Home page > Research Groups > G Lutfalla, K Kissa > G Lutfalla > Cytokines, evolution and the onset of immunity > The Spb1p methyltransferase and the methylation of the 25S ribosomal RNA in S. cerevisiae yeast
The Spb1p methyltransferase and the methylation of the 25S ribosomal ARN in S cerevisiae yeast. I Introduction :
I-A The ribosomal RNAs are highly modified : The function of snoRNAs
Ribosome biogenesis is a complex and energy-consuming process occurring predominantly in the nucleolus. It requires the coordinated action of nucleolar proteins and the three RNA polymerases: RNA polymerase I for the transcription of 25S, 18S and 5.8S rRNA, RNA polymerase III for transcription of 5S rRNA, and RNA polymerase II for transcription of the genes encoding ribosomal proteins. The process starts with the transcription by RNA polymerase I of an rRNA precursor (pre-rRNA), which undergoes a rapid series of exo- and endonucleolytic cleavages concomitantly with chemical modification of the rRNA molecule, mainly by methylation (2’-O-ribose methylation and base-methylation) and pseudouridylation. During this process, many of the ribosomal proteins are assembled onto rRNA and numerous non-ribosomal proteins transiently associate with pre-rRNAs. These processing steps ultimately lead to the synthesis and export of the large 60S (LSU) and of the small 40S (SSU) ribosomal subunits to the cytoplasm (Kressler et al., 1999a; Venema and Tollervey, 1999; Fatica and Tollervey, 2002). While progress has been made on the identification and characterization of new factors involved in this process, several questions remain unsolved. Particularly, the function of the chemical modifications of the rRNA remains elusive. Almost all of the modified nucleotides that have been localized so far lie in evolutionary conserved regions of the rRNA, that are brought together in the functional center of the ribosome, arguing that these modifications play an important role either in biogenesis or in faithful function of the ribosome (Decatur and Fournier, 2002). In the last few years, several enzymes responsible for methylation of the rRNA have been identified both in bacteria and eukaryotes. In bacteria, site-specific enzymes perform these modifications (Bugl et al., 2000; Caldas et al., 2000; Gustafsson and Persson, 1998; Helser et al., 1972; Tscherne et al., 1999a; Tscherne et al., 1999b). In eukaryotes, this is also likely the case of base modification (Lafontaine et al., 1995). In contrast, 2’-O-ribose methylation and pseudouridylation are directed by small ribonucleoprotein particles (snoRNPs): snoRNP C/D for 2’-O-ribose methylation and snoRNP H/ACA for pseudouridylation (Kiss, 2001). Within the snoRNP, the RNA component confers site-specificity through base pairing (guide function) while catalysis is mediated by a specific protein component. For instance, within the snoRNP C/D, several lines of evidence suggest that Nop1 (also known as fibrillarin) displays the methyltransferase (MTase) activity (Galardi et al., 2002).
I-B Non guided 2’-O-ribose methylation: the hint toward one of the functions of Spb1 :
In S. cerevisiae, 51 out of the 55 2’-O-ribose-methylated sites that contain the cytoplasmic rRNA were shown to be directed by 41 snoRNAs C/D (Lowe and Eddy, 1999). Disruption of a specific snoRNA gene results in the absence of methylation on the corresponding position(s) but surprisingly, does not impair cell growth on rich media nor exhibit any defects in ribosome biogenesis or function. However, the absence of most of the 2’O-methylations, as observed in nop1-3, a temperature-sensitive mutant of NOP1, is lethal for the cells (Tollervey et al., 1993). LSU-U2918 is one of the four nucleotides that could be methylated by a different process. Indeed, snR52 has been predicted to direct the methylation of LSU-U2918 but this prediction has not been experimentally verified (Lowe and Eddy, 1999). Importantly, this residue is one of the five universally conserved residues of the A-loop in the peptidyl transferase center (PTC) of the ribosome that directly interacts with the tRNA aminoacyl (A)-site. It has been shown to be ribose methylated in the majority of organisms that have been investigated so far (Hansen et al., 2002). The equivalent residue is also methylated in the 21S mitochondrial rRNA (U2791). In E. coli, RrmJ has been identified as the site-specific MTase for this position (U2552 in E.coli 23S rRNA) (Bugl et al., 2000) (Caldas et al., 2000). Three yeast proteins exhibit striking sequence similarities to RrmJ: Spb1, Trm7 and Mrm2. Mrm2 is a mitochondrial protein which is responsible for the formation of Um2791 of the 21S mitochondrial rRNA (Pintard et al., 2002a), and Trm7 is a protein that catalyses the formation of 2’-O-methylribose in the tRNA anticodon loop (Pintard et al., 2002b). Therefore, we have hypothesized that Spb1, an essential nucleolar protein involved in ribosome biogenesis (Pintard et al., 2000) (Kressler et al., 1999b), may target methylation of LSU-U2918.
