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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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Rcl1p and Bms1p are both recruited and released at similar stages of the pre-ribosome maturation pathway. (A) Pre-rRNAs associated with Rcl1p-TAP or Bms1p-3HA. The pre-ribosomal particles containing Rcl1p-TAP or Bms1p-3HA were immunoprecipitated by affinity chromatography. The associated RNAs were purified and analysed by northern blot using specific probes (indicated on the right) allowing detection of the indicated pre-rRNAs. (B) Association of Rcl1p-TAP with early pre-ribosomal particles in presence or absence of Nan1p (UTP-A module), Pwp2p (UTP-B module) or Rrp5p. Strains GAL::NAN1, GAL::PWP2 and GAL::RRP5 expressing Rcl1p-TAP were shifted from a galactose- to a glucose-containing medium to deplete the corresponding proteins. A strain expressing Rcl1p-TAP but otherwise wild type (WT) was used as a positive control and strain BY4741 (no tag) was used to reveal background immunoprecipitation signals. Total extracts were prepared from these different strains and the pre-ribosomal particles containing Rcl1p-TAP were isolated. The co-immunoprecipitated RNAs were analysed by northern blot using specific probes (mentioned on the right) allowing detection of the indicated pre-rRNAs. (C) Association of Bms1p and Rcl1p with a series of pre-ribosomal particles corresponding to different stages of the maturation pathway. Total extracts were prepared from strains expressing Bms1p-3HA and either Enp1p-TAP, Pwp2p-TAP, Noc4p-TAP, Rio2p-TAP, Npa1p-ZZ, Ssf1p-TAP or otherwise WT and the pre-ribosomal particles containing the TAP-tagged proteins were isolated. The co-immunoprecipitated proteins were analysed by western blot using anti-Rcl1p and anti-HA antibodies to detect endogenous Rcl1p and Bms1p-3HA, respectively. *, Bms1p-3HA signal remaining from the previous incubation with anti-HA antibodies. **, Pwp2p-TAP detected with the anti-HA antibodies.
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Figure 6: Rcl1p and Bms1p are both recruited and released at similar stages of the pre-ribosome maturation pathway. (A) Pre-rRNAs associated with Rcl1p-TAP or Bms1p-3HA. The pre-ribosomal particles containing Rcl1p-TAP or Bms1p-3HA were immunoprecipitated by affinity chromatography. The associated RNAs were purified and analysed by northern blot using specific probes (indicated on the right) allowing detection of the indicated pre-rRNAs. (B) Association of Rcl1p-TAP with early pre-ribosomal particles in presence or absence of Nan1p (UTP-A module), Pwp2p (UTP-B module) or Rrp5p. Strains GAL::NAN1, GAL::PWP2 and GAL::RRP5 expressing Rcl1p-TAP were shifted from a galactose- to a glucose-containing medium to deplete the corresponding proteins. A strain expressing Rcl1p-TAP but otherwise wild type (WT) was used as a positive control and strain BY4741 (no tag) was used to reveal background immunoprecipitation signals. Total extracts were prepared from these different strains and the pre-ribosomal particles containing Rcl1p-TAP were isolated. The co-immunoprecipitated RNAs were analysed by northern blot using specific probes (mentioned on the right) allowing detection of the indicated pre-rRNAs. (C) Association of Bms1p and Rcl1p with a series of pre-ribosomal particles corresponding to different stages of the maturation pathway. Total extracts were prepared from strains expressing Bms1p-3HA and either Enp1p-TAP, Pwp2p-TAP, Noc4p-TAP, Rio2p-TAP, Npa1p-ZZ, Ssf1p-TAP or otherwise WT and the pre-ribosomal particles containing the TAP-tagged proteins were isolated. The co-immunoprecipitated proteins were analysed by western blot using anti-Rcl1p and anti-HA antibodies to detect endogenous Rcl1p and Bms1p-3HA, respectively. *, Bms1p-3HA signal remaining from the previous incubation with anti-HA antibodies. **, Pwp2p-TAP detected with the anti-HA antibodies.

Mentions: Since Bms1p is required for the import of Rcl1p into the nucleus, we next investigated the chronology of recruitment and release of each protein in the maturation pathway. To get an overview of the pre-ribosomal particles containing Bms1p and Rcl1p, we purified Rcl1p-TAP or Bms1p-3HA from total cellular extracts and analysed the co-precipitated pre-rRNAs by northern blot. As shown in Figure 6A, the early 35S, 33S/32S and 23S precursors are precipitated above background levels with both Bms1p and Rcl1p. The 22S and 21S precursors are barely detectable in the total extracts, suggesting that these intermediates display a short half-life at steady state as compared to other species. Surprisingly, however, these species are best co-immunoprecipitated with both Rcl1p and Bms1p. Very low levels of 27SA2 intermediate are co-precipitated with Bms1p and Rcl1p suggesting that these proteins are not incorporated into early pre-60S particles following A2 cleavage. In contrast, significant amounts of the 20S precursor are associated with Bms1p and Rcl1p, indicating that both proteins are not immediately released from the particles following A2 cleavage but remain associated to some extent with pre-40S pre-ribosomal particles.


