Identities and Phylogenetic Comparisons of Posttranscriptional Modifications in 16 S Ribosomal RNA from Haloferax volcanii

Abstract

Small subunit (16 S) rRNA from the archaeon Haloferax volcanii, for which sites of modification were previously reported, was examined using mass spectrometry. A census of all modified residues was taken by liquid chromatography/electrospray ionization-mass spectrometry analysis of a total nucleoside digest of the rRNA. Following rRNA hydrolysis by RNase T 1 , accurate molecular mass values of oligonucleotide products were measured using liquid chromatography/electrospray ionization-mass spectrometry and compared with values predicted from the corresponding gene sequence. Three modified nucleosides, distributed over four conserved sites in the decoding region of the molecule, were characterized: 3-(3-amino-3-carboxypropyl)uridine-966, N 6 -methyladenosine-1501, and N 6 ,N 6 -dimethyladenosine-1518 and -1519 (all Escherichia coli numbering). Nucleoside 3-(3-amino-3-carboxypropyl)uridine, previously unknown in rRNA, occurs at a highly conserved site of modification in all three evolutionary domains but for which no structural assignment in archaea has been previously reported. Nucleoside N 6 -methyladenosine, not previously placed in archaeal rRNAs, frequently occurs at the analogous location in eukaryotic small subunit rRNA but not in bacteria. H. volcanii small subunit rRNA appears to reflect the phenotypically low modification level in the Crenarchaeota kingdom and is the only cytoplasmic small subunit rRNA shown to lack pseudouridine.

The archaeal COG1901/DUF358 SPOUT-methyltransferase members, together with pseudouridine synthase Pus10, catalyze the formation of 1-methylpseudouridine at position 54 of tRNA

“…1, A and B) present in nearly all tRNAs sequenced so far (Marck and Grosjean 2002), Nep1 is certainly not the missing tRNA N1-pseudouridine methyltransferase. The reasons are: (1) The genome of S. acidocaldarius contains a Nep1 homolog (Saci_0034), but its bulk tRNA lacks m 1 C, and its only sequenced tRNA harbors U m 54 (Table 1; Gupta and Woese 1980;Kuchino et al 1982); (2) conversely, the genome of H. volcanii lacks the gene coding for Nep1, while nearly all of its tRNAs harbor m 1 C54 (Table 1; Gupta 1984Gupta , 1986; (3) this absence is consistent with the fact that helix 35 of H. volcanii 16S rRNA harbors an acp 3 U and not the ''hypermodified'' m 1 acp 3 C as in eukaryotes (Kowalak et al 2000). A better candidate for the missing m 1 C54 methyltransferase came from a bioinformatics analysis of a large variety of orphan genes coding for putative AdoMet-dependent methyltransferases in genomes of microorganisms belonging to the three domains of life.…”

Loss of rRNA modifications in the decoding center of the ribosome impairs translation and strongly delays pre-rRNA processing

“…We favor the latter possibility, since depleting the hypermodified C in different combinations with other modification depletions has different effects on processing. Consistent with a special role, this site has unusual modifications in all three kingdoms of life, although with different types: m 2 G966 in bacteria, acp3U966 in archaea, and m1acp3C in yeast and human (Brand et al 1978;Kowalak et al 2000). Based on present knowledge about the hypermodification process, we propose that in yeast C formation is blocked by depletion of the C guide snoRNA and this disruption prevents N1 methylation from occurring.…”

The identification and characterization of non-coding and coding RNAs and their modified nucleosides by mass spectrometry

“…The number of characterized bovine mitochondrial tRNAs was increased from 11 to 22 tRNAs, and 5 tRNAs that were previously characterized were found to contain www.landesbioscience.comadditional modifications that were originally not reported or reported as unknown modifications. 50 McCloskey and coworkers, building extensively on their RNA modification mapping protocol, 37 have mapped modified nucleosides onto the sequences of 16S rRNA from Haloferax volcanii, 51 Thermus thermophilus, 39 and Thermotoga maritima 40 by LC-MS/MS. Also using LC-MS/MS, Rozenski and coworkers mapped modifications onto the 16S rRNA sequences of Clostridium acetobutylicum 52 and Legionella pneumophila.…”