Interaction Of Ribosomal Subunits Research Articles

Abstract 3719: Targeting the eIF6 and 60S ribosomal subunit interaction interface in cancers

Abstract Translational control is integral to cancer initiation and progression. A subset of translation initiation factors are deregulated in cancers to facilitate their rampant growth and proliferation. Eukaryotic translation initiation factor 6 (eIF6) is one such factor that is over expressed in many cancers including lung, colon, ovarian and breast cancers, and deregulation of eIF6 is correlated with a poor cancer prognosis. Association of eIF6 with 60S is critical for 60S assembly. However, eIF6 must be released from 60S prior to translation to permit inter subunit interactions between 60S and 40S and to facilitate the formation of translationally proficient 80S complex. Release of eIF6 is deregulated in the ribosomopathy- Shwachman Diamond Syndrome that is predisposed to leukemias. Also, loss of eIF6 markedly delays tumorigenesis without affecting normal growth, which presents eIF6 as a viable therapeutic target. A potential therapeutic strategy is to target the eIF6 and 60S ribosomal interaction interface. Through extensive biophysical analyses, we had identified residues that are critical for the interaction between eIF6 and 60S ribosomal subunit. We show that targeting these key residues in the eIF6-60S interaction interface markedly delays colonic cancer growth and inhibits protein synthesis. Interestingly, targeting eIF6 leads to an upregulation of p53 independent of the DNA damage response, suggesting that ribosomal stress contributes to the stabilization of p53. Future studies will determine the mRNA targets that are translationally deregulated by inactivation of eIF6. We will also determine the effect of targeting eIF6 function in inhibiting tumor growth and progression in vivo. Citation Format: Kavya Meena Harish, Aparna Biswas, Poonam Roshan, Sofia Origanti. Targeting the eIF6 and 60S ribosomal subunit interaction interface in cancers. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3719.

Diverse relationships between metal ions and the ribosome.

The ribosome requires metal ions for structural stability and translational activity. These metal ions are important for stabilizing the secondary structure of ribosomal RNA, binding of ribosomal proteins to the ribosome, and for interaction of ribosomal subunits. In this review, various relationships between ribosomes and metal ions, especially Mg2+ and Zn2+, are presented. Mg2+ regulates gene expression by modulating the translational stability and synthesis of ribosomes, which in turn contribute to the cellular homeostasis of Mg2+. In addition, Mg2+ can partly complement the function of ribosomal proteins. Conversely, a reduction in the cellular concentration of Zn2+ induces replacement of ribosomal proteins, which mobilizes free-Zn2+ in the cell and represses translation activity. Evolutional relationships between these metal ions and the ribosome are also discussed.

Eukaryotic initiation factor 6 (eif6) overexpression affects eye development in Xenopus laevis

The translation initiation factor eif6 has been implicated as a regulator of ribosome assembly, selective mRNA translation and apoptosis. Many of these activities depend upon the phosphorylation of eif6 serine 235 by PKC. Previous data showed that eif6 binds to the 60S ribosomal subunit when unphosphorylated, inhibiting assembly with the 40S subunit. Phosphorylation of Ser235 releases eif6 from the 60S subunit and allows assembly. eif6 acts as an anti-apoptotic factor via regulation of the bcl2/bax balance and acts selectively upstream of bcl2. This activity also depends upon phosphorylation of eif6 Ser235. One of the consequences of eif6 overexpression in Xenopus embryos is aberrant eye development. Here we evaluate the eye phenotype and show that it is transient. We show that the whole eye, particularly the retina layers, of the embryos injected with eif6-encoding mRNA recover by stage 42. Embryos over-expressing eif6 have normal expression of anterior- and brain-specific markers, indicating that outside the eye field, other neural regions appear unaffected by the eif6 injection. No eye defect was detected when morpholinos were used to reduce eif6 protein synthesis. We tested how two known pathways of eif6 function with respect to alteration of eye development. We found that injection of bcl2 did not produce the eye phenotype and eif6-bax co-injection did not rescue the eye defect, suggesting that the eye phenotype is not bearing on the anti-apoptotic role played by eif6 is not linked to its role as an anti-apoptotic factor. We also determined that PKC-dependant phosphorylation of Ser235 in eif6 is not required to produce defective eye development. These results indicate that the aberrant eye phenotype, produced by eif6 overexpression, is not directly linked to the PKC-regulated effects of eif6 on translation and ribosomal subunit interaction or on eif6 anti-apoptotic properties.

