Dual function of eIF3j/Hcr1p in processing 20 S pre-rRNA and translation initiation.

eIF3j/Hcr1p, a protein associated with eIF3, was shown to bind to, and stabilize, the multifactor complex containing eIFs 1, 2, 3, and 5 and Met-tRNA(i)(Met), whose formation is required for an optimal rate of translation initiation. Here we present evidence that eIF3j/Hcr1p is an RNA binding protein that enhances a late step in 40 S ribosome maturation involving cleavage of the 20 S precursor of 18 S rRNA in the cytoplasm. Immunofluorescence staining shows that eIF3j/Hcr1p is localized predominantly in the cytoplasm. The hcr1Delta mutant exhibits a decreased amount of 40 S subunits, hypersensitivity to paromomycin, and increased levels of 20 S pre-rRNA. Combining the hcr1Delta mutation with drs2Delta or rps0aDelta, deletions of two other genes involved in the same step of 40 S subunit biogenesis, produced a synthetic growth defect. p35, the human ortholog of eIF3j/Hcr1p, partially complemented the slow growth phenotype conferred by hcr1Delta when overexpressed in yeast. heIF3j/p35 was found physically associated with yeast eIF3 and 43 S initiation complexes in vitro and in vivo. Because it did not complement the 40 S biogenesis defect of hcr1Delta, it appears that heIF3j can substitute for eIF3j/Hcr1p only in translation initiation. We conclude that eIF3j/Hcr1p is required for rapid processing of 20 S to 18 S rRNA besides its role in translation initiation, providing an intriguing link between ribosome biogenesis and translation.

Saccharomyces cerevisiae protein Pci8p and human protein eIF3e/Int-6 interact with the eIF3 core complex by binding to cognate eIF3b subunits.

Mammalian, plant, and Schizosaccharomyces pombe eukaryotic initiation factor-3 (eIF3) contains a protein homologous to the product of int-6 (eIF3e), a frequent integration site of mouse mammary tumor viruses. By contrast, Saccharomyces cerevisiae does not encode a protein closely related to eIF3e/Int-6. Here, we characterize a novel S. cerevisiae protein (Pci8p, Yil071cp) that contains a PCI (proteasome-COP9 signalosome-eIF3) domain conserved in eIF3e/Int-6. We show that both Pci8p and human eIF3e/Int-6 expressed in budding yeast interact with the yeast eIF3 complex in vivo and in vitro by binding to a discrete segment of its eIF3b subunit Prt1p and that human eIF3e/Int-6 interacts with the human eIF3b segment homologous to the Pci8p-binding site of yeast Prt1p. These results refine our understanding of subunit interactions in the eIF3 complex and suggest structural similarity between human eIF3e/Int-6 and yeast Pci8p. However, deletion of PCI8 had no discernible effect on cell growth or translation initiation as judged by polysome analysis, suggesting that Pci8p is not required for the essential function of eIF3 in translation initiation. Motivated by the involvement of Int-6 in transcriptional control, we investigated the effects of deleting PCI8 on the total mRNA expression profile by oligonucleotide microarray analysis and found reduced mRNA levels for a subset of heat shock proteins in the pci8Delta mutant. We discuss possible dual functions of Pci8p and Int-6 in transcriptional and translational control.

A subcomplex of three eIF3 subunits binds eIF1 and eIF5 and stimulates ribosome binding of mRNA and tRNA(i)Met.

Yeast translation initiation factor 3 contains five core subunits (known as TIF32, PRT1, NIP1, TIF34 and TIF35) and a less tightly associated component known as HCR1. We found that a stable subcomplex of His8-PRT1, NIP1 and TIF32 (PN2 subcomplex) could be affinity purified from a strain overexpressing these eIF3 subunits. eIF5, eIF1 and HCR1 co-purified with this subcomplex, but not with distinct His8-PRT1- TIF34-TIF35 (P45) or His8-PRT1-TIF32 (P2) sub complexes. His8-PRT1 and NIP1 did not form a stable binary subcomplex. These results provide in vivo evidence that TIF32 bridges PRT1 and NIP1, and that eIFs 1 and 5 bind to NIP1, in native eIF3. Heat-treated prt1-1 extracts are defective for Met-tRNA(i)Met binding to 40S subunits, and we also observed defective 40S binding of mRNA, eIFs 1 and 5 and eIF3 itself in these extracts. We could rescue 40S binding of Met- tRNA(i)Met and mRNA, and translation of luciferase mRNA, in a prt1-1 extract almost as well with purified PN2 subcomplex as with five-subunit eIF3, whereas the P45 subcomplex was nearly inactive. Thus, several key functions of eIF3 can be carried out by the PRT1-TIF32-NIP1 subcomplex.

