Hrr25/CK1δ-directed release of Ltv1 from pre-40S ribosomes is necessary for ribosome assembly and cell growth.
Bottom Line: Conversely, phosphomimetic Ltv1 variants rescued viability after Hrr25 depletion.Finally, Ltv1 knockdown in human breast cancer cells impaired apoptosis induced by CK1δ/ε inhibitors, establishing that the antiproliferative activity of these inhibitors is due, at least in part, to disruption of ribosome assembly.These findings validate the ribosome assembly pathway as a novel target for the development of anticancer therapeutics.
Affiliation: Department of Cancer Biology and Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458.Show MeSH
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Mentions: Maturation of pre-40S involves the joining of a large 60S ribosomal subunit and the formation of 80S-like ribosomes in a quality control cycle (Lebaron et al., 2012; Strunk et al., 2012). Depletion of the AF Fap7 leads to accumulation of 80S-like ribosomes. Hence, a block in the joining of 60S and pre-40S subunits can be monitored by the loss of 80S-like ribosomes when Fap7 is depleted (Strunk et al., 2012). To test the role of the Hrr25-Ltv1 circuit for entering this maturation cascade, we constructed a yeast strain where both Hrr25 and Fap7 are galactose-inducible and thus can be simultaneously depleted by growth in glucose. This strain was supplemented with a plasmid encoding the 1NA-PP1–sensitive mutant of Hrr25 (Hrr25-I82G). Inhibition of Hrr25 by addition of 1NA-PP1 to cultures grown in the presence of glucose resulted in loss of 80S-like assembly intermediates, whereas 80S-like ribosomes were observed in vehicle-treated cells (Fig. 5, A and B). Further, ΔLtv1/Gal::Fap7 cells supplemented with a plasmid encoding the dominant-negative Ltv1-S/A failed to form 80S-like ribosomes containing the 20S rRNA precursor in the absence of Fap7, whereas those with wild-type Ltv1 formed this key intermediate during 40S maturation (Fig. 5, C and D). These effects were not due to effects on export of pre-40S ribosomes from the nucleus, as in both experiments only the cytoplasmic fractions were analyzed (Fig. S5 C). Thus, Hrr25-mediated release of Ltv1 from pre-40S subunits is required for joining of 60S subunits and for entering into the translation-like cycle.
Affiliation: Department of Cancer Biology and Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458.