Specific interaction of one subunit of eukaryotic initiation factor eIF-3 with 18S ribosomal RNA within the binary complex, eIF-3.small ribosomal subunit, as shown by cross-linking experiments

AbstractRibosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AF) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate “handover” from one highly related AF to another remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the activities of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1. Inactive Tsr3 blocks Rio1, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

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Hypusine Is Required for a Sequence-specific Interaction of Eukaryotic Initiation Factor 5A with Postsystematic Evolution of Ligands by Exponential Enrichment RNA

Journal of Biological Chemistry ◽

10.1074/jbc.m008982200 ◽

2000 ◽

Vol 276 (4) ◽

pp. 2555-2561 ◽

Cited By ~ 49

Author(s):

Aiguo Xu ◽

Kuang Yu Chen

Keyword(s):

Specific Interaction ◽

Initiation Factor ◽

Eukaryotic Initiation Factor ◽

Exponential Enrichment

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Inactivation of eukaryotic initiation factor 2B in vitro by heat shock

Biochemical Journal ◽

10.1042/bj3340463 ◽

1998 ◽

Vol 334 (2) ◽

pp. 463-467 ◽

Cited By ~ 14

Author(s):

Gert C. SCHEPER ◽

Adri A. M. THOMAS ◽

van Roel WIJK

Keyword(s):

Heat Shock ◽

Thermal Inactivation ◽

Ribosomal Subunit ◽

Initiation Factor ◽

Eukaryotic Initiation Factor ◽

Nucleotide Exchange ◽

Mild Heat ◽

Eif2α Phosphorylation ◽

Cell Extracts

Protein synthesis in rat H35 Reuber hepatoma cells is rapidly inhibited on heat shock. At mild heat-shock temperatures the main cause for inhibition is the inactivation of the guanine nucleotide exchange factor eukaryotic initiation factor 2B (eIF2B); under more severe heat-shock conditions the activity of several initiation factors is compromised. eIF2B is required for GDP/GTP exchange on eIF2, which delivers methionyl-tRNA to the 40 S ribosomal subunit. We have tried to elucidate the mechanism underlying the inactivation of eIF2B by assays in vitro. Incubation of cell extracts at 41 °C or higher led to the inactivation of eIF2B. In agreement with observations in cells exposed to mild heat shocks, the thermal inactivation of eIF2B could be ascribed to neither eIF2α phosphorylation nor the induction of another inhibitor. With the use of glycerol gradients we show that eIF2B forms aggregates in heat-treated extracts. Furthermore eIF2B activity is protected against heat shock in thermotolerant cells. Taken together, these results suggest a role for chaperones in the control of eIF2B activity.

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A region rich in aspartic acid, arginine, tyrosine, and glycine (DRYG) mediates eukaryotic initiation factor 4B (eIF4B) self-association and interaction with eIF3.

Molecular and Cellular Biology ◽

10.1128/mcb.16.10.5328 ◽

1996 ◽

Vol 16 (10) ◽

pp. 5328-5334 ◽

Cited By ~ 118

Author(s):

N Méthot ◽

M S Song ◽

N Sonenberg

Keyword(s):

Aspartic Acid ◽

Ribosomal Subunit ◽

Direct Interaction ◽

Initiation Factor ◽

Eukaryotic Initiation Factor ◽

Ribosome Binding ◽

Initiation Factors ◽

Self Association

The binding of mRNA to the ribosome is mediated by eukaryotic initiation factors eukaryotic initiation factor 4F (eIF4F), eIF4B, eIF4A, and eIF3, eIF4F binds to the mRNA cap structure and, in combination with eIF4B, is believed to unwind the secondary structure in the 5' untranslated region to facilitate ribosome binding. eIF3 associates with the 40S ribosomal subunit prior to mRNA binding. eIF4B copurifies with eIF3 and eIF4F through several purification steps, suggesting the involvement of a multisubunit complex during translation initiation. To understand the mechanism by which eIF4B promotes 40S ribosome binding to the mRNA, we studied its interactions with partner proteins by using a filter overlay (protein-protein [far Western]) assay and the two-hybrid system. In this report, we show that eIF4B self-associates and also interacts directly with the p170 subunit of eIF3. A region rich in aspartic acid, arginine, tyrosine, and glycine, termed the DRYG domain, is sufficient for self-association of eIF4B, both in vitro and in vivo, and for interaction with the p170 subunit of eIF3. These experiments suggest that eIF4B participates in mRNA-ribosome binding by acting as an intermediary between the mRNA and eIF3, via a direct interaction with the p170 subunit of eIF3.

