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Yeast ribosomal protein L7 and its homologue Rlp7 are simultaneously present at distinct sites on pre-60S ribosomal particles.

Babiano R, Badis G, Saveanu C, Namane A, Doyen A, Díaz-Quintana A, Jacquier A, Fromont-Racine M, de la Cruz J - Nucleic Acids Res. (2013)

Bottom Line: However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles.Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles.Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética, Universidad de Sevilla, E-41012 Seville, Spain, Institut Pasteur, Génétique des Interactions Macromoléculaires, CNRS UMR-3525, Paris, France and Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Seville, Spain.

ABSTRACT
Ribosome biogenesis requires >300 assembly factors in Saccharomyces cerevisiae. Ribosome assembly factors Imp3, Mrt4, Rlp7 and Rlp24 have sequence similarity to ribosomal proteins S9, P0, L7 and L24, suggesting that these pre-ribosomal factors could be placeholders that prevent premature assembly of the corresponding ribosomal proteins to nascent ribosomes. However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles. Cross-linking and cDNA analysis experiments showed that Rlp7 binds to the ITS2 region of 27S pre-rRNAs, at two sites, in helix III and in a region adjacent to the pre-rRNA processing sites C1 and E. However, L7 binds to mature 25S and 5S rRNAs and cross-linked predominantly to helix ES7(L)b within 25S rRNA. Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles. Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation.

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Rlp7 and L7 association with pre-ribosomal particles is not mutually exclusive. Extracts were prepared from cells co-expressing Rlp7-HA and L7B-TAP or, as a control, Rlp7-HA and untagged L7B. Total extracts (T) and L7B-TAP affinity-purified samples (IP) were analysed by western blotting. Co-precipitation of Rlp7-HA, Has1, Nop1 and r-proteins L1 and L35 was tested with specific antibodies. The asterisk corresponds to a L7B-TAP cross-reaction.
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gkt726-F2: Rlp7 and L7 association with pre-ribosomal particles is not mutually exclusive. Extracts were prepared from cells co-expressing Rlp7-HA and L7B-TAP or, as a control, Rlp7-HA and untagged L7B. Total extracts (T) and L7B-TAP affinity-purified samples (IP) were analysed by western blotting. Co-precipitation of Rlp7-HA, Has1, Nop1 and r-proteins L1 and L35 was tested with specific antibodies. The asterisk corresponds to a L7B-TAP cross-reaction.

Mentions: To confirm these results, we performed IgG-Sepharose purification with extracts of cells expressing both C-terminal TAP-tagged L7B and HA-tagged Rlp7 proteins. Analysis of purified complexes by SDS–PAGE and western blotting demonstrated the association of Rlp7-HA with L7B-TAP, the r-proteins L1 and L35 and the trans-acting factor Has1 (Figure 2). This association seems to be specific, as no co-purification was observed when a strain harbouring a non-tagged L7B was used as a control. In contrast, the trans-acting factor Nop1 did not associate with L7B-TAP (Figure 2). This result correlates well with the identification of Has1 in Group 1 and Nop1 in Group 2 of Rlp7-TAP associated proteins. We conclude that both Rlp7 and L7 are able to simultaneously bind to the same particles.Figure 2.


Yeast ribosomal protein L7 and its homologue Rlp7 are simultaneously present at distinct sites on pre-60S ribosomal particles.

Babiano R, Badis G, Saveanu C, Namane A, Doyen A, Díaz-Quintana A, Jacquier A, Fromont-Racine M, de la Cruz J - Nucleic Acids Res. (2013)

Rlp7 and L7 association with pre-ribosomal particles is not mutually exclusive. Extracts were prepared from cells co-expressing Rlp7-HA and L7B-TAP or, as a control, Rlp7-HA and untagged L7B. Total extracts (T) and L7B-TAP affinity-purified samples (IP) were analysed by western blotting. Co-precipitation of Rlp7-HA, Has1, Nop1 and r-proteins L1 and L35 was tested with specific antibodies. The asterisk corresponds to a L7B-TAP cross-reaction.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814368&req=5

gkt726-F2: Rlp7 and L7 association with pre-ribosomal particles is not mutually exclusive. Extracts were prepared from cells co-expressing Rlp7-HA and L7B-TAP or, as a control, Rlp7-HA and untagged L7B. Total extracts (T) and L7B-TAP affinity-purified samples (IP) were analysed by western blotting. Co-precipitation of Rlp7-HA, Has1, Nop1 and r-proteins L1 and L35 was tested with specific antibodies. The asterisk corresponds to a L7B-TAP cross-reaction.
Mentions: To confirm these results, we performed IgG-Sepharose purification with extracts of cells expressing both C-terminal TAP-tagged L7B and HA-tagged Rlp7 proteins. Analysis of purified complexes by SDS–PAGE and western blotting demonstrated the association of Rlp7-HA with L7B-TAP, the r-proteins L1 and L35 and the trans-acting factor Has1 (Figure 2). This association seems to be specific, as no co-purification was observed when a strain harbouring a non-tagged L7B was used as a control. In contrast, the trans-acting factor Nop1 did not associate with L7B-TAP (Figure 2). This result correlates well with the identification of Has1 in Group 1 and Nop1 in Group 2 of Rlp7-TAP associated proteins. We conclude that both Rlp7 and L7 are able to simultaneously bind to the same particles.Figure 2.

Bottom Line: However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles.Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles.Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Genética, Universidad de Sevilla, E-41012 Seville, Spain, Institut Pasteur, Génétique des Interactions Macromoléculaires, CNRS UMR-3525, Paris, France and Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Seville, Spain.

ABSTRACT
Ribosome biogenesis requires >300 assembly factors in Saccharomyces cerevisiae. Ribosome assembly factors Imp3, Mrt4, Rlp7 and Rlp24 have sequence similarity to ribosomal proteins S9, P0, L7 and L24, suggesting that these pre-ribosomal factors could be placeholders that prevent premature assembly of the corresponding ribosomal proteins to nascent ribosomes. However, we found L7 to be a highly specific component of Rlp7-associated complexes, revealing that the two proteins can bind simultaneously to pre-ribosomal particles. Cross-linking and cDNA analysis experiments showed that Rlp7 binds to the ITS2 region of 27S pre-rRNAs, at two sites, in helix III and in a region adjacent to the pre-rRNA processing sites C1 and E. However, L7 binds to mature 25S and 5S rRNAs and cross-linked predominantly to helix ES7(L)b within 25S rRNA. Thus, despite their predicted structural similarity, our data show that Rlp7 and L7 clearly bind at different positions on the same pre-60S particles. Our results also suggest that Rlp7 facilitates the formation of the hairpin structure of ITS2 during 60S ribosomal subunit maturation.

Show MeSH
Related in: MedlinePlus