis factors. Moreover, sahy1 mutants are less sensitive to protein translation inhibitors and show altered expression of structural constituents of ribosomal genes and ribosome subunit profiles, reflecting the involvement of SAHY1 in ribosome composition and ribosome biogenesis. Analyses of ploidy, S-phase cell cycle progression, and auxin transport and signaling indicated the impairment of mitotic activity, translation of auxin transport carrier proteins, and expression of the auxin-responsive marker DR5::GFP in the root tips or embryos of sahy1 plants. Collectively, these data demonstrate that SAHY1, a nucleolar protein involved in ribosome biogenesis, plays critical roles in normal plant growth in association with auxin transport and signaling.

SALT-HYPERSENSITIVE MUTANT 1, a nucleolar protein involved in ribosome biogenesis, regulates the auxin-mediated development of vegetative and reproductive tissues.     

Fluorescence-tagged transgenic lines reveal genetic defects in pollen growth--application to the eIF3 complex

Background

Mutations in several subunits of eukaryotic translation initiation factor 3 (eIF3) cause male transmission defects in Arabidopsis thaliana. To identify the stage of pollen development at which eIF3 becomes essential it is desirable to examine viable pollen and distinguish mutant from wild type. To accomplish this we have developed a broadly applicable method to track mutant alleles that are not already tagged by a visible marker gene through the male lineage of Arabidopsis.

Methodology/Principal Findings

Fluorescence tagged lines (FTLs) harbor a transgenic fluorescent protein gene (XFP) expressed by the pollen-specific LAT52 promoter at a defined chromosomal position. In the existing collection of FTLs there are enough XFP marker genes to track nearly every nuclear gene by virtue of its genetic linkage to a transgenic marker gene. Using FTLs in a quartet mutant, which yields mature pollen tetrads, we determined that the pollen transmission defect of the eif3h-1 allele is due to a combination of reduced pollen germination and reduced pollen tube elongation. We also detected reduced pollen germination for eif3e. However, neither eif3h nor eif3e, unlike other known gametophytic mutations, measurably disrupted the early stages of pollen maturation.

Conclusion/Significance

eIF3h and eIF3e both become essential during pollen germination, a stage of vigorous translation of newly transcribed mRNAs. These data delimit the end of the developmental window during which paternal rescue is still possible. Moreover, the FTL collection of mapped fluorescent protein transgenes represents an attractive resource for elucidating the pollen development phenotypes of any fine-mapped mutation in Arabidopsis.     

The Saccharomyces cerevisiae Homologue of Mammalian Translation Initiation Factor 6 Does Not Function as a Translation Initiation Factor

Eukaryotic translation initiation factor 6 (eIF6) binds to the 60S ribosomal subunit and prevents its association with the 40S ribosomal subunit. The Saccharomyces cerevisiae gene that encodes the 245-amino-acid eIF6 (calculated M_r 25,550), designated TIF6, has been cloned and expressed in Escherichia coli. The purified recombinant protein prevents association between 40S and 60S ribosomal subunits to form 80S ribosomes. TIF6 is a single-copy gene that maps on chromosome XVI and is essential for cell growth. eIF6 expressed in yeast cells associates with free 60S ribosomal subunits but not with 80S monosomes or polysomal ribosomes, indicating that it is not a ribosomal protein. Depletion of eIF6 from yeast cells resulted in a decrease in the rate of protein synthesis, accumulation of half-mer polyribosomes, reduced levels of 60S ribosomal subunits resulting in the stoichiometric imbalance in the 40S/60S subunit ratio, and ultimately cessation of cell growth. Furthermore, lysates of yeast cells depleted of eIF6 remained active in translation of mRNAs in vitro. These results indicate that eIF6 does not act as a true translation initiation factor. Rather, the protein may be involved in the biogenesis and/or stability of 60S ribosomal subunits.     