Multiple roles for the C-terminal domain of eIF5 in translation initiation complex assembly and GTPase activation.

eIF5 stimulates the GTPase activity of eIF2 bound to Met-tRNA(i)(Met), and its C-terminal domain (eIF5-CTD) bridges interaction between eIF2 and eIF3/eIF1 in a multifactor complex containing Met-tRNA(i)(Met). The tif5-7A mutation in eIF5-CTD, which destabilizes the multifactor complex in vivo, reduced the binding of Met-tRNA(i)(Met) and mRNA to 40S subunits in vitro. Interestingly, eIF5-CTD bound simultaneously to the eIF4G subunit of the cap-binding complex and the NIP1 subunit of eIF3. These interactions may enhance association of eIF4G with eIF3 to promote mRNA binding to the ribosome. In vivo, tif5-7A eliminated eIF5 as a stable component of the pre-initiation complex and led to accumulation of 48S complexes containing eIF2; thus, conversion of 48S to 80S complexes is the rate-limiting defect in this mutant. We propose that eIF5-CTD stimulates binding of Met-tRNA(i)(Met) and mRNA to 40S subunits through interactions with eIF2, eIF3 and eIF4G; however, its most important function is to anchor eIF5 to other components of the 48S complex in a manner required to couple GTP hydrolysis to AUG recognition during the scanning phase of initiation.

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.

Rpg1p, the subunit of the Saccharomyces cerevisiae eIF3 core complex, is a microtubule-interacting protein.

The essential gene RPG1/TIF32 of Saccharomyces cerevisiae encodes the 110-kDa subunit of the translation initiation factor 3 (eIF3) core complex. In this study, the Rpg1p-specific monoclonal antibody PK1/1 was used to analyse the cellular distribution of Rpg1p by epifluorescence and confocal laser scanning microscopy (CLSM). In budded cells, a portion of Rpg1p was obviously co-localised with microtubules. In addition, CLSM revealed an accumulation of Rpg1p in a patch at the very end of cytoplasmic microtubules reaching the bud tip. A punctate fluorescence pattern was typical for separated unbudded cells. Distribution of Rpg1p was confirmed using a strain expressing exclusively a hemaglutinin-tagged version of Rpg1p. In nocodazole-treated cells, the pattern of the PK1/1 staining was disturbed. No staining was observed in Rpg1p-depleted cells. In vitro experiments revealed that Rpg1p was specifically co-immunoprecipitated with alpha-tubulin from the yeast cell free extract and this observation was further supported by showing that Rpg1p co-sedimented with hog brain microtubules. We conclude that Rpg1p is a microtubule-interacting protein that indicates an interesting connection between the translation initiation machinery and cytoskeleton in yeast Saccharomyces cerevisiae.

The Saccharomyces cerevisiae HCR1 gene encoding a homologue of the p35 subunit of human translation initiation factor 3 (eIF3) is a high copy suppressor of a temperature-sensitive mutation in the Rpg1p subunit of yeast eIF3.

The complex eukaryotic initiation factor 3 (eIF3) was shown to promote the formation of the 43 S preinitiation complex by dissociating 40 S and 60 S ribosomal subunits, stabilizing the ternary complex, and aiding mRNA binding to 40 S ribosomal subunits. Recently, we described the identification of RPG1 (TIF32), the p110 subunit of the eIF3 core complex in yeast. In a screen for Saccharomyces cerevisiae multicopy suppressors of the rpg1-1 temperature-sensitive mutant, an unknown gene corresponding to the open reading frame YLR192C was identified. When overexpressed, the 30-kDa gene product, named Hcr1p, was able to support, under restrictive conditions, growth of the rpg1-1 temperature-sensitive mutant, but not of a Rpg1p-depleted mutant. An hcr1 null mutant was viable, but showed slight reduction of growth when compared with the wild-type strain. Physical interaction between the Hcr1 and Rpg1 proteins was shown by co-immunoprecipitation analysis. The combination of Deltahcr1 and rpg1-1 mutations resulted in a synthetic enhancement of the slow growth phenotype at a semipermissive temperature. In a computer search, a significant homology to the human p35 subunit of the eIF3 complex was found. We assume that the yeast Hcr1 protein participates in translation initiation likely as a protein associated with the eIF3 complex.

