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: To assess the relevance of the Bms1p–Rcl1p interaction in yeast cells, we introduced pFL46-derived plasmids encoding the wild-type or C277R, R327A or C277R/R327A altered versions of Rcl1p into a haploid yeast strain bearing a deletion of the chromosomal RCL1 gene by plasmid shuffling. These strains did not display significant growth defects at 30°C on a rich medium, indicating that the altered proteins complement the essential function of Rcl1p in these conditions. To assess the effect of these mutations on pre-rRNA processing (see Supplementary Figure S2 for a detailed description of the pre-rRNA processing pathway in yeast), total RNAs extracted from these strains were analysed by northern blot (Figure 4). Expression of wild-type RCL1 from the pFL46 plasmid (lane 2) results in subtle processing defects as compared to the wild-type BY4741 strain (lane 1) probably due to the overexpression of the protein (Supplementary Figure S3). In comparison, the R327A and C277R/R327A mutants (lanes 4 and 5, respectively) display stronger pre-rRNA processing defects characterized by an accumulation of the 35S, 33S and 23S pre-rRNAs and a decrease in the production of the 27SA2 and 20S pre-rRNAs (Figure 4B). These defects typically result from altered cleavages at sites A0, A1 and A2. However, we also observed a mild accumulation of the combined 21S + 22S pre-rRNAs and of the 22S intermediate detected alone, which may suggest that cleavage defects at sites A1 and A2 are stronger than that at site A0. The C277R substitution (lane 3) reproducibly restores a pre-rRNA accumulation profile comparable to that observed in the BY4741 wild-type cells (lane 1). The corresponding altered Rcl1p protein accumulates at slightly lower levels than the overexpressed wild-type protein (Supplementary Figure S3). We therefore hypothesized that this substitution per se does not affect significantly pre-rRNA processing (consistent with the fact that it does not affect the interaction between Rcl1p and Bms1p in vitro) and that the beneficial effect comes from the decreased expression level.
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.