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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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Localization of the CRAC interaction sites of Rlp7 with pre-rRNA sequences displayed on the ‘hairpin model’ (A) and on the ‘ring model’ (B) for yeast ITS2 secondary structure [for a reference, see (45)]. The CRAC sites are highlighted in yellow; blue circles indicate frequently mutated residues found in the experiments (see Supplementary Figure S8). The Nsa3 and Nop15 CRAC sites, as described in (7), are represented as purple and green, respectively. The location of the CRAC sites cleavage sites C1, C2 and E are also indicated.
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gkt726-F5: Localization of the CRAC interaction sites of Rlp7 with pre-rRNA sequences displayed on the ‘hairpin model’ (A) and on the ‘ring model’ (B) for yeast ITS2 secondary structure [for a reference, see (45)]. The CRAC sites are highlighted in yellow; blue circles indicate frequently mutated residues found in the experiments (see Supplementary Figure S8). The Nsa3 and Nop15 CRAC sites, as described in (7), are represented as purple and green, respectively. The location of the CRAC sites cleavage sites C1, C2 and E are also indicated.

Mentions: L7 is a RNA binding protein (19) and due to its homology with L7, Rlp7 is also predicted to bind RNA. To find out whether Rlp7 and L7 share the same binding site on pre-60S ribosome, we attempted to identify their in vivo binding sites by using the CRAC method (9). The Rlp7-HTP strain did not show any growth phenotype at 30°C (Supplementary Figure S5). We also analysed L7B-HTP tagged strains, with or without the RPL7A endogenous copy, both presenting a phenotype consistent with the non-tagged corresponding strain (Supplementary Figure S5). We found that Rlp7-HTP directly and specifically contacts two regions in ITS2 (Figures 4 and 5, Supplementary Figure S7A, S8 and S9), whereas the non-ultraviolet cross-linked Rlp7-HTP protein did not significantly cross-link detectable rRNA (Supplementary Figure S7A). These two regions of ITS2 overlap the boundaries of 25S 5′ end and 5.8S 3′ end. Previous CRAC experiments with other A3 factors, Erb1, Nop7, Nop15 and Nsa3 revealed binding sites close and even overlapping these boundaries, consistent with the described collective role of these factors for processing of the 27SA3 pre-rRNA (6,7) (Figure 5 and Supplementary Figure S7A). Indeed, loss-of-function of the A3 factors (including Rlp7) leads to the accumulation of the 27SA3 pre-rRNA, reduced formation of 27SBS relative to 27SBL pre-rRNA and loss of cleavage at site C2 in ITS2 (6,7,20,21). Interestingly, the nucleotide substitutions analysis at specific positions in the sequence reads allowed us to precisely identify cross-link sites of Rlp7-HTP as nucleotides distributed in two groups, one adjacent to the pre-rRNA processing sites C1 and E that define the 5′ end and 3′ end of mature 25S and 5.8S rRNAs, respectively, and another at helix III at the 3′ end of ITS2, between nucleotides 200 and 225 (Figure 5 and Supplementary Figure S8).Figure 4.


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)

Localization of the CRAC interaction sites of Rlp7 with pre-rRNA sequences displayed on the ‘hairpin model’ (A) and on the ‘ring model’ (B) for yeast ITS2 secondary structure [for a reference, see (45)]. The CRAC sites are highlighted in yellow; blue circles indicate frequently mutated residues found in the experiments (see Supplementary Figure S8). The Nsa3 and Nop15 CRAC sites, as described in (7), are represented as purple and green, respectively. The location of the CRAC sites cleavage sites C1, C2 and E are also indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt726-F5: Localization of the CRAC interaction sites of Rlp7 with pre-rRNA sequences displayed on the ‘hairpin model’ (A) and on the ‘ring model’ (B) for yeast ITS2 secondary structure [for a reference, see (45)]. The CRAC sites are highlighted in yellow; blue circles indicate frequently mutated residues found in the experiments (see Supplementary Figure S8). The Nsa3 and Nop15 CRAC sites, as described in (7), are represented as purple and green, respectively. The location of the CRAC sites cleavage sites C1, C2 and E are also indicated.
Mentions: L7 is a RNA binding protein (19) and due to its homology with L7, Rlp7 is also predicted to bind RNA. To find out whether Rlp7 and L7 share the same binding site on pre-60S ribosome, we attempted to identify their in vivo binding sites by using the CRAC method (9). The Rlp7-HTP strain did not show any growth phenotype at 30°C (Supplementary Figure S5). We also analysed L7B-HTP tagged strains, with or without the RPL7A endogenous copy, both presenting a phenotype consistent with the non-tagged corresponding strain (Supplementary Figure S5). We found that Rlp7-HTP directly and specifically contacts two regions in ITS2 (Figures 4 and 5, Supplementary Figure S7A, S8 and S9), whereas the non-ultraviolet cross-linked Rlp7-HTP protein did not significantly cross-link detectable rRNA (Supplementary Figure S7A). These two regions of ITS2 overlap the boundaries of 25S 5′ end and 5.8S 3′ end. Previous CRAC experiments with other A3 factors, Erb1, Nop7, Nop15 and Nsa3 revealed binding sites close and even overlapping these boundaries, consistent with the described collective role of these factors for processing of the 27SA3 pre-rRNA (6,7) (Figure 5 and Supplementary Figure S7A). Indeed, loss-of-function of the A3 factors (including Rlp7) leads to the accumulation of the 27SA3 pre-rRNA, reduced formation of 27SBS relative to 27SBL pre-rRNA and loss of cleavage at site C2 in ITS2 (6,7,20,21). Interestingly, the nucleotide substitutions analysis at specific positions in the sequence reads allowed us to precisely identify cross-link sites of Rlp7-HTP as nucleotides distributed in two groups, one adjacent to the pre-rRNA processing sites C1 and E that define the 5′ end and 3′ end of mature 25S and 5.8S rRNAs, respectively, and another at helix III at the 3′ end of ITS2, between nucleotides 200 and 225 (Figure 5 and Supplementary Figure S8).Figure 4.

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