Random pseuoduridylation in vivo reveals critical region of Escherichia coli 23S rRNA for ribosome assembly.
Pseudouridine is the most common modified nucleoside in RNA, which is found in stable RNA species and in eukaryotic mRNAs. Functional analysis of pseudouridine is complicated by marginal effect of its absence. We demonstrate that excessive pseudouridines in rRNA inhibit ribosome assembly. Ten-fold increase of pseudouridines in the 16S and 23S rRNA made by a chimeric pseudouridine synthase leads to accumulation of the incompletely assembled large ribosome subunits. Hyper modified 23S rRNA is found in the r-protein assembly defective particles and are selected against in the 70S and polysome fractions showing modification interference. Eighteen positions of 23S rRNA were identified where isomerization of uridines interferes with ribosome assembly. Most of the interference sites are located in the conserved core of the large subunit, in the domain 0 of 23S rRNA, around the peptide exit tunnel. A plausible reason for pseudouridine-dependent inhibition of ribosome assembly is stabilization of rRNA structure, which leads to the folding traps of rRNA and to the retardation of the ribosome assembly.
Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia.
Margus
M
Leppik
Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia.
Aivar
A
Liiv
Institute of Molecular and Cell Biology, University of Tartu, Riia 23, 51010 Tartu, Estonia.
Jaanus
J
Remme
D001426:Bacterial Proteins / Q000032:analysis; D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D006836:Hydro-Lyases / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D001678:Organelle Biogenesis; D011560:Pseudouridine / Q000737:chemistry*; D059370:RNA Folding; D012323:RNA Processing, Post-Transcriptional; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D012338:RNA, Ribosomal, 23S / Q000737:chemistry / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012269:Ribosomal Proteins / Q000032:analysis; D012270:Ribosomes / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; D012338:RNA, Ribosomal, 23S; D011993:Recombinant Fusion Proteins; D012269:Ribosomal Proteins; D011560:Pseudouridine; D006836:Hydro-Lyases; C115603:RluD protein, E coli; C115994:rluC protein, E coli