CLN3 expression is sufficient to restore G1-to-S-phase progression in Saccharomyces cerevisiae mutants defective in translation initiation factor eIF4E

The essential cap-binding protein (eIF4E) of Saccharomycescerevisiae is encoded by the CDC33 (wild-type) gene, originally isolated as a mutant, cdc33-1, which arrests growth in the G1 phase of the cell cycle at 37 °C. We show that other cdc33 mutants also arrest in G1. One of the first events required for G1-to-S-phase progression is the increased expression of cyclin 3. Constructs carrying the 5ʹ-untranslated region of CLN3 fused to lacZ exhibit weak reporter activity, which is significantly decreased in a cdc33-1 mutant, implying that CLN3 mRNA is an inefficiently translated mRNA that is sensitive to perturbations in the translation machinery. A cdc33-1 strain expressing either stable Cln3p (Cln3-1p) or a hybrid UBI4 5ʹ-CLN3 mRNA, whose translation displays decreased dependence on eIF4E, arrested randomly in the cell cycle. In these cells CLN2 mRNA levels remained high, indicating that Cln3p activity is maintained. Induction of a hybrid UBI4 5ʹ-CLN3 message in a cdc33-1 mutant previously arrested in G1 also caused entry into a new cell cycle. We conclude that eIF4E activity in the G1-phase is critical in allowing sufficient Cln3p activity to enable yeast cells to enter a new cell cycle.

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Translation initiation factor 4A from Saccharomyces cerevisiae: analysis of residues conserved in the D-E-A-D family of RNA helicases.

Molecular and Cellular Biology ◽

10.1128/mcb.11.7.3463 ◽

1991 ◽

Vol 11 (7) ◽

pp. 3463-3471 ◽

Cited By ~ 62

Author(s):

S R Schmid ◽

P Linder

Keyword(s):

Saccharomyces Cerevisiae ◽

Translation Initiation ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Clear Correlation ◽

Temperature Sensitive ◽

Rna Helicases ◽

Initiation Of Translation

The eukaryotic translation initiation factor 4A (eIF-4A) possesses an in vitro helicase activity that allows the unwinding of double-stranded RNA. This activity is dependent on ATP hydrolysis and the presence of another translation initiation factor, eIF-4B. These two initiation factors are thought to unwind mRNA secondary structures in preparation for ribosome binding and initiation of translation. To further characterize the function of eIF-4A in cellular translation and its interaction with other elements of the translation machinery, we have isolated mutations in the TIF1 and TIF2 genes encoding eIF-4A in Saccharomyces cerevisiae. We show that three highly conserved domains of the D-E-A-D protein family, encoding eIF-4A and other RNA helicases, are essential for protein function. Only in rare cases could we make a conservative substitution without affecting cell growth. The mutants show a clear correlation between their growth and in vivo translation rates. One mutation that results in a temperature-sensitive phenotype reveals an immediate decrease in translation activity following a shift to the nonpermissive temperature. These in vivo results confirm previous in vitro data demonstrating an absolute dependence of translation on the TIF1 and TIF2 gene products.

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Fal1p is an essential DEAD-box protein involved in 40S-ribosomal-subunit biogenesis in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.17.12.7283 ◽

1997 ◽

Vol 17 (12) ◽

pp. 7283-7294 ◽

Cited By ~ 108

Author(s):

D Kressler ◽

J de la Cruz ◽

M Rojo ◽

P Linder

Keyword(s):

Saccharomyces Cerevisiae ◽

Translation Initiation ◽

18S Rrna ◽

Amino Acid Level ◽

Ribosomal Subunit ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Northern Hybridization ◽

Ribosomal Subunits ◽

Dead Box

A previously uncharacterized Saccharomyces cerevisiae gene, FAL1, was found by sequence comparison as a homolog of the eukaryotic translation initiation factor 4A (eIF4A). Fal1p has 55% identity and 73% similarity on the amino acid level to yeast eIF4A, the prototype of ATP-dependent RNA helicases of the DEAD-box protein family. Although clearly grouped in the eIF4A subfamily, the essential Fal1p displays a different subcellular function and localization. An HA epitope-tagged Fal1p is localized predominantly in the nucleolus. Polysome analyses in a temperature-sensitive fal1-1 mutant and a Fal1p-depleted strain reveal a decrease in the number of 40S ribosomal subunits. Furthermore, these strains are hypersensitive to the aminoglycoside antibiotics paromomycin and neomycin. Pulse-chase labeling of pre-rRNA and steady-state-level analysis of pre-rRNAs and mature rRNAs by Northern hybridization and primer extension in the Fal1p-depleted strain show that Fal1p is required for pre-rRNA processing at sites A0, A1, and A2. Consequently, depletion of Fal1p leads to decreased 18S rRNA levels and to an overall deficit in 40S ribosomal subunits. Together, these results implicate Fal1p in the 18S rRNA maturation pathway rather than in translation initiation.

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