Targeting capacity and conservation of PreP homologues localization in mitochondria of different species

Mitochondrial presequences and other unstructured peptides are degraded inside mitochondria by presequence proteases (PrePs) identified in Arabidopsis thaliana (AtPreP), humans (hPreP), and yeast (Cym1/Mop112). The presequences of A. thaliana and human PreP are predicted to consist of 85 and 29 amino acids, respectively, whereas the Saccharomyces cerevisiae Cym1/Mop112 presequence contains only 7 residues. These differences may explain the reported targeting of homologous proteins to different mitochondrial subcompartments. Here we have investigated the targeting capacity of the PreP homologues' presequences. We have produced fusion constructs containing N-terminal portions of AtPreP(1-125), hPreP(1-69), and Cym1(1-40) coupled to green fluorescent protein (GFP) and studied their import into isolated plant, mammalian, and yeast mitochondria, followed by mitochondrial subfractionation. Whereas the AtPreP presequence has the capacity to target GFP into the mitochondrial matrix of all three species, the hPreP presequence only targets GFP to the matrix of mammalian and yeast mitochondria. The Cym1/Mop112 presequence has an overall much weaker targeting capacity and only ensures mitochondrial sorting in its host species yeast. Revisiting the submitochondrial localization of Cym1 revealed that endogenous Cym1/Mop112 is localized to the matrix space, as has been previously reported for the plant and human homologues. Moreover, complementation studies in yeast show that native AtPreP restores the growth phenotype of yeast cells lacking Cym1, demonstrating functional conservation.     

40S Ribosome Biogenesis Co-Factors Are Essential for Gametophyte and Embryo Development

Ribosome biogenesis is well described in Saccharomyces cerevisiae. In contrast only very little information is available on this pathway in plants. This study presents the characterization of five putative protein co-factors of ribosome biogenesis in Arabidopsis thaliana, namely Rrp5, Pwp2, Nob1, Enp1 and Noc4. The characterization of the proteins in respect to localization, enzymatic activity and association with pre-ribosomal complexes is shown. Additionally, analyses of T-DNA insertion mutants aimed to reveal an involvement of the plant co-factors in ribosome biogenesis. The investigated proteins localize mainly to the nucleolus or the nucleus, and atEnp1 and atNob1 co-migrate with 40S pre-ribosomal complexes. The analysis of T-DNA insertion lines revealed that all proteins are essential in Arabidopsis thaliana and mutant plants show alterations of rRNA intermediate abundance already in the heterozygous state. The most significant alteration was observed in the NOB1 T-DNA insertion line where the P-A3 fragment, a 23S-like rRNA precursor, accumulated. The transmission of the T-DNA through the male and female gametophyte was strongly inhibited indicating a high importance of ribosome co-factor genes in the haploid stages of plant development. Additionally impaired embryogenesis was observed in some mutant plant lines. All results support an involvement of the analyzed proteins in ribosome biogenesis but differences in rRNA processing, gametophyte and embryo development suggested an alternative regulation in plants.     

Genome-wide identification and characterization of the RIO atypical kinase family in plants

Members of the right open reading frame (RIO) atypical kinase family are present in all three domains of life. In eukaryotes, three subfamilies have been identified: RIO1, RIO2, and RIO3. Studies have shown that the yeast and human RIO1 and RIO2 kinases are essential for the biogenesis of small ribosomal subunits. Thus far, RIO3 has been found only in multicellular eukaryotes. In this study, we systematically identified members of the RIO gene family in 37 species representing the major evolutionary lineages in Viridiplantae. A total of 84 RIO genes were identified; among them, 41 were classified as RIO1 and 43 as RIO2. However, no RIO3 gene was found in any of the species examined. Phylogenetic trees constructed for plant RIO1 and RIO2 proteins were generally congruent with the species phylogeny. Subcellular localization analyses showed that the plant RIO proteins were localized mainly in the nucleus and/or cytoplasm. Expression profile analysis of rice, maize, and Arabidopsis RIO genes in different tissues revealed similar expression patterns between RIO1 and RIO2 genes, and their expression levels were high in certain tissues. In addition, the expressions of plant RIO genes were regulated by two drugs: mycophenolic acid and actinomycin D. Function prediction using genome-wide coexpression analysis revealed that most plant RIO genes may be involved in ribosome biogenesis. Our results will be useful for the evolutionary analysis of the ancient RIO kinase family and provide a basis for further functional characterization of RIO genes in plants.     

