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
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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.
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.