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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-associated pre-ribosomal particles contain the L7 r-protein. Wild-type and Rlp7-TAP cells were mixed in equal proportions prior to complex purification. The log2 of SILAC ratios (median value of Wild-type/Rlp7-TAP peptide ratio) were plotted against the sum of the intensity of all the peptides for each protein. Dots are coloured according to protein function: pre-60S factors (red), 60S r-proteins (Rpl, blue), r-stalk proteins (P0/P1/P2, light blue), 40S r-proteins (Rps, green) and proteins of other different functions (grey). Yellow stars indicate the Rlp7 and L7 values. The identity of pre-60S factors specifically enriched (Group 1) or not (Group 2) is indicated below the graph. These factors are listed from their highest to lowest intensity values (see Supplementary Data Set S1).
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gkt726-F1: Rlp7-associated pre-ribosomal particles contain the L7 r-protein. Wild-type and Rlp7-TAP cells were mixed in equal proportions prior to complex purification. The log2 of SILAC ratios (median value of Wild-type/Rlp7-TAP peptide ratio) were plotted against the sum of the intensity of all the peptides for each protein. Dots are coloured according to protein function: pre-60S factors (red), 60S r-proteins (Rpl, blue), r-stalk proteins (P0/P1/P2, light blue), 40S r-proteins (Rps, green) and proteins of other different functions (grey). Yellow stars indicate the Rlp7 and L7 values. The identity of pre-60S factors specifically enriched (Group 1) or not (Group 2) is indicated below the graph. These factors are listed from their highest to lowest intensity values (see Supplementary Data Set S1).

Mentions: To determine whether Rlp7 and L7 co-exist in the same pre-ribosomal particles, we performed a SILAC experiment combined with LC-MS/MS mass spectrometry to monitor the relative amount of L7 present in affinity purified pre-ribosomal complexes from a strain expressing TAP-tagged Rlp7. We have identified 60 pre-60S factors and most r-proteins from 60S (Rpl) and 40S (Rps) r-subunits (Figure 1 and Supplementary Data Set S1). Proteins clearly arranged in two groups. Group 1 (specifically enriched proteins; log2 of SILAC ratio between −6 and −1) comprises most Rpl proteins (including L7) and 31 pre-60S factors, in addition to Rlp7, which were strongly enriched in the Rlp7-TAP associated sample; among them, early-acting pre-60S factors including several A3 factors (Erb1, Nop7, Nsa3, Ytm1 and Nop15) (6) were specifically abundant as judged from the total MS signal intensity. Group 2 (non-specific proteins; log2 of SILAC ratio between −1 and 0) includes 28 pre-60S factors, among them late-acting pre-60S factors (e.g. Arb1, Bud20, Arx1 and Alb1) and components of snoRNPs (e.g. Nop56, Cbf5, Nop1, Nhp2, Nop58, Gar1 and Snu13). Interestingly, all detected Rps proteins, the Rpl proteins from the r-stalk (P0, P1 and P2) and L10 appeared at values considered as contamination (log2 of SILAC ratio > 0). These results are consistent with the fact that the assembly of the r-stalk proteins and L10 into 60S r-subunits occurs predominantly in the cytoplasm (42,43). These results, confirmed by a second independent SILAC experiment, clearly identify Rlp7 as an early pre-60S assembly factor and strongly suggest that Rlp7 and L7 bind to the same pre-ribosomal particles (Supplementary Data Set S1).Figure 1.


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-associated pre-ribosomal particles contain the L7 r-protein. Wild-type and Rlp7-TAP cells were mixed in equal proportions prior to complex purification. The log2 of SILAC ratios (median value of Wild-type/Rlp7-TAP peptide ratio) were plotted against the sum of the intensity of all the peptides for each protein. Dots are coloured according to protein function: pre-60S factors (red), 60S r-proteins (Rpl, blue), r-stalk proteins (P0/P1/P2, light blue), 40S r-proteins (Rps, green) and proteins of other different functions (grey). Yellow stars indicate the Rlp7 and L7 values. The identity of pre-60S factors specifically enriched (Group 1) or not (Group 2) is indicated below the graph. These factors are listed from their highest to lowest intensity values (see Supplementary Data Set S1).
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Related In: Results  -  Collection

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

gkt726-F1: Rlp7-associated pre-ribosomal particles contain the L7 r-protein. Wild-type and Rlp7-TAP cells were mixed in equal proportions prior to complex purification. The log2 of SILAC ratios (median value of Wild-type/Rlp7-TAP peptide ratio) were plotted against the sum of the intensity of all the peptides for each protein. Dots are coloured according to protein function: pre-60S factors (red), 60S r-proteins (Rpl, blue), r-stalk proteins (P0/P1/P2, light blue), 40S r-proteins (Rps, green) and proteins of other different functions (grey). Yellow stars indicate the Rlp7 and L7 values. The identity of pre-60S factors specifically enriched (Group 1) or not (Group 2) is indicated below the graph. These factors are listed from their highest to lowest intensity values (see Supplementary Data Set S1).
Mentions: To determine whether Rlp7 and L7 co-exist in the same pre-ribosomal particles, we performed a SILAC experiment combined with LC-MS/MS mass spectrometry to monitor the relative amount of L7 present in affinity purified pre-ribosomal complexes from a strain expressing TAP-tagged Rlp7. We have identified 60 pre-60S factors and most r-proteins from 60S (Rpl) and 40S (Rps) r-subunits (Figure 1 and Supplementary Data Set S1). Proteins clearly arranged in two groups. Group 1 (specifically enriched proteins; log2 of SILAC ratio between −6 and −1) comprises most Rpl proteins (including L7) and 31 pre-60S factors, in addition to Rlp7, which were strongly enriched in the Rlp7-TAP associated sample; among them, early-acting pre-60S factors including several A3 factors (Erb1, Nop7, Nsa3, Ytm1 and Nop15) (6) were specifically abundant as judged from the total MS signal intensity. Group 2 (non-specific proteins; log2 of SILAC ratio between −1 and 0) includes 28 pre-60S factors, among them late-acting pre-60S factors (e.g. Arb1, Bud20, Arx1 and Alb1) and components of snoRNPs (e.g. Nop56, Cbf5, Nop1, Nhp2, Nop58, Gar1 and Snu13). Interestingly, all detected Rps proteins, the Rpl proteins from the r-stalk (P0, P1 and P2) and L10 appeared at values considered as contamination (log2 of SILAC ratio > 0). These results are consistent with the fact that the assembly of the r-stalk proteins and L10 into 60S r-subunits occurs predominantly in the cytoplasm (42,43). These results, confirmed by a second independent SILAC experiment, clearly identify Rlp7 as an early pre-60S assembly factor and strongly suggest that Rlp7 and L7 bind to the same pre-ribosomal particles (Supplementary Data Set S1).Figure 1.

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