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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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Bms1p depletion results in cytoplasmic accumulation of Rcl1p in yeast cells. (A) Subcellular localization of Rcl1p-GFP in the presence or absence of Bms1p. Upper 3 rows: yeast cells expressing a chromosomal GFP-tagged version of Rcl1p were grown in a glucose-containing rich medium. Lower 3 rows: yeast cells expressing Rcl1p-GFP and harbouring a chromosomal GAL1::BMS1 construct were shifted from a galactose- to a glucose-containing medium and grown for 11 h. In both cases, cells were harvested and processed for fluorescence microscopy. From left to right: Rcl1p-GFP fluorescence signal (green), DAPI staining (blue), merged images. (B) Accumulation levels of Rcl1p-GFP in the presence or absence of Bms1p. Yeast cells expressing Rcl1p-GFP and harbouring the chromosomal GAL1::BMS1 construct were shifted from a galactose- to a glucose-containing medium and grown for 11 h. Cells were harvested, total proteins were extracted and analysed by western blot using the indicated antibodies. The RCL1::GFP and BY4741 strains were grown on a glucose-containing medium and processed in parallel to determine the accumulation levels of Rcl1p-GFP and the endogenous Rcl1p protein, respectively, in these conditions. The nucleolar protein Nhp2p was detected as a loading control. * and **, non-specific proteins detected with the anti-GFP antibodies.
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Figure 5: Bms1p depletion results in cytoplasmic accumulation of Rcl1p in yeast cells. (A) Subcellular localization of Rcl1p-GFP in the presence or absence of Bms1p. Upper 3 rows: yeast cells expressing a chromosomal GFP-tagged version of Rcl1p were grown in a glucose-containing rich medium. Lower 3 rows: yeast cells expressing Rcl1p-GFP and harbouring a chromosomal GAL1::BMS1 construct were shifted from a galactose- to a glucose-containing medium and grown for 11 h. In both cases, cells were harvested and processed for fluorescence microscopy. From left to right: Rcl1p-GFP fluorescence signal (green), DAPI staining (blue), merged images. (B) Accumulation levels of Rcl1p-GFP in the presence or absence of Bms1p. Yeast cells expressing Rcl1p-GFP and harbouring the chromosomal GAL1::BMS1 construct were shifted from a galactose- to a glucose-containing medium and grown for 11 h. Cells were harvested, total proteins were extracted and analysed by western blot using the indicated antibodies. The RCL1::GFP and BY4741 strains were grown on a glucose-containing medium and processed in parallel to determine the accumulation levels of Rcl1p-GFP and the endogenous Rcl1p protein, respectively, in these conditions. The nucleolar protein Nhp2p was detected as a loading control. * and **, non-specific proteins detected with the anti-GFP antibodies.

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)

Bms1p depletion results in cytoplasmic accumulation of Rcl1p in yeast cells. (A) Subcellular localization of Rcl1p-GFP in the presence or absence of Bms1p. Upper 3 rows: yeast cells expressing a chromosomal GFP-tagged version of Rcl1p were grown in a glucose-containing rich medium. Lower 3 rows: yeast cells expressing Rcl1p-GFP and harbouring a chromosomal GAL1::BMS1 construct were shifted from a galactose- to a glucose-containing medium and grown for 11 h. In both cases, cells were harvested and processed for fluorescence microscopy. From left to right: Rcl1p-GFP fluorescence signal (green), DAPI staining (blue), merged images. (B) Accumulation levels of Rcl1p-GFP in the presence or absence of Bms1p. Yeast cells expressing Rcl1p-GFP and harbouring the chromosomal GAL1::BMS1 construct were shifted from a galactose- to a glucose-containing medium and grown for 11 h. Cells were harvested, total proteins were extracted and analysed by western blot using the indicated antibodies. The RCL1::GFP and BY4741 strains were grown on a glucose-containing medium and processed in parallel to determine the accumulation levels of Rcl1p-GFP and the endogenous Rcl1p protein, respectively, in these conditions. The nucleolar protein Nhp2p was detected as a loading control. * and **, non-specific proteins detected with the anti-GFP antibodies.
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Figure 5: Bms1p depletion results in cytoplasmic accumulation of Rcl1p in yeast cells. (A) Subcellular localization of Rcl1p-GFP in the presence or absence of Bms1p. Upper 3 rows: yeast cells expressing a chromosomal GFP-tagged version of Rcl1p were grown in a glucose-containing rich medium. Lower 3 rows: yeast cells expressing Rcl1p-GFP and harbouring a chromosomal GAL1::BMS1 construct were shifted from a galactose- to a glucose-containing medium and grown for 11 h. In both cases, cells were harvested and processed for fluorescence microscopy. From left to right: Rcl1p-GFP fluorescence signal (green), DAPI staining (blue), merged images. (B) Accumulation levels of Rcl1p-GFP in the presence or absence of Bms1p. Yeast cells expressing Rcl1p-GFP and harbouring the chromosomal GAL1::BMS1 construct were shifted from a galactose- to a glucose-containing medium and grown for 11 h. Cells were harvested, total proteins were extracted and analysed by western blot using the indicated antibodies. The RCL1::GFP and BY4741 strains were grown on a glucose-containing medium and processed in parallel to determine the accumulation levels of Rcl1p-GFP and the endogenous Rcl1p protein, respectively, in these conditions. The nucleolar protein Nhp2p was detected as a loading control. * and **, non-specific proteins detected with the anti-GFP antibodies.
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