Crucial role of the Rcl1p-Bms1p interaction for yeast pre-ribosomal RNA processing.
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.
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.Show MeSH
Mentions: On the basis of the structure, we identified and mutated some Rcl1p residues located at the interface with Bms1p. The C277R and R327A substitutions altering one or the other binding site and the double mutation C277R/R327A were designed (Figure 2B and C). The various point mutants were co-expressed in bacteria with His-fused Bms1p (1–705) and pull-down experiments using a cobalt affinity resin were performed and analysed by SDS-PAGE (Figure 3). Interaction between wild-type Rcl1p and Bms1p as well as control experiments in which either Bms1p or Rcl1p was omitted are shown in lanes 1–3. The C277R single point mutation does not detectably affect Bms1p–Rcl1p interaction (lane 5), whereas, in contrast, the R327A mutation reduces the amount of Rcl1p pulled down by Bms1p (lane 4). The combination of C277R and R327A mutations seems to affect more drastically the interaction between the proteins (lane 6). Altogether we show that both Bms1p binding sites on Rcl1p contribute to the interaction with Bms1p and their modification affects the interaction between Bms1p and Rcl1p in vitro.
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.