II Technical approach of 2’-O-ribose methylated residues
II-A The direct analysis of 2’-O-ribose methyled nucleotides
2’-O-ribose methylated nucleotides were first discovered in rRNA as a result of their conferring resistance to alkaline hydrolysis upon the phosphodiester bond (Singh and Lane, 1964). The first major advance for the analysis of rRNA methylation came from the introduction of oligonucleotide fingerprinting that allowed the counting of the methylated residues (Maden and Salim, 1974). The second major advance came with the localization of the methylated nucleotides : Methyl-labelled rRNA from cultured cells were hybridized to restriction fragments of rDNA and the methylated oligonucleotides that were recovered from the various hybridized regions were identified (Salim and Maden, 1981). For yeasts, the analysis was performed with S calbergensis. For this reason, the community has chosen to use the S calbergensis numbering for the methylated residues in yeast. The article Grosjean et col.(2004) describes the up to date methods for these analysis.
II-B The reverse transcriptase approach:
The last major advance for the detection of 2’-O-ribose methylated nucleotides in RNA was introduced by Maden (1995). It takes advantage of the relative difficulty for the RT to copy matrices with 2’-O-ribose methylated nucleotides. This phenomenon is evident when comparing reverse transcriptions of rRNAs performed with low (4microM) or high (1mM) concentrations of nucleotides and running them side by side on sequencing gels. They are called the “concentration dependent pauses”. Lowe and Eddy (1999) have systematically analyzed the contribution of snoRNAs for the 2’-O-ribose methylation of yeast rRNAs using this method. Their results with all the gels are available on their web site(http://lowelab.ucsc.edu/snoRNAdb/). This systematic analysis has raised some drawbacks of this technique. First, some 2’-O-ribose methylated nucleotides do not create “concentration dependent pauses”. Second, in each cases where two adjacent nucleotides are 2’-O-ribose methylated, the “concentration dependent pause” is not observed when the polymerase reaches the first modified nucleotides, but only when it reaches the second. For instance, in the case of the 5 universally conserved residues of the A loop in the peptidyl transferase center (PTC) (U2918GUUC2922, community numbering), U2918 and G2919 are 2’-O-ribose methylated. In reverse transcription experiments, the polymerase reaches G2919 first, but no obvious “concentration dependent pause” is observed; the obvious “concentration dependent pause” is at the level of U2918(http://lowelab.ucsc.edu/snoRNAdb/Sc...).
Figure 1 : The reverse transription block at LSU2278-2279 Despite the fact that the polymerase reaches LSU2279 first, the pause is observed on the second position (LSU2278)(http://lowelab.ucsc.edu/snoRNAdb/)
This is also observed for the other adjacent 2’-O-ribose methylated nucleotides present in the large (LSU) or small (SSU) sub-units rRNAs : SSU1265-1267(Fig 2); LSU 647-648 (Fig 3); LSU2278-2279 (http://lowelab.ucsc.edu/snoRNAdb/Sc...). The reverse transcription method therefore cannot be used for the study of the modification of the second modified base in such as those described here. In the case of the A site of the PTC, it can be used to study the modification of U2918 but not for the study of the modification of G2919 (see figure 1) . The modification of the latter requires direct analysis (see below). Figure 2 : The reverse transription block at SSU1265-1267(http://lowelab.ucsc.edu/snoRNAdb/Sc...)
Figure 3 : The reverse transription block at SSU1265-1267 (http://lowelab.ucsc.edu/snoRNAdb/Sc...)
III The 2’-O-ribose methylase activity of Spb1p :
Two contribution, one in 2003 and one in 2004 have addressed the 2’-O-ribose methylation of the residues of the A site of the PTC by Spb1p. One has addressed the methylation of U2918, the other has addressed the methylation of G2919 and confirmed the results of the first contribution on the methylation of U2918. The second contribution also addressed the question of the timing of the methylations of Um2918 and Gm2919.