Translational control by 80S formation and 60S availability: The central role of eIF6, a rate limiting factor in cell cycle progression and tumorigenesis

Ribosome biogenesis and translation can be simplified as the processes of generating ribosomes and their use for decoding mRNA into a protein. Ribosome biogenesis has been efficiently studied in unicellular organisms like the budding yeast, allowing us a deep and basic knowledge of this process in growing cells. Translation has been modeled in vitro and in unicellular organisms. These studies have given us an important insight into the mechanisms and evolutionarily conserved aspects of ribosome biology. However, we advocate the need of the direct study of these processes in multicellular organisms. Analysis of ribosome biogenesis and translation in vivo in Metazoa and mammalian models is emerging and unveils the unexpected consequences of perturbed ribosome biogenesis and translation. Here, we will describe how one factor, eIF6, plays a crucial role both in the generation of the large ribosomal subunit and its availability for translation. From there, we will make specific conclusions on the physiological relevance of eIF6 in 80S formation, cell cycle progression and disease, raising the point that the control of gene expression may occur at the unexpected level of the large ribosomal subunit. In the future, the modulation of eIF6 binding to the 60S may be pharmacologically exploited to reduce the growth of cancer cells or ameliorate the phenotype of SDS syndrome.     

Related eIF3 subunits TIF32 and HCR1 interact with an RNA recognition motif in PRT1 required for eIF3 integrity and ribosome binding

eIF3 binds to 40S ribosomal subunits and stimulates recruitment of Met-tRNAiMet and mRNA to the pre-initiation complex. Saccharomyces cerevisiae contains an ortholog of human eIF3 subunit p35, HCR1, whose interactions with yeast eIF3 are not well defined. We found that HCR1 has a dual function in translation initiation: it binds to, and stabilizes, the eIF3-eIF5- eIF1-eIF2 multifactor complex and is required for the normal level of 40S ribosomes. The RNA recognition motif (RRM) of eIF3 subunit PRT1 interacted simultaneously with HCR1 and with an internal domain of eIF3 subunit TIF32 that has sequence and functional similarity to HCR1. PRT1, HCR1 and TIF32 were also functionally linked by genetic suppressor analysis. We propose that HCR1 stabilizes or modulates interaction between TIF32 and the PRT1 RRM. Removal of the PRT1 RRM resulted in dissociation of TIF32, NIP1, HCR1 and eIF5 from eIF3 in vivo, and destroyed 40S ribosome binding by the residual PRT1-TIF34-TIF35 subcomplex. Hence, the PRT1 RRM is crucial for the integrity and ribosome-binding activity of eIF3.     

Optimized expression of the dirigent protein AtDIR6 in Pichia pastoris and impact of glycosylation on protein structure and function

Phenoxy radical coupling reactions are involved in the biosynthesis of lignans in planta. Interestingly, the reaction can be guided by dirigent proteins, which mediate the stereoselective formation of either (+) or (?)-pinoresinol from coniferyl alcohol. So far, the mechanism is poorly understood, and for detailed mechanistic studies, a heterologous expression platform which allows the cost-effective, fast, and robust expression in high yields is needed. We established a reliable, high-yield fed-batch fermentation process with Pichia pastoris resulting in 47 mg?L^?1 of the dirigent protein AtDIR6, which represents a more than 250-fold increase compared to previous studies. Biochemical characterization of AtDIR6 produced with P. pastoris showed an overall agreement in protein structure, N-glycosylation sites, and dirigent activity compared to AtDIR6 produced by plant cell cultures of Solanum peruvianum. CD spectroscopy verified the β-barrel structure proposed by earlier studies and bioconversion experiments revealed similar activities to plant-derived protein, validating P. pastoris as a suitable expression system for dirigent proteins. Compared to the complex glycan structures of most plant cells, proteins produced with P. pastoris have the advantage that they can be enzymatically deglycosylated under non-denaturating conditions. With this study, we demonstrate that the glycan structures of AtDIR6 are essential for structure, solubility, and function of the protein as deglycosylation induced conformational changes leading to the complete loss in dirigent activity and subsequent protein aggregation.