Inhibition of Antiassociation Activity of Translation Initiation Factor 3 by Paromomycin

The effect of paromomycin on the interaction of ribosomal subunits was studied. Paromomycin inhibited the antiassociation activity of initiation factor 3 (IF3). Furthermore, ribosomal subunits were associated to form 70S ribosomes by paromomycin even in the presence of 1 mM Mg(2+). Paromomycin did not inhibit the binding of IF3 to the 30S ribosomal subunits. On the other hand, IF3 bound to the 30S subunits was expelled by paromomycin-induced subunit association (70S formation). These results indicate that the stabilization of 70S ribosomes by paromomycin may in part be responsible for its inhibitory effects on translocation and ribosome recycling.

Characterization of GE82832, a peptide inhibitor of translocation interacting with bacterial 30S ribosomal subunits

GE82832, a secondary metabolite produced by Streptosporangium cinnabarinum (strain GE82832), has been identified as a translational inhibitor by in vitro screening of a library of natural products. Secondary functional tests specific for individual steps of the translational pathway demonstrated that translocation is the specific target of GE82832. Chemical probing in situ demonstrated that this antibiotic protects bases A1324 and A1333 and exposes C1336 of 16S rRNA, thereby indicating that its binding site is located on the head of the 30S ribosomal subunit. The ribosomal location of GE82832, near ribosomal protein S13 and G1338, two elements of the small subunit that are part of or close to the B1a intrasubunit bridge, suggests that translocation inhibition results from an altered dynamics of 30S-50S ribosomal subunit interaction.

Characterization of dFMR1, a Drosophila melanogaster homolog of the fragile X mental retardation protein.

Fragile X syndrome is the most common inherited form of mental retardation. It is caused by loss of FMR1 gene activity due to either lack of expression or expression of a mutant form of the protein. In mammals, FMR1 is a member of a small protein family that consists of FMR1, FXR1, and FXR2. All three members bind RNA and contain sequence motifs that are commonly found in RNA-binding proteins, including two KH domains and an RGG box. The FMR1/FXR proteins also contain a 60S ribosomal subunit interaction domain and a protein-protein interaction domain which mediates homomer and heteromer formation with each family member. Nevertheless, the specific molecular functions of FMR1/FXR proteins are unknown. Here we report the cloning and characterization of a Drosophila melanogaster homolog of the mammalian FMR1/FXR gene family. This first invertebrate homolog, termed dfmr1, has a high degree of amino acid sequence identity/similarity with the defined functional domains of the FMR1/FXR proteins. The dfmr1 product binds RNA and is similar in subcellular localization and embryonic expression pattern to the mammalian FMR1/FXR proteins. Overexpression of dfmr1 driven by the UAS-GAL4 system leads to apoptotic cell loss in all adult Drosophila tissues examined. This phenotype is dependent on the activity of the KH domains. The ability to induce a dominant phenotype by overexpressing dfmr1 opens the possibility of using genetic approaches in Drosophila to identify the pathways in which the FMR1/FXR proteins function.

Binding sites of the antibiotics pactamycin and celesticetin on ribosomal RNAs

The binding sites of the antibiotics pactamycin and celesticetin on the rRNAs of Escherichia coli ribosomes were investigated by a chemical footprinting procedure. Pactamycin protected residues G-693 and C-795 in 16S RNA which are located in an improtant functional region of the 30S subunit participating in initiation complex formation and ribosomal subunit interaction. Celesticetin altered the reactivities of 5 residues A-2058, A-2059, A-2062, A-2451 and G-2505 within the central loop of domain V of 23S RNA which has been implicated in peptidyltransferase activity. Inferences are drawn concerning the mode of action of the antibiotic.

Studies on the function of two adjacent N6,N6-dimethyladenosines near the 3' end of 16S ribosomal RNA of Escherichia coli. IV. The effect of the methylgroups on ribosomal subunit interaction.

