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Hrr25/CK1δ-directed release of Ltv1 from pre-40S ribosomes is necessary for ribosome assembly and cell growth.

Ghalei H, Schaub FX, Doherty JR, Noguchi Y, Roush WR, Cleveland JL, Stroupe ME, Karbstein K - J. Cell Biol. (2015)

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Biology and Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458.

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ΔLtv1 pre-40S ribosomes reconstituted with Yar1–Rps3 and Ltv1/Enp1 are structurally identical to native pre-40S ribosomes. (A) The solvent face of the reconstituted pre-40S ribosome shows the features expected from the presence of Enp1/Ltv1 and Rps3 near the beak. (B) Identical view of the ΔLtv1 pre-40S ribosomes used as a starting material in reconstitutions (EMD1924; Strunk et al., 2011). (C) Natively purified pre-40S ribosome (EMD1927; Strunk et al., 2011). (D) A superimposition of reconstituted (yellow) and ΔLtv1 pre-40S (pink) shows the repositioning of the beak upon addition of Enp1–Ltv1–Rps3, which suggests a structural recapitulation of the biochemically characterized pre-40S state. (E) Superimposition of reconstituted (yellow) and natively purified (blue) pre-40S ribosomes shows that reconstituted pre-40S ribosomes are structurally identical to purified native pre-40S ribosomes.
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fig2: ΔLtv1 pre-40S ribosomes reconstituted with Yar1–Rps3 and Ltv1/Enp1 are structurally identical to native pre-40S ribosomes. (A) The solvent face of the reconstituted pre-40S ribosome shows the features expected from the presence of Enp1/Ltv1 and Rps3 near the beak. (B) Identical view of the ΔLtv1 pre-40S ribosomes used as a starting material in reconstitutions (EMD1924; Strunk et al., 2011). (C) Natively purified pre-40S ribosome (EMD1927; Strunk et al., 2011). (D) A superimposition of reconstituted (yellow) and ΔLtv1 pre-40S (pink) shows the repositioning of the beak upon addition of Enp1–Ltv1–Rps3, which suggests a structural recapitulation of the biochemically characterized pre-40S state. (E) Superimposition of reconstituted (yellow) and natively purified (blue) pre-40S ribosomes shows that reconstituted pre-40S ribosomes are structurally identical to purified native pre-40S ribosomes.

Mentions: To test if reconstituted ribosomes accurately mimic native pre-40S ribosome intermediates purified from yeast, we used cryo-EM (Strunk et al., 2011). ΔLtv1 subunits were reconstituted with Enp1–Ltv1–Rps3 as above, before preparation of specimens for cryo-EM data acquisition (see Materials and methods). Comparison of the reconstituted pre-40S subunits with the ΔLtv1 subunits established additional density in the beak area where the Enp1–Ltv1–Rps3 complex is located (Figs. 2 and S1). Furthermore, a side view shows that the beak is retracted after the addition of Enp1–Ltv1–Rps3, as observed in the native purified pre-40S assembly intermediates (Strunk et al., 2011). Indeed, a comparison of the reconstituted pre-40S subunits with native pre-40S subunits shows that these structures are superimposable (Fig. 2). Thus, the in vitro delivery of Rps3 produces a molecule indistinguishable from native assembly intermediates, thereby validating the in vitro reconstitution.


Hrr25/CK1δ-directed release of Ltv1 from pre-40S ribosomes is necessary for ribosome assembly and cell growth.

Ghalei H, Schaub FX, Doherty JR, Noguchi Y, Roush WR, Cleveland JL, Stroupe ME, Karbstein K - J. Cell Biol. (2015)

ΔLtv1 pre-40S ribosomes reconstituted with Yar1–Rps3 and Ltv1/Enp1 are structurally identical to native pre-40S ribosomes. (A) The solvent face of the reconstituted pre-40S ribosome shows the features expected from the presence of Enp1/Ltv1 and Rps3 near the beak. (B) Identical view of the ΔLtv1 pre-40S ribosomes used as a starting material in reconstitutions (EMD1924; Strunk et al., 2011). (C) Natively purified pre-40S ribosome (EMD1927; Strunk et al., 2011). (D) A superimposition of reconstituted (yellow) and ΔLtv1 pre-40S (pink) shows the repositioning of the beak upon addition of Enp1–Ltv1–Rps3, which suggests a structural recapitulation of the biochemically characterized pre-40S state. (E) Superimposition of reconstituted (yellow) and natively purified (blue) pre-40S ribosomes shows that reconstituted pre-40S ribosomes are structurally identical to purified native pre-40S ribosomes.
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4362465&req=5

fig2: ΔLtv1 pre-40S ribosomes reconstituted with Yar1–Rps3 and Ltv1/Enp1 are structurally identical to native pre-40S ribosomes. (A) The solvent face of the reconstituted pre-40S ribosome shows the features expected from the presence of Enp1/Ltv1 and Rps3 near the beak. (B) Identical view of the ΔLtv1 pre-40S ribosomes used as a starting material in reconstitutions (EMD1924; Strunk et al., 2011). (C) Natively purified pre-40S ribosome (EMD1927; Strunk et al., 2011). (D) A superimposition of reconstituted (yellow) and ΔLtv1 pre-40S (pink) shows the repositioning of the beak upon addition of Enp1–Ltv1–Rps3, which suggests a structural recapitulation of the biochemically characterized pre-40S state. (E) Superimposition of reconstituted (yellow) and natively purified (blue) pre-40S ribosomes shows that reconstituted pre-40S ribosomes are structurally identical to purified native pre-40S ribosomes.
Mentions: To test if reconstituted ribosomes accurately mimic native pre-40S ribosome intermediates purified from yeast, we used cryo-EM (Strunk et al., 2011). ΔLtv1 subunits were reconstituted with Enp1–Ltv1–Rps3 as above, before preparation of specimens for cryo-EM data acquisition (see Materials and methods). Comparison of the reconstituted pre-40S subunits with the ΔLtv1 subunits established additional density in the beak area where the Enp1–Ltv1–Rps3 complex is located (Figs. 2 and S1). Furthermore, a side view shows that the beak is retracted after the addition of Enp1–Ltv1–Rps3, as observed in the native purified pre-40S assembly intermediates (Strunk et al., 2011). Indeed, a comparison of the reconstituted pre-40S subunits with native pre-40S subunits shows that these structures are superimposable (Fig. 2). Thus, the in vitro delivery of Rps3 produces a molecule indistinguishable from native assembly intermediates, thereby validating the in vitro reconstitution.

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer Biology and Department of Chemistry, The Scripps Research Institute, Jupiter, FL 33458.

Show MeSH
Related in: MedlinePlus