III-A The methylation of U2918 by both Spb1p and a snR52-dependent mechanism: The study of the methylation of A2918 was performed using the method described by Lowe and Eddy (1999) using the same primers. A N-terminal HA tagged Spb1p mutant (HASpb1DA) devoided of Mtase activity was expressed in a strain with a deleted endogenous Spb1 gene. Controls indicate that the mutated tagged protein is expressed to the same level as a non mutated HA tagged version of Spb1p (HASpb1) that fully complements the deletion (Figure 4A). The first result is that the doubling time of the HASpb1DA strain is 160 minutes as compared to the 100 minutes for the HASpb1 strain (Figure 4B). The second is that the reverse transcription pattern of the rRNA around the A site is not modified. The reverse transcription block at the level of U2918 is unchanged (Figure 4C). The primer used in this study is the same as the one used in the study of Lowe and Eddy. Gels can therefore be directly compared to those of their Web page (http://lowelab.ucsc.edu/snoRNAdb/Sc...). Figure 4 : Phenotypic analysis of a strain expressing a methylase dead version of Spb1 (A) The methylase-dead and wild-type Spb1 proteins are produced at similar levels. Western blot analysis of spb1depleted cells complemented with a wild-type untagged protein (no tag), a wild-type tagged protein (Spb1), or a methylase-dead tagged protein (Spb1DA). (B) The methylase-dead Spb1 strain displays a growth defect. Growth of strains complemented by the WT tagged Spb1 (open squares) or complemented by Spb1DA (shaded diamonds) in YPD at 30C was monitored with a CASY-counter. (C) Expression of the methylase-dead version of Spb1 does not abrogate methylation of U2918. Methylation status of U2918 in 25S rRNA extracted from spb1depleted cells complemented with a plasmid expressing a wild-type (Spb1, lanes 2 & 3) or a mutant (Spb1DA, lanes 4 & 5) Spb1. O, unextended primer (lane 1).
As stressed by Eddy and Lowe (1999), methylation of Um2918 could be directed by snR52. snR52 could direct methylation at two different loci : SSU-Am420 and LSU-Um2918. The snR52 gene was therefore deleted and the methylation at both SSU-Am420 and LSU-Um2918 was tested in the context of a wt HASpb1p or in the context of a mutated HASpb1DAp. The first result is that the doubling time of the snR52d strain expressing the mutated HASpb1DAp has a doubling time of 250 minutes. The results of the analysis of the methylation status of SSU-Am420 and LSU-Um2918 are shown in next figure (Figure 5). It appears that, as already published by Eddy and Lowe, deletion of snR52 in the context of a wt HASpb1p impairs methylation at SSU-Am420 but does not change the reverse transcription pattern around LSU A site. On the other hand, yeasts with a deleted snR52 gene and a mutated HASpb1DAp do not show any “concentration dependent pause” at the level of Um2918. This proves that Um2918 is no more methylated. Bonnerot at al (2003) have shown that LSU-Um2918 can be methylated by the immunopurified HASpb1p protein in vitro. Figure 5 : Both snR52 and Spb1p are required for the methylation of Um2918
In the second contribution, by studying the methylation status of LSU-Um2918 (Um2921 with the S cerevisiae numbering) the authors have shown that the snR52 dependent methylation is an early mechanism taking place at the level of the 35S pre-rRNA but that Spb1p acts on later steps of rRNA biogenesis. Unfortunately, they have not used the same primer as Lowe and Eddy(1999), their ladders are difficult to calibrated and they have reached a wrong conclusion on the identity of the “concentration dependent pause” they report: They report that the “concentration dependent pause” is at the level of Gm2919 (Gm2922 with S cerevisiae numbering) when methylation is due to Spb1p and at the level of Am2918 when methylation is due snR52 alone. Careful examination of the ladders in their figure 4 (Lapeyre and Purushotaman, 2004) show that the “concentration dependent pause” they attribute to Gm2922 and the “concentration dependent pause” they attribute to Um2921 are in fact the same. Their figure 4 is therefore clearly mis-interpreted. They have missed the fact that when the reverse transcriptase encounters two successive methylated bases, it stops at the second position (see section II-B and figures 1 to 3 for illustration). The other reason why they can display such misinterpreted data is that the primer they use for the reverse transcription is too close to the methylated positions and does not allow callibration of the scales of the gel using LSU-Gm2945 and LSU-Am2943 as internal callibrators. They also inverted stains and reached wrong conclusions as regard to the doubling time of the different mutants they studied. Due to this inversion, their figure 1C is not correctly annotated and their conclusions are wrong.