The effect of the presence or absence of the methylgroups of the m2(6)Am2(6)A sequence near the 3' end of 16S rRNA of Escherichia coli on the interaction of the ribosomal subunits has been studied, using wild-type (methylated) and mutant (unmethylated) ribosomes. Subunit exchange experiments and competitive association experiments show a strong preference of the 50S subunit for association with methylated 30S subunits. The results indicate that the equilibrium constant of the reaction 70S in equilibrium with 30S + 50S is dependent on the methylgroups; mutant 30S.50S couples are less stable than wild-type 30S.50S couples. It is postulated that the methylgroups also stimulate the interaction between 30S subunits and initiation factor IF-3.

Ribosomal subunit interaction as studied by light scattering. Evidence of different classes of ribosome preparations.

The Mg2+-dependent equilibrium of ribosomal subunits was studied by light scattering in the absence of any other factor of protein synthesis. The difference of intensity in the light scattered by dissociated 30S-50S couples and by 70S particles was used as a method to measure the percentage of association as a function of Mg2+ under conditions of nonperturbation of the equilibrium. We found that pressure-resistant (type A) and non-pressure-resistant (type B) ribosomes may be characterized by the different behavior of their association equilibrium curves. The thermodynamic parameters of this equilibrium and their comparison for the two classes of ribosomes were calculated from experiments at different temperatures.

Protein-protein proximity in the association of ribosomal subunits of Escherichia coli: Crosslinking of 30 S protein S16 to 50 S proteins by glutaraldehyde or formaldehyde

The interaction of ribosomal subunits from Escherichia coli has been studied using crosslinking reagents. Radioactive 35S-labeled 50 S subunits and non-radioactive 30 S subunits were allowed to reassociate to form 70 S ribosomes. The 70 S particles, containing radioactivity only in the 50 S protein moiety, were incubated with glutaraldehyde or formaldehyde. As a result of this treatment a substantial fraction of the 70 S particles did not dissociate at 1 m m-Mg 2+. This fraction was isolated and the ribosomal proteins were extracted. The protein mixture was analyzed by the Ouchterlony double diffusion technique by using eighteen antisera prepared against single 30 S ribosomal proteins (all except those against S3, S15 and S17). As a result of the crosslinking procedure it was found that only anti-S16 co-precipitated 35S-labeled 50 S protein. It is concluded that the 30 S protein S16 is at or near the site of interaction between subunits and can become crosslinked to one or more 50 S ribosomal proteins.

The distribution of radioactive peptides synthesized by polysomes and ribosomal subunits combined in vitro with microsomal membranes

1. Rat liver endoplasmic reticulum membranes were conditioned in various ways to bind exogenous polysomes in vitro. The puromycin-mediated release of nascent peptides from polysomes bound to such membranes was then studied, in efforts to determine whether the complexes functioned as secretory units, directing released peptides toward the inside of the membrane vesicle rather than outward toward the incubation medium. The distribution of released peptides between the incubation medium and a detergent-soluble fraction, which represents the vesicle membrane and contents, was examined. 2. The following combinations of ribosomes and microsomal membranes were tested: (a) The hormone-mediated binding of free polysomes to smooth endoplasmic reticulum membranes; (b) the binding of polysomes to membranes conditioned with citrate and pyrophosphate, with EDTA and ribonuclease, or with puromycin at high concentrations of KCl; and (c) the interaction of ribosomal subunits and polyuridylic acid with puromycin-KCl-conditioned membranes. 3. There is no evidence of vectorial transport of nascent peptides across membranes in in vitro recombination systems. It is concluded that these systems do not duplicate the type of binding observed in naturally occurring rough-surfaced endoplasmic reticulum.

Sedimentation studies on the interaction of ribosomal subunits from liver

1. The 50-S and 60-S components which appear in the intermediate stage of dissociation of hepatic ribosomes were isolated by sucrose density-gradient centrifugation. Sedimentation analyses have shown that these two components are each composed of large and small subunits. 2. The release of the small subunit from the isolated 50-S and 60-S components was found to be exclusively dependent on Mg 2+ concentration. 3. Stable small and large subunits were isolated in the presence of 1 mM Mg 2+ and 0.1 mM Mg 2+, respectively. Association of the isolated small subunit as well as of the isolated large subunit was observed in the presence of more than 2 mM Mg 2+. It has not been possible to show any preferential association between the small subunit particles as compared with the large subunit particles, nor has any selective interaction between the small and the large subunit particles been demonstrated.