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Crucial role of the Rcl1p-Bms1p interaction for yeast pre-ribosomal RNA processing.

Delprato A, Al Kadri Y, Pérébaskine N, Monfoulet C, Henry Y, Henras AK, Fribourg S - Nucleic Acids Res. (2014)

Bottom Line: We demonstrate that Rcl1p nuclear import depends on Bms1p and that the two proteins are loaded into pre-ribosomes at a similar stage of the maturation pathway and remain present within pre-ribosomes after cleavage at A2.Importantly, GTP binding to Bms1p is not required for the import in the nucleus nor for the incorporation of Rcl1p into pre-ribosomes, but is essential for early pre-rRNA processing.We propose that GTP binding to Bms1p and/or GTP hydrolysis may induce conformational rearrangements within the Bms1p-Rcl1p complex allowing the interaction of Rcl1p with its RNA substrate.

View Article: PubMed Central - PubMed

Affiliation: Institut Européen de Chimie et Biologie, ARNA laboratory, Université de Bordeaux, F-33607 Pessac, France Institut National de la Santé Et de la Recherche Médicale, INSERM - U869, ARNA laboratory, F-33000 Bordeaux, France.

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GTP binding to Bms1p is not required for incorporation of Rcl1p into pre-ribosomes. (A) Subcellular localization of Rcl1p-3HA in cells expressing Bms1pK82A or in ‘wild-type’ or Bms1p-depleted cells as controls. A yeast strain expressing Rcl1p-3HA and harbouring the chromosomal GAL::BMS1 construct was transformed with vectors expressing wild-type Bms1p (left panel), Bms1pK82A (right panel) or with the empty vector as a control (central panel). The resulting strains were shifted from a galactose- to a glucose-containing medium and grown for 20 h. Cells were harvested and processed for immunofluorescence microscopy using anti-HA antibodies. From left to right: DAPI (4',6'-diamidino-2-phénylindole) staining (blue), Rcl1p-3HA immunofluorescence signal (red) and the merged images. (B) Sucrose gradient sedimentation profile of Rcl1p using extracts from cells expressing ‘wild-type’ Bms1p (upper panel), Bms1pK82A (lower panel) or from Bms1p-depleted cells (central panel). The yeast strains grown as described in panel A were treated with cycloheximide and harvested. Total extracts were prepared and sedimented through 4.5–45% sucrose gradients. Twenty fractions were collected from which RNAs and proteins were extracted. Rcl1p-3HA was detected in the protein samples by western blot using anti-HA antibodies. The 35S, 23S and 20S pre-rRNAs were detected by northern blot using probe ‘a’. (C) Pre-rRNAs associated with Rcl1p-3HA in cells expressing Bms1pK82A or in ‘wild-type’ or Bms1p-depleted cells as controls. The pre-ribosomal particles containing Rcl1p-3HA were immunoprecipitated from extracts corresponding to the different strains grown as described in (A). The associated RNAs were analysed by northern blot using probe ‘a’ and probe ‘U3’.
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Figure 7: GTP binding to Bms1p is not required for incorporation of Rcl1p into pre-ribosomes. (A) Subcellular localization of Rcl1p-3HA in cells expressing Bms1pK82A or in ‘wild-type’ or Bms1p-depleted cells as controls. A yeast strain expressing Rcl1p-3HA and harbouring the chromosomal GAL::BMS1 construct was transformed with vectors expressing wild-type Bms1p (left panel), Bms1pK82A (right panel) or with the empty vector as a control (central panel). The resulting strains were shifted from a galactose- to a glucose-containing medium and grown for 20 h. Cells were harvested and processed for immunofluorescence microscopy using anti-HA antibodies. From left to right: DAPI (4',6'-diamidino-2-phénylindole) staining (blue), Rcl1p-3HA immunofluorescence signal (red) and the merged images. (B) Sucrose gradient sedimentation profile of Rcl1p using extracts from cells expressing ‘wild-type’ Bms1p (upper panel), Bms1pK82A (lower panel) or from Bms1p-depleted cells (central panel). The yeast strains grown as described in panel A were treated with cycloheximide and harvested. Total extracts were prepared and sedimented through 4.5–45% sucrose gradients. Twenty fractions were collected from which RNAs and proteins were extracted. Rcl1p-3HA was detected in the protein samples by western blot using anti-HA antibodies. The 35S, 23S and 20S pre-rRNAs were detected by northern blot using probe ‘a’. (C) Pre-rRNAs associated with Rcl1p-3HA in cells expressing Bms1pK82A or in ‘wild-type’ or Bms1p-depleted cells as controls. The pre-ribosomal particles containing Rcl1p-3HA were immunoprecipitated from extracts corresponding to the different strains grown as described in (A). The associated RNAs were analysed by northern blot using probe ‘a’ and probe ‘U3’.

Mentions: In Figure 5, strain RCL1::GFP was grown in a glucose-containing YP medium and strain [GAL::BMS1, RCL1::GFP] was shifted from a galactose- to a glucose-containing YP medium and grown for 11 h. In Figure 7, strain [GAL::BMS1, RCL1::3HA] transformed with vectors expressing wild-type Bms1p, Bms1pK82A or with the empty vector as a control were shifted from a galactose- to a glucose-containing synthetic medium (with all the required amino acids except histidine and leucine) and grown for 20 h to deplete the chromosome-encoded Bms1p protein. Fluorescence and immunofluorescence microscopy experiments were performed as described (21) with some minor modifications described in Supplementary Materials and Methods.