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)

Rcl1p and Bms1p are both recruited and released at similar stages of the pre-ribosome maturation pathway. (A) Pre-rRNAs associated with Rcl1p-TAP or Bms1p-3HA. The pre-ribosomal particles containing Rcl1p-TAP or Bms1p-3HA were immunoprecipitated by affinity chromatography. The associated RNAs were purified and analysed by northern blot using specific probes (indicated on the right) allowing detection of the indicated pre-rRNAs. (B) Association of Rcl1p-TAP with early pre-ribosomal particles in presence or absence of Nan1p (UTP-A module), Pwp2p (UTP-B module) or Rrp5p. Strains GAL::NAN1, GAL::PWP2 and GAL::RRP5 expressing Rcl1p-TAP were shifted from a galactose- to a glucose-containing medium to deplete the corresponding proteins. A strain expressing Rcl1p-TAP but otherwise wild type (WT) was used as a positive control and strain BY4741 (no tag) was used to reveal background immunoprecipitation signals. Total extracts were prepared from these different strains and the pre-ribosomal particles containing Rcl1p-TAP were isolated. The co-immunoprecipitated RNAs were analysed by northern blot using specific probes (mentioned on the right) allowing detection of the indicated pre-rRNAs. (C) Association of Bms1p and Rcl1p with a series of pre-ribosomal particles corresponding to different stages of the maturation pathway. Total extracts were prepared from strains expressing Bms1p-3HA and either Enp1p-TAP, Pwp2p-TAP, Noc4p-TAP, Rio2p-TAP, Npa1p-ZZ, Ssf1p-TAP or otherwise WT and the pre-ribosomal particles containing the TAP-tagged proteins were isolated. The co-immunoprecipitated proteins were analysed by western blot using anti-Rcl1p and anti-HA antibodies to detect endogenous Rcl1p and Bms1p-3HA, respectively. *, Bms1p-3HA signal remaining from the previous incubation with anti-HA antibodies. **, Pwp2p-TAP detected with the anti-HA antibodies.
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Figure 6: Rcl1p and Bms1p are both recruited and released at similar stages of the pre-ribosome maturation pathway. (A) Pre-rRNAs associated with Rcl1p-TAP or Bms1p-3HA. The pre-ribosomal particles containing Rcl1p-TAP or Bms1p-3HA were immunoprecipitated by affinity chromatography. The associated RNAs were purified and analysed by northern blot using specific probes (indicated on the right) allowing detection of the indicated pre-rRNAs. (B) Association of Rcl1p-TAP with early pre-ribosomal particles in presence or absence of Nan1p (UTP-A module), Pwp2p (UTP-B module) or Rrp5p. Strains GAL::NAN1, GAL::PWP2 and GAL::RRP5 expressing Rcl1p-TAP were shifted from a galactose- to a glucose-containing medium to deplete the corresponding proteins. A strain expressing Rcl1p-TAP but otherwise wild type (WT) was used as a positive control and strain BY4741 (no tag) was used to reveal background immunoprecipitation signals. Total extracts were prepared from these different strains and the pre-ribosomal particles containing Rcl1p-TAP were isolated. The co-immunoprecipitated RNAs were analysed by northern blot using specific probes (mentioned on the right) allowing detection of the indicated pre-rRNAs. (C) Association of Bms1p and Rcl1p with a series of pre-ribosomal particles corresponding to different stages of the maturation pathway. Total extracts were prepared from strains expressing Bms1p-3HA and either Enp1p-TAP, Pwp2p-TAP, Noc4p-TAP, Rio2p-TAP, Npa1p-ZZ, Ssf1p-TAP or otherwise WT and the pre-ribosomal particles containing the TAP-tagged proteins were isolated. The co-immunoprecipitated proteins were analysed by western blot using anti-Rcl1p and anti-HA antibodies to detect endogenous Rcl1p and Bms1p-3HA, respectively. *, Bms1p-3HA signal remaining from the previous incubation with anti-HA antibodies. **, Pwp2p-TAP detected with the anti-HA antibodies.
Mentions: Since Bms1p is required for the import of Rcl1p into the nucleus, we next investigated the chronology of recruitment and release of each protein in the maturation pathway. To get an overview of the pre-ribosomal particles containing Bms1p and Rcl1p, we purified Rcl1p-TAP or Bms1p-3HA from total cellular extracts and analysed the co-precipitated pre-rRNAs by northern blot. As shown in Figure 6A, the early 35S, 33S/32S and 23S precursors are precipitated above background levels with both Bms1p and Rcl1p. The 22S and 21S precursors are barely detectable in the total extracts, suggesting that these intermediates display a short half-life at steady state as compared to other species. Surprisingly, however, these species are best co-immunoprecipitated with both Rcl1p and Bms1p. Very low levels of 27SA2 intermediate are co-precipitated with Bms1p and Rcl1p suggesting that these proteins are not incorporated into early pre-60S particles following A2 cleavage. In contrast, significant amounts of the 20S precursor are associated with Bms1p and Rcl1p, indicating that both proteins are not immediately released from the particles following A2 cleavage but remain associated to some extent with pre-40S pre-ribosomal particles.

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