III-B The methylation of G2919 by Spb1p:
As we have stressed in the previous part (“The reverse transcriptasse approach”) the methylation status of G2919 can not be studied by reverse transcription. In the second contribution, Lapeyre and Purushotaman have directly addressed the methylation status of U2918 and G2919. They confirmed the conclusions from the first study that U2918 can be mtethylated by both Spb1p and snR52. More importantly, they have schown that G2919 can only be methylated by Spb1p. This is an “a posteriori” confirmation that its methylation status cannot be investigated by reverse transcription : In figure 4C of this web page, lane 2, Gm2919 and Um2918 are both methylated (activity of Spb1p) and the reverse transcription pattern is the same as that in lane 4 where only Um2918 is methylated (mediated by snR52). Figure 6 here in, is the same as Figure 4C but with methylation status of residues highlighted. Figure 6: Concentration dependant poses and methylation status of Gm2919 and Um2918
Baudin, A., Ozier-Kalogeropoulos, O., Denouel, A., Lacroute, F., and Cullin, C. (1993). A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res. 21, 3329-3330.
Bonnerot, C., Pintard, L., and Lutfalla, G. (2003). Functional redundancy of Spb1p and a snR52-dependent mechanism for the 2’-O-ribose methylation of a conserved rRNA position in yeast. Mol Cell 12, 1309-1315.
Brand, R. C., Klootwijk, J., Van Steenbergen, T. J., De Kok, A. J., and Planta, R. J. (1977). Secondary methylation of yeast ribosomal precursor RNA. Eur. J. Biochem. 75, 311-318.
Bugl, H., Fauman, E. B., Staker, B. L., Zheng, F., Kushner, S. R., Saper, M. A., Bardwell, J. C., and Jakob, U. (2000). RNA methylation under heat shock control. Mol. Cell 6, 349-360.
Caldas, T., Binet, E., Bouloc, P., Costa, A., Desgres, J., and Richarme, G. (2000). The FtsJ/RrmJ heat shock protein of Escherichia coli is a 23 S ribosomal RNA methyltransferase. J. Biol. Chem. 275, 16414-16419.
Decatur, W. A., and Fournier, M. J. (2002). rRNA modifications and ribosome function. Trends Biochem. Sci. 27, 344-351. Fatica, A., and Tollervey, D. (2002). Making ribosomes. Curr. Opin. Cell Biol. 14, 313-318. Galardi, S., Fatica, A., Bachi, A., Scaloni, A., Presutti, C., and Bozzoni, I. (2002). Purified box C/D snoRNPs are able to reproduce site-specific 2’-O-methylation of target RNA in vitro. Mol. Cell. Biol. 22, 6663-6668.
Giaever, G., Chu, A. M., Ni, L., Connelly, C., Riles, L., Veronneau, S., Dow, S., Lucau-Danila, A., Anderson, K., Andre, B., et al. (2002). Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387-391.
Grandi, P., Rybin, V., Bassler, J., Petfalski, E., Strauss, D., Marzioch, M., Schafer, T., Kuster, B., Tschochner, H., Tollervey, D., et al. (2002). 90S pre-ribosomes include the 35S pre-rRNA, the U3 snoRNP, and 40S subunit processing factors but predominantly lack 60S synthesis factors. Mol. Cell 10, 105-115.
Green, R., and Noller, H. F. (1999). Reconstitution of functional 50S ribosomes from in vitro transcripts of Bacillus stearothermophilus 23S rRNA. Biochemistry 38, 1772-1779.
Grosjean, H., Keith, G., and Droogmans, L. (2004). Detection and quantification of modified nucleotides in RNA using thin-layer chromatography. Methods Mol Biol 265, 357-391.
Gustafsson, C., and Persson, B. C. (1998). Identification of the rrmA gene encoding the 23S rRNA m1G745 methyltransferase in Escherichia coli and characterization of an m1G745-deficient mutant. J. Bacteriol. 180, 359-365.
Guthrie, C., and Fink, R. G. (1991). Guide to yeast genetics and molecular biology. Methods Enzymol. 194, 1-863.
Hansen, M. A., Kirpekar, F., Ritterbusch, W., and Vester, B. (2002). Posttranscriptional modifications in the A-loop of 23S rRNAs from selected archaea and eubacteria. Rna 8, 202-213.
Helser, T. L., Davies, J. E., and Dahlberg, J. E. (1972). Mechanism of kasugamycin resistance in Escherichia coli. Nat. New Biol. 235, 6-9.
