Abstract
The nucleolus of the yeast Saccharomyces cerevisiae contains the small nucleolar RNA snR30 (snoRNA), that is found associated with at least two proteins, NOP1 and GAR1. All three of these molecules are essential for the cell's viability and have been implicated in pre-rRNA maturation. NOP1 and GAR1 are believed to be general rRNA-processing factors or, alternatively, integral protein components of the small nucleolar ribonucleoprotein particle snR30 (snoRNP). In this paper, we describe procedures for the biochemical isolation of snR30 RNP, and we identify seven snR30 RNP proteins of molecular masses of 10, 23, 25, 38, 46, 48, and 65 kDa, including the previously reported GAR1 protein. Additional proteins, including NOP1, may also be components of snR30 RNP but are lost during our stringent isolation procedure. The 10-, 23-, and 25-kDa (GAR1) and 65-kDa proteins remain tightly associated with the snR30 RNA even after isopycnic sedimentation in cesium sulfate gradients. Electron microscopy of Mono Q-purified snR30 RNPs show a slightly elongated two-domain structure approximately 20 nm long and 14 nm wide.
Highlights
From the Institut fur Molekularbiologie und Tumorforschung, Philipps Universitat Marburg, Emil-Mannkopff-Stra/3e 2, D-35037 Marburg, Federal Republic of Germany
Nucleolar snRNPs Are Enriched in the m 7G Fraction of an Anti-Me.1G Immunoaffinity Column-We have previously demonstrated that both spliceosomal snRNPs and the nucleolar U3 snRNP can be isolated from yeast whole cell extracts by antiMe3G-immunoaffinity chromatography [23]
Isolation of snR30 RNP by Mono Q Anion-exchange Chromatography-We investigated whether individual snR30 particles could be isolated using Mono Q anion-exchange chromatography in the way previously described for mammalian snRNPs [22, 29]
Summary
From the Institut fur Molekularbiologie und Tumorforschung, Philipps Universitat Marburg, Emil-Mannkopff-Stra/3e 2, D-35037 Marburg, Federal Republic of Germany. The nucleolus of the yeast Saccharomyces cerevisiae contains the small nucleolar RNA snR30 (snoRNA), that is found associated with at least two proteins, NOPI and GAR! In addition to the snRNAs that mediate mRNA splicing, at least 14 snRNA species appear to be associated with the nucleolus and are implicated in ribosome biogenesis This has been elegantly proven by genetic approaches for the small nucleolar RNAs (snoRNAs) U3 (formerly termed snR17), U14 (formerly snR128), snR10, snR30 and, more recently, for snR31 [3,4,5,6,7,8].
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