We report here the characterization of Spb1p, a novel nucleolar protein of S. cerevisiae
, that is required for normal pre-rRNA processing. Spb1p copurifies with Nop1p through size chromatography and cation-exchange chromatography. Coimmunoprecipitation experiments demonstrate the association of Spb1p with Nop1p and Nop5/58p, another nucleolar protein previously shown to be associated with Nop1p (21
). A protein that migrates at 120 kDa was previously found to be associated with Nop1p and Nop5/58p (81
). This size is in good agreement with the observation that Spb1p migrates on SDS-PAGE as a protein with a size of ~120 kDa.
We present here evidence that the low level of 60S particles in the spb1-2
mutant is due to the inhibition of the processing of the 27S pre-rRNA into mature 25S and 5.8S rRNAs. We also detected a partial inhibition of the cleavage at sites A0, A1, and A2 that is observed in other yeast mutants that affect primarily 60S synthesis (42
). However, we cannot conclude whether Spb1p is directly involved in this process, particularly since we have not been able to detect a specific association of Spb1p with the C+D snoRNAs. Spb1p could exert its effect on rRNA synthesis through the proteins with which it interacts. This view is supported by the observation that depletion of Spb1p is accompanied by a delocalization of Nop1p in most cells. However, one cannot conclude at this point whether this delocalization is a direct consequence of the depletion of Spb1p or if it is a consequence of a general disorganization of the nucleolus due to an arrest of ribosome synthesis. It is striking that the spb1-2
strain has a very low 18S and 25S rRNA content. This could result from the very slow synthesis of the 60S subunits in the mutant, with the 40S being then adjusted by a feedback mechanism. Otherwise, in the light of its isolation as an essential gene involved in silencing (54
), Spb1p could have an additional function such as regulating rDNA transcription or recombination.
Comparing the sequence of Spb1p with the available databases reveals that the most striking feature is the presence of a putative AdoMet-binding domain, the cofactor of many methyltransferase enzymes. The presence of this motif in Spb1p was first reported (41
) due to similarities to another putative AdoMet-binding protein encoded by the ORF YCR47c (9
), both proteins being related to Escherichia coli
). We set up an in vitro assay to assess the AdoMet-binding activity of immunoprecipitated proteins. We found that Spb1p is able to bind in vitro to [3
H]AdoMet, supporting the view that it could be a novel methylase. Native extracts prepared from a strain expressing HA-Spb1p contained three major peptides that derived from Spb1p by proteolysis and contained the same N-terminal domain, since they were all detected by the anti-HA antibodies (Fig. ). These peptides were all able to bind [3
H]AdoMet, thus demonstrating that they all contain the AdoMet-binding domain. This finding is in agreement with the observation that the putative AdoMet-binding domain lies within the first 160 residues of the protein.
Considering that Spb1p is a putative methylase, we searched for its potential substrates. By comparing the incorporation of [5,6-3
H]uracil with l
H]methionine, we found that global methylation of the pre-rRNA was not decreased in the mutant cells, in contrast to the defect observed in the nop1-3
). This result indicates that Spb1p is not the major methylase of the pre-rRNA, even though we cannot exclude that Spb1p could be required to methylate only a subset of the methyl groups, as has been proposed for Nop2p (32
Another possibility would be that Spb1p is a protein methylase. Protein methylation is one of the numerous protein modifications that can modulate protein activity in vivo. Some modifications that appear to be reversible, such as the formation of methyl esters on the carboxyl groups, may participate in signal transduction (2
). In contrast, methylation of the amino group of the side chain of lysine or arginine residues seems to be irreversible but leads to the formation of modified amino acid groups that extend the repertoire of biochemical reactions that a protein can perform (13
). It has been known for a long time that proteins involved in RNA metabolism contain asymmetric dimethylarginines (19
). Among these, there is a subset of proteins that are located within the nucleolus and that share a common domain known as the GAR domain, in which arginine residues are dimethylated (25
). It has been suggested that the GAR domain could be involved in RNA binding (22
) or in protein-protein interactions (10
). A recent report indicates that methylation of the arginines could reduce their affinity for RNA (39
). Other workers have found that methylation of the nuclear protein Hrp1, which is involved in mRNA 3′-end cleavage and export, is inhibited by its cognate RNA (17
The major activity for arginine methylation in yeast has been characterized as the product of the gene RMT1
). In an rmt1Δ
strain, the amount of dimethylarginine is reduced to less than 15% compared to the wild type. However, an rmt1Δ
strain is viable, indicating that the function of this methylase is not essential or that it is partially redundant with another enzyme. Since there is some arginine dimethylation activity left in an rmt1Δ
strain, it is conceivable that Spb1p could be responsible for that activity, allowing the cell to survive. If Spb1p is truly a protein methylase, one obvious substrate would be the GAR protein Nop1p to which it is associated. Methylating the GAR domain of Nop1p could possibly control the activity of the protein, providing the cell with a tool for regulating ribosome synthesis.
AdoMet is used as a cofactor in a very broad spectrum of biochemical reactions. Its methyl group can be transferred to more than 40 different molecules. In addition to the AdoMet-binding domain, the broad variety of enzymes that utilize AdoMet as a cofactor must contain other motifs that should be characteristic of the classes to which they belong, as DNA-methylase, rRNA-methylase, protein-methylase, decarboxylase, etc. It is striking that the best similarity between the potential homologues of Spb1p is observed outside the four motifs that are proposed to form the AdoMet-binding domain. This might reveal that the AdoMet-binding domain has evolved to acquire a more specialized function. So far, sequence analyses do not readily detect the additional motifs that would allow us to sort these proteins and to predict their activity. However, data are now accumulating rapidly that may allow us in the near future to make this prediction. It is also conceivable that there is a second domain, interlaced with the AdoMet-binding one, that is specific for the Spb1p function and not for a class of methylases. We also are considering the possibility that Spb1p may not be a methyltransferase. Its ability to bind AdoMet could serve to regulate the availability and/or accessibility of AdoMet to other methylases such as Nop1p in order to regulate their activity. Experiments are in progress to assess the activity of a recombinant Spb1p protein in vitro and to identify its substrates in vivo.