Crucial role of the Rcl1p-Bms1p interaction for yeast pre-ribosomal RNA processing.

Delprato A, Al Kadri Y, Pérébaskine N, Monfoulet C, Henry Y, Henras AK, Fribourg S - Nucleic Acids Res. (2014)

GTP binding to Bms1p is not required for incorporation of Rcl1p into pre-ribosomes. (A) Subcellular localization of Rcl1p-3HA in cells expressing Bms1pK82A or in ‘wild-type’ or Bms1p-depleted cells as controls. A yeast strain expressing Rcl1p-3HA and harbouring the chromosomal GAL::BMS1 construct was transformed with vectors expressing wild-type Bms1p (left panel), Bms1pK82A (right panel) or with the empty vector as a control (central panel). The resulting strains were shifted from a galactose- to a glucose-containing medium and grown for 20 h. Cells were harvested and processed for immunofluorescence microscopy using anti-HA antibodies. From left to right: DAPI (4',6'-diamidino-2-phénylindole) staining (blue), Rcl1p-3HA immunofluorescence signal (red) and the merged images. (B) Sucrose gradient sedimentation profile of Rcl1p using extracts from cells expressing ‘wild-type’ Bms1p (upper panel), Bms1pK82A (lower panel) or from Bms1p-depleted cells (central panel). The yeast strains grown as described in panel A were treated with cycloheximide and harvested. Total extracts were prepared and sedimented through 4.5–45% sucrose gradients. Twenty fractions were collected from which RNAs and proteins were extracted. Rcl1p-3HA was detected in the protein samples by western blot using anti-HA antibodies. The 35S, 23S and 20S pre-rRNAs were detected by northern blot using probe ‘a’. (C) Pre-rRNAs associated with Rcl1p-3HA in cells expressing Bms1pK82A or in ‘wild-type’ or Bms1p-depleted cells as controls. The pre-ribosomal particles containing Rcl1p-3HA were immunoprecipitated from extracts corresponding to the different strains grown as described in (A). The associated RNAs were analysed by northern blot using probe ‘a’ and probe ‘U3’.
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Figure 7: GTP binding to Bms1p is not required for incorporation of Rcl1p into pre-ribosomes. (A) Subcellular localization of Rcl1p-3HA in cells expressing Bms1pK82A or in ‘wild-type’ or Bms1p-depleted cells as controls. A yeast strain expressing Rcl1p-3HA and harbouring the chromosomal GAL::BMS1 construct was transformed with vectors expressing wild-type Bms1p (left panel), Bms1pK82A (right panel) or with the empty vector as a control (central panel). The resulting strains were shifted from a galactose- to a glucose-containing medium and grown for 20 h. Cells were harvested and processed for immunofluorescence microscopy using anti-HA antibodies. From left to right: DAPI (4',6'-diamidino-2-phénylindole) staining (blue), Rcl1p-3HA immunofluorescence signal (red) and the merged images. (B) Sucrose gradient sedimentation profile of Rcl1p using extracts from cells expressing ‘wild-type’ Bms1p (upper panel), Bms1pK82A (lower panel) or from Bms1p-depleted cells (central panel). The yeast strains grown as described in panel A were treated with cycloheximide and harvested. Total extracts were prepared and sedimented through 4.5–45% sucrose gradients. Twenty fractions were collected from which RNAs and proteins were extracted. Rcl1p-3HA was detected in the protein samples by western blot using anti-HA antibodies. The 35S, 23S and 20S pre-rRNAs were detected by northern blot using probe ‘a’. (C) Pre-rRNAs associated with Rcl1p-3HA in cells expressing Bms1pK82A or in ‘wild-type’ or Bms1p-depleted cells as controls. The pre-ribosomal particles containing Rcl1p-3HA were immunoprecipitated from extracts corresponding to the different strains grown as described in (A). The associated RNAs were analysed by northern blot using probe ‘a’ and probe ‘U3’.
Mentions: In Figure 5, strain RCL1::GFP was grown in a glucose-containing YP medium and strain [GAL::BMS1, RCL1::GFP] was shifted from a galactose- to a glucose-containing YP medium and grown for 11 h. In Figure 7, strain [GAL::BMS1, RCL1::3HA] transformed with vectors expressing wild-type Bms1p, Bms1pK82A or with the empty vector as a control were shifted from a galactose- to a glucose-containing synthetic medium (with all the required amino acids except histidine and leucine) and grown for 20 h to deplete the chromosome-encoded Bms1p protein. Fluorescence and immunofluorescence microscopy experiments were performed as described (21) with some minor modifications described in Supplementary Materials and Methods.

Bottom Line: We demonstrate that Rcl1p nuclear import depends on Bms1p and that the two proteins are loaded into pre-ribosomes at a similar stage of the maturation pathway and remain present within pre-ribosomes after cleavage at A2.Importantly, GTP binding to Bms1p is not required for the import in the nucleus nor for the incorporation of Rcl1p into pre-ribosomes, but is essential for early pre-rRNA processing.We propose that GTP binding to Bms1p and/or GTP hydrolysis may induce conformational rearrangements within the Bms1p-Rcl1p complex allowing the interaction of Rcl1p with its RNA substrate.

View Article: PubMed Central - PubMed

Affiliation: Institut Européen de Chimie et Biologie, ARNA laboratory, Université de Bordeaux, F-33607 Pessac, France Institut National de la Santé Et de la Recherche Médicale, INSERM - U869, ARNA laboratory, F-33000 Bordeaux, France.

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Related in: MedlinePlus