Stepwise dissociation of yeast 60S ribosomal subunits by LiCl and identification of L25 as a primary 26S rRNA binding protein

Abstract

Treatment of yeast 60s ribosomal subunits with 0.5 M LiCl was found to remove all but six of the ribosomal proteins. The proteins remaining associated with the (26s + 5.8s) rRNA complex were identified as L4, L8, LIO, L12, L16 and L25. These core proteins were split off sequentially in the order (L16 + L12), L10, (L4 + L8), L25by further increasing the LiCl concentration. At 1 .O M LiCl only ribosomal protein L25 remains bound to the rRNA. Upon lowering the LiCl concentration the core proteins reassociate with the rRNA in the reverse order of their removal. The susceptibility of the ribosomal proteins to removal by LiCl corresponds quite well with their order of assembly into the 60s subunit in viva as determined earlier [Kruiswijk et al. (1978) Biochim. Biophys. Acta 517,378 -3891. Binding studies in vitro using partially purified L25 showed that this protein binds specifically to 26s rRNA. Therefore our experiments for the first time directly identify a eukaryotic ribosomal protein capable of binding to high-molecular-mass rRNA. Binding studies in vitro using a blot technique demonstrated that core proteins L8 and L16 as well as protein L21, though not present in any of the core particles, are also capable of binding to 26s rRNA to approximately the same extent as L25. About nine additional 60s proteins appeared to interact with the 26s rRNA, though to a lesser extent.Structural and functional studies on prokaryotic ribosomes have benefited enormously from our ability to take ribosomal subunits apart and reassemble the components again into biologically active particles. A widely used technique for controlled dissociation of ribosomal constituents is treatment of ribosomal subunits with high concentrations of monovalent cations, notably LiC1. Such treatment results in removal of a portion of the ribosomal proteins, leaving discrete core particles on which a well-defined subset of the ribosomal proteins remains [I -31. Such cores have played an important role in establishing structure-function relationships for various prokaryotic ribosomal components (see for instance [4]) and continue to be used for this purpose [5, 61. Moreover, the split protein fractions obtained by LiCl treatment are an excellent source of partially purified ribosomal proteins, the conformation of which appears not to be unduly disturbed by this extraction method [7].

Yeast Ribosomal Protein L12 Is a Substrate of Protein-arginine Methyltransferase 2

“…It contains dimethyllysine(s), trimethyllysines(s) (30), and methylarginine(s) (31). It can complex with rRNA in the presence of 0.5 M LiCl (32). Incubating the ribosome with 0.5 M NH 4 Cl generated the ribosome salt wash fraction in this study.…”

Factors Affecting Nuclear Export of the 60S Ribosomal Subunit In Vivo

“…Although the differences between these two 60S localization assays may seem minor, they have the potential to identify a different set of factors required for ribosomal subunit assembly and export. Because Rpl25 binds directly to the rRNA, it is likely to be buried within the 60S ribosomal subunit (El-Baradi et al, 1984, 1987Yeh and Lee, 1998). Rpl11 binds later in ribosome assembly and appears to be located at the surface of the 60S ribosomal subunit (Tsay et al, 1994).…”

Protein trans-Acting Factors Involved in Ribosome Biogenesis in Saccharomyces cerevisiae

“…It has also been shown that three large-subunit r-proteins on mature 60S subunits can exchange in vivo, and the exchangeability of Rpl10p/Qsr1p, in particular, which is required for 60S to 40S subunit joining, suggests the possibility of an additional translational regulatory mechanism (46,209). Due to the lack of in vitro reconstitution assays for eukaryotic ribosomes, the abovementioned studies could only be complemented by approaches that examined the abilities of the different r-proteins to either bind to pre-rRNA molecules in vitro (204) or dissociate from purified ribosomal particles by increasing concentration of ions (52,106). The affinity of r-proteins for pre-rRNAs and the release of r-proteins by increasing concentration of ions are in general agreement with the order of their association with the preribosomal particles during in vivo ribosomal subunit assembly.…”