Molecular cloning and characterisation of a maize cDNA for a homologue of the large subunit of the eukaryotic initiation factor 3 (eIF3).

In order to identify genes that are specifically expressed in distinct cell populations of the maize root apex, we have constructed PCR-directed cDNA libraries from microdissected populations of cells, and screened them by differential hybridisation. A meristem-specific cDNA was isolated and characterised. This cDNA, termed ZmeIF3A, encodes a protein homologous to the large subunit of the eukaryotic translation Initiation Factor 3 (eIF3), which is an essential multi-protein complex for the initiation of protein synthesis. The ZmeIF3A protein is most similar to the yeast homologue RPG1, lacking the repeated C-terminal domain characteristic of its mammalian counterparts. However, despite this similarity, it fails to replace the RPG1 protein in complementation experiments on yeast mutants. Analysis of gene expression in situ showed that the ZmeIF3A transcript is expressed in the region of the root meristem surrounding the central stele. ZmeIF3A mRNA is also expressed in the young root, the male inflorescence, and the developing cob and seed. In maize, ZmeIF3A is encoded by one or two genomic sequences. This is the first report on the isolation and characterisation of a cDNA from higher plants that encodes a product homologous to a component of the eIF3 complex.

Rpg1, the Saccharomyces cerevisiae homologue of the largest subunit of mammalian translation initiation factor 3, is required for translational activity.

Eukaryotic initiation factor 3 (eIF3) consists of at least eight subunits and plays a key role in the formation of the 43 S preinitiation complex by dissociating 40 and 60 S ribosomal subunits, stabilizing the ternary complex, and promoting mRNA binding to 40 S ribosomal subunits. The product of the Saccharomyces cerevisiae RPG1 gene has been described as encoding a protein required for passage through the G1 phase of the cell cycle and exhibiting significant sequence similarity to the largest subunit of human eIF3. Here we show that under nondenaturing conditions, Rpg1p copurifies with a known yeast eIF3 subunit, Prt1p. An anti-Rpg1p antibody co-immunoprecipitates Prt1p, and an antibody directed against the Myc tag of a tagged version of Prt1p co-immunoprecipitates Rpg1p, demonstrating that both proteins are present in the same complex. A cell-free translation system derived from the temperature-sensitive rpg1-1 mutant strain becomes inactivated by incubation at 37 degreesC, and its activity can be restored by the addition of the Rpg1-containing protein complex. Finally, the rpg1-1 temperature-sensitive mutant strain shows a dramatic reduction of the polysome/monosome ratio upon shift to the restrictive temperature. These data show that Rpg1p is an authentic eIF3 subunit and plays an important role in the initiation step of translation.

RPG1: an essential gene of saccharomyces cerevisiae encoding a 110-kDa protein required for passage through the G1 phase.

In Saccharomyces cerevisiae cells a number of genes are required for progression through, or else to pass beyond, the G1 phase. We characterized a novel gene, RPG1, which is also involved in this phase. RPG1 is an essential gene encoding a 110-kDa evolutionarily conserved protein. Elutriated or alpha-factor-synchronized cells of the rpg1-1 temperature-sensitive mutant were arrested in the first cell cycle when shifted to a non-permissive temperature. The cells remained unbudded and neither grew nor duplicated DNA. rpg1-1 cells synchronized in S phase completed mitosis and arrested as unseparated G1 cells after a shift to a non-permissive temperature. Similarly, the asynchronous rpg1-1 cells accumulated in G1 at the non-permissive temperature, but mother and daughter cells did not separate. A bulk of Calcofluor-stained material was localized in the region adjacent to the cell septum. Our data show that Rpg1p is required for passage through the G1 phase and may be involved in growth control. Data published recently indicate that Rpg1p exhibits significant sequence similarity to a subunit of the mammalian translation initiation factor 3.