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Cap-dependent eukaryotic initiation factor-mRNA interactions probed by cross-linking

RNA ◽

10.1261/rna.971208 ◽

2008 ◽

Vol 14 (5) ◽

pp. 960-969 ◽

Cited By ~ 30

Author(s):

L. Lindqvist ◽

H. Imataka ◽

J. Pelletier

Keyword(s):

Initiation Factor ◽

Cross Linking ◽

Eukaryotic Initiation Factor

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The modifying enzyme Tsr3 establishes the hierarchy of Rio kinase activity in 40S ribosome assembly

RNA ◽

10.1261/rna.078994.121 ◽

2022 ◽

pp. rna.078994.121

Author(s):

Haina Huang ◽

Melissa D Parker ◽

Katrin Karbstein

Keyword(s):

Quality Control ◽

Binding Site ◽

Ribosomal Rna ◽

Ribosomal Proteins ◽

Kinase Activity ◽

Ribosomal Subunit ◽

Ribosome Assembly ◽

Small Ribosomal Subunit ◽

Yeast Strains

Ribosome assembly is an intricate process, which in eukaryotes is promoted by a large machinery comprised of over 200 assembly factors (AF) that enable the modification, folding, and processing of the ribosomal RNA (rRNA) and the binding of the 79 ribosomal proteins. While some early assembly steps occur via parallel pathways, the process overall is highly hierarchical, which allows for the integration of maturation steps with quality control processes that ensure only fully and correctly assembled subunits are released into the translating pool. How exactly this hierarchy is established, in particular given that there are many instances of RNA substrate “handover” from one highly related AF to another remains to be determined. Here we have investigated the role of Tsr3, which installs a universally conserved modification in the P-site of the small ribosomal subunit late in assembly. Our data demonstrate that Tsr3 separates the activities of the Rio kinases, Rio2 and Rio1, with whom it shares a binding site. By binding after Rio2 dissociation, Tsr3 prevents rebinding of Rio2, promoting forward assembly. After rRNA modification is complete, Tsr3 dissociates, thereby allowing for recruitment of Rio1. Inactive Tsr3 blocks Rio1, which can be rescued using mutants that bypass the requirement for Rio1 activity. Finally, yeast strains lacking Tsr3 randomize the binding of the two kinases, leading to the release of immature ribosomes into the translating pool. These data demonstrate a role for Tsr3 and its modification activity in establishing a hierarchy for the function of the Rio kinases.

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Contrasting mechanisms of regulating translation of specific Drosophila germline mRNAs at the level of 5′-cap structure binding

Biochemical Society Transactions ◽

10.1042/bst0331544 ◽

2005 ◽

Vol 33 (6) ◽

pp. 1544-1546 ◽

Cited By ~ 2

Author(s):

P. Lasko ◽

P. Cho ◽

F. Poulin ◽

N. Sonenberg

Keyword(s):

Translational Control ◽

Binding Proteins ◽

Regulatory Mechanism ◽

Ribosomal Subunit ◽

Drosophila Embryo ◽

Initiation Factor ◽

Control Mechanisms ◽

Eukaryotic Initiation Factor ◽

Eukaryotic Initiation Factor 4E ◽

Cognate Protein

Translational control is a key genetic regulatory mechanism underlying the initial establishment of the major spatial axes of the Drosophila embryo. Many translational control mechanisms target eIF4E (eukaryotic initiation factor 4E), an initiation factor that recognizes the 5′-cap structure of the mRNA. Cap recognition by eIF4E, in complex with eIF4G, is essential for recruitment of the mRNA to the small ribosomal subunit. One established mechanism for repressing translation involves eIF4E-binding proteins, which competitively inhibit the eIF4E–eIF4G interaction. Our group has uncovered a novel mechanism for repression in which an eIF4E cognate protein called d4EHP, which cannot bind eIF4G, binds to the 5′-cap structure of cad mRNA thus rendering it translationally inactive. These two related, but distinct, mechanisms are discussed and contrasted in this review.

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