While E. coli cells only have one RrmJ homologue, eukaryotic cells usually have several. Yeast cells, for instance, have been found to harbor three RrmJ homologues; Trm7p in the cytosol (Pintard et al., 2002b), Mrm2p in mitochondria (Pintard et al., 2002a) and Spb1p in the nucleus (Pintard et al., 2000). Whereas the mitochondrial and nuclear RrmJ homologues function as rRNA methyltransferases, the cytosolic Trm7p has been shown to be responsible for two 2’-O-ribose methylations at position 32 (Cm32) and 34 (Gm34) in the anticodon loop of certain yeast tRNAs.
1.2.1 Spb1p – A rRNA methyltransferase in the yeast nucleolus
Spb1p has been shown by Pintard et al. to be located in the nucleolus of yeast cells, where it methylates 25S rRNA. Spb1p is almost three times the size of Mrm2p and Trm7p, which are 37.4 kDa and 34.7 kDa, respectively, and about ~100 amino acids longer than E.
coli RrmJ (Pintard et al., 2000). The main methylation target of Spb1 appears to be the essential G2922, whose equivalent nucleotide in E. coli 23S rRNA is G2553. Methylation of the
U2552 equivalent nucleotide U2591 in yeast rRNA, on the other hand, is guided by the snoRNA snR52 (Lapeyre and Purushothaman, 2004). Although, Spb1p seems to have acquired a new specificity, it is still able to methylate U2591 when the snoRNA snR52 is deleted (Lapeyre and Purushothaman, 2004). G2922 in yeast and its counterpart in E. coli, G2553, are the docking sites for aminoacyl tRNA and are critical for protein translation. In E. coli however, this highly conserved nucleotide G2553 is not modified.
Similar to the RrmJ mediated methylation of U2552, the modification of G2922 occurs at a very late state of ribosome maturation (Lapeyre and Purushothaman, 2004). This late recruitment of the enzyme to the ribosome suggests that the protein itself rather than the methylation is important for ribosome processing (Lapeyre and Purushothaman, 2004).
However, so far it was not possible to separate these potentially diverse functions of Spb1p.
Deletion of Spb1p is lethal. A point mutation that disrupts AdoMet binding and leaves Spb1p methyltranferase inactive, however, shows a dramatic growth phenotype and ribosome defect. Impaired pre-rRNA processing leads to a decrease of the 60S peak and to a relative increase of the 40S peak (Lapeyre and Purushothaman, 2004).
1.2.2 Mrm2p – A rRNA methyltransferase in yeast mitochondria
The RrmJ homologue Mrm2p is located in the mitochondria and methylates U2791 in the 21S rRNA. This is the equivalent nucleotide to U2552, the methylation target of E. coli RrmJ (Pintard et al., 2002a). Similar to RrmJ and Spb1p, this methylation occurs at a late stage of ribosome maturation. Unlike RrmJ, however, deletion of Mrm2p does not cause any ribosomal defect. However, a strain lacking the mrm2 gene becomes unable to respire at 37°C and rapidly loses its mitochondrial DNA when grown in YPD at 30°C. Therefore, Mrm2p appears to have an important function, which might be involved in altering the mitochondrial translation ability. Noteworthy, Pintard and coworkers show that Mrm2p co-fractionates with its ribosomal substrate, the 60S ribosomal subunit, on sucrose gradients (Pintard et al., 2002a). This was a rather unexpected finding considering the fact that Mrm2p works as an enzyme where fast release of the substrate is usually necessary to provide efficient catalytic activity.
1.2.3 Trm7p – A tRNA methyltransferase in the yeast cytosol
Trm7p is localized in the cytoplasm of S. cerevisiae where it methylates tRNA instead of rRNA. Pintard et al. demonstrated that Trm7p methylates C32 and G34 in the anticodon loop of tRNAPhe, tRNATrp and possibly tRNALeu (Pintard et al., 2002b). Both methylations occur at a late stage after the removal of the intron and are not dependent on other modifications in the respective tRNAs (Pintard et al., 2002b). Pintard et al. hypothesize that C32 is methylated in the conformational flexible, hypomodified anticodon hairpin, where its 2’hydroxyl can be presented to the active site of Trm7p. G34, on the other hand, is being methylated in a rigid, hypomodified loop. A trm7 deletion mutant exerts a low rate of protein synthesis, which is reflected by a decrease in polysomes as well as a significant growth defect. A point mutation in the AdoMet binding region of Trm7p was unable to rescue this growth phenotype, indicating that the loss of methylation activity is responsible for the impaired growth of the trm7 deletion strain.
The ability of yeast RrmJ homologues to recognize either rRNA or tRNA corroborates reports about a possible dual substrate specificity of E. coli RrmJ, which has been found to also be capable of methylating tRNAs in vitro in addition to 23S rRNA (Bügl et al., 2000).
1.2.4 Comparison of the RNA substrates
Structural comparison of the A-loop of the 23S rRNA, which harbors the target nucleotide of RrmJ, to the RNA substrates of RrmJ’s homologues in yeast revealed significant similarities (Fig. 1). Only the tRNA anticodon loop, where the target nucleotides of Trm7p reside, seems to slightly differ from the three rRNA loops (Pintard et al., 2002b).
However, although the tRNA anticodon loop is formed by seven nucleotides instead of five, the target nucleotide (C32) is in the same 5’ position as U2552 in the A-loop of the 23S rRNA.
Furthermore, the anticodon loop can be reduced to a five nucleotide-long loop due to a base pairing between positions 32 and 38. Therefore, all four proteins, RrmJ in E. coli, as well as Spb1p, Mrm2p and Trm7p in yeast, recognize a very similar loop structure suggesting that all three yeast proteins, which are present in distinct compartments have evolved from a common ancestor (Pintard et al., 2002b).
Figure 1: Comparison of the RNA substrates of E. coli RrmJ and its yeast homologues (Pintard et al., 2002b)
Schematic representation of the secondary structure of the target sites of E. coli RrmJ and each of the three enzymes in yeast that belong to the 2’-O-RNA methyltransferases (Pintard et al., 2002b).