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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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Interaction mapping of Rcl1p binding site on Bms1p. (A) Bms1p harbours from the N-terminus to the C-terminus a GTPase domain, a U3 snoRNA binding site (15), two coiled coil domains as defined by HHPRED server flanking a GAP domain and a C-terminal NLS. Several truncated versions of Bms1p have been produced in order to define the Rcl1p binding site on Bms1p. (B) Truncated derivatives of Bms1p tagged with 6 histidines at the N-terminus have been co-expressed with untagged Rcl1p. Cell lysates were incubated with cobalt-affinity resin and the bound fractions were analysed by SDS-PAGE and Coomassie-blue staining. (*) represents a known E. coli contaminating protein (Bifunctional polymyxin resistance protein Arn).
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Figure 1: Interaction mapping of Rcl1p binding site on Bms1p. (A) Bms1p harbours from the N-terminus to the C-terminus a GTPase domain, a U3 snoRNA binding site (15), two coiled coil domains as defined by HHPRED server flanking a GAP domain and a C-terminal NLS. Several truncated versions of Bms1p have been produced in order to define the Rcl1p binding site on Bms1p. (B) Truncated derivatives of Bms1p tagged with 6 histidines at the N-terminus have been co-expressed with untagged Rcl1p. Cell lysates were incubated with cobalt-affinity resin and the bound fractions were analysed by SDS-PAGE and Coomassie-blue staining. (*) represents a known E. coli contaminating protein (Bifunctional polymyxin resistance protein Arn).

Mentions: As shown in Figure 1A, the GTP binding domain of Bms1p covers residues 69–320 while the GAP domain (residues 737–1120) and two coiled coil regions, extending between residues 705–737 and 1120–1159, can be identified using the HHPRED server (23). Previous studies have shown that the Bms1p GAP domain is dispensable for the interaction with Rcl1p (15). In addition, yeast two hybrid experiments suggested that a minimal fragment of Bms1p, encompassing residues 535–661, interacts with Rcl1p (14). To confirm and further characterize these interactions, several truncated versions of Bms1p have been generated and tested for their ability to interact with Rcl1p (Figure 1B). No background Rcl1p binding is detected upon incubation with affinity resin while formation of the His-Bms1p (1–705)-Rcl1p complex is visualized (lane 1). In contrast, the GTPase domain (residues 1–433) is not able to retain Rcl1p (lane 4). The 433–705 region of Bms1p has been reduced to a shorter fragment through the observation and the characterization of proteolytic fragments generated during the purification procedure. The fragment encompassing residues 523–670 is sufficient to retain Rcl1p upon pull down (lane 5).


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)

Interaction mapping of Rcl1p binding site on Bms1p. (A) Bms1p harbours from the N-terminus to the C-terminus a GTPase domain, a U3 snoRNA binding site (15), two coiled coil domains as defined by HHPRED server flanking a GAP domain and a C-terminal NLS. Several truncated versions of Bms1p have been produced in order to define the Rcl1p binding site on Bms1p. (B) Truncated derivatives of Bms1p tagged with 6 histidines at the N-terminus have been co-expressed with untagged Rcl1p. Cell lysates were incubated with cobalt-affinity resin and the bound fractions were analysed by SDS-PAGE and Coomassie-blue staining. (*) represents a known E. coli contaminating protein (Bifunctional polymyxin resistance protein Arn).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4150785&req=5

Figure 1: Interaction mapping of Rcl1p binding site on Bms1p. (A) Bms1p harbours from the N-terminus to the C-terminus a GTPase domain, a U3 snoRNA binding site (15), two coiled coil domains as defined by HHPRED server flanking a GAP domain and a C-terminal NLS. Several truncated versions of Bms1p have been produced in order to define the Rcl1p binding site on Bms1p. (B) Truncated derivatives of Bms1p tagged with 6 histidines at the N-terminus have been co-expressed with untagged Rcl1p. Cell lysates were incubated with cobalt-affinity resin and the bound fractions were analysed by SDS-PAGE and Coomassie-blue staining. (*) represents a known E. coli contaminating protein (Bifunctional polymyxin resistance protein Arn).
Mentions: As shown in Figure 1A, the GTP binding domain of Bms1p covers residues 69–320 while the GAP domain (residues 737–1120) and two coiled coil regions, extending between residues 705–737 and 1120–1159, can be identified using the HHPRED server (23). Previous studies have shown that the Bms1p GAP domain is dispensable for the interaction with Rcl1p (15). In addition, yeast two hybrid experiments suggested that a minimal fragment of Bms1p, encompassing residues 535–661, interacts with Rcl1p (14). To confirm and further characterize these interactions, several truncated versions of Bms1p have been generated and tested for their ability to interact with Rcl1p (Figure 1B). No background Rcl1p binding is detected upon incubation with affinity resin while formation of the His-Bms1p (1–705)-Rcl1p complex is visualized (lane 1). In contrast, the GTPase domain (residues 1–433) is not able to retain Rcl1p (lane 4). The 433–705 region of Bms1p has been reduced to a shorter fragment through the observation and the characterization of proteolytic fragments generated during the purification procedure. The fragment encompassing residues 523–670 is sufficient to retain Rcl1p upon pull down (lane 5).

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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