Khaitovich, P., Tenson, T., Kloss, P., and Mankin, A. S. (1999). Reconstitution of functionally active Thermus aquaticus large ribosomal subunits with in vitro-transcribed rRNA. Biochemistry 38, 1780-1788.
Kiss, T. (2001). Small nucleolar RNA-guided post-transcriptional modification of cellular RNAs. Embo J. 20, 3617-3622.
Kressler, D., Linder, P., and de La Cruz, J. (1999a). Protein trans-acting factors involved in ribosome biogenesis in Saccharomyces cerevisiae. Mol.Cell. Biol. 19, 7897-7912.
Kressler, D., Rojo, M., Linder, P., and Cruz, J. (1999b). Spb1p is a putative methyltransferase required for 60S ribosomal subunit biogenesis in Saccharomyces cerevisiae. Nucleic Acids Res. 27, 4598-4608.
Lafontaine, D., Vandenhaute, J., and Tollervey, D. (1995). The 18S rRNA dimethylase Dim1p is required for pre-ribosomal RNA processing in yeast. Genes Dev 9, 2470-2481.
Lapeyre, B, and Purushotaman, S. (2004) Spb1p-directed formation of Gm2922 in the ribosome catalytic center occurs at late processing stage. Mol. Cell 16, 663-669.
Lovgren, J. M., and Wikstrom, P. M. (2001). The rlmB gene is essential for formation of Gm2251 in 23S rRNA but not for ribosome maturation in Escherichia coli. J. Bacteriol. 183, 6957-6960.
Lowe, T. M., and Eddy, S. R. (1999). A computational screen for methylation guide snoRNAs in yeast. Science 283, 1168-1171.
Pintard, L., Bujnicki, J. M., Lapeyre, B., and Bonnerot, C. (2002a). MRM2 encodes a novel yeast mitochondrial 21S rRNA methyltransferase. Embo J. 21, 1139-1147.
Pintard, L., Kressler, D., and Lapeyre, B. (2000). Spb1p is a yeast nucleolar protein associated with Nop1p and Nop58p that is able to bind S-adenosyl-L-methionine in vitro. Mol. Cell. Biol. 20, 1370-1381.
Pintard, L., Lecointe, F., Bujnicki, J. M., Bonnerot, C., Grosjean, H., and Lapeyre, B. (2002b). Trm7p catalyses the formation of two 2’-O-methylriboses in yeast tRNA anticodon loop. Embo J. 21, 1811-1820.
Puig, O., Caspary, F., Rigaut, G., Rutz, B., Bouveret, E., Bragado-Nilsson, E., Wilm, M., and Seraphin, B. (2001). The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24, 218-229.
Qu, L. H., Henras, A., Lu, Y. J., Zhou, H., Zhou, W. X., Zhu, Y. Q., Zhao, J., Henry, Y., Caizergues-Ferrer, M., and Bachellerie, J. P. (1999). Seven novel methylation guide small nucleolar RNAs are processed from a common polycistronic transcript by Rat1p and RNase III in yeast. Mol. Cell. Biol. 19, 1144-1158.
Sirum-Connolly, K., and Mason, T. L. (1993). Functional requirement of a site-specific ribose methylation in ribosomal RNA. Science 262, 1886-1889. Tan, J., Jakob, U., and Bardwell, J. C. (2002). Overexpression of two different GTPases rescues a null mutation in a heat-induced rRNA methyltransferase. J. Bacteriol. 184, 2692-2698.
Tollervey, D., Lehtonen, H., Jansen, R., Kern, H., and Hurt, E. C. (1993). Temperature-sensitive mutations demonstrate roles for yeast fibrillarin in pre-rRNA processing, pre-rRNA methylation, and ribosome assembly. Cell 72, 443-457.
Tscherne, J. S., Nurse, K., Popienick, P., Michel, H., Sochacki, M., and Ofengand, J. (1999a). Purification, cloning, and characterization of the 16S RNA m5C967 methyltransferase from Escherichia coli. Biochemistry 38, 1884-1892.
Tscherne, J. S., Nurse, K., Popienick, P., and Ofengand, J. (1999b). Purification, cloning, and characterization of the 16 S RNA m2G1207 methyltransferase from Escherichia coli. J. Biol. Chem. 274, 924-929.
Venema, J., and Tollervey, D. (1999). Ribosome synthesis in Saccharomyces cerevisiae. Annu. Rev. Genet. 33, 261-311.