Modified nucleotides in eukaryotic ribosomal RNA

Hughes, David Geoffrey (1976) Modified nucleotides in eukaryotic ribosomal RNA. PhD thesis, University of Glasgow.

Abstract

Eukaryotic ribosomal RNA contains a considerable number of modified nucleotides. The most common types of modification are 2'-0-methylation and pseudouridylation. This thesis describes studies on pseudouridine in eukaryotic rRNA and its possible relationship to 2'-0-methylation. A simple two-dimensional chromatographic system was developed for separating Up and Up from digests of 32P- labelled RNA. This system was used to quantitate up in HeLa rRNA,. The number of pseudouridines was found to be very close to the number of 2'-0-methyl groups. HeLa 28S RNA contains some 62 2'-0--methyl groups and 60 pseudouridines whilst 18S RNA. contains approximately 38 2'-0-methylations and 37 up residues. The up content of rRNA from mouse L-cells, Xenopus laevis, and Dicty ostelium discoidium was also quantitated. The number of pseudouridines and the number of 2'0-methyl groups was approximately equal in L-cell rRNA and in Xenopus 28S RNA, There was an appreciable excess of pseudouridine over 2'-0-methylation in Xenopus 1 8S RNA, The up content of Dictyostelium rRNA was markedly lower than in vertebrate rRNA's. Data on the number of 2'-0-methyl groups in this species are not accurate but it appears that methylation is also lower than in vertebrates. The pseudouridine content of HeLa 32S and 45S r pre RNA's was investigated. Considerable numbers of up residues were present in both precursors. 32 S RNA was found to contain slightly more pseudouridines than 28S RNA, The estimated number of up residues in 45S RNA (70) was less than the sum of the numbers found in 1 8S and 28S RNA,'s. Possible alternative explanations for this are discussed. Techniques for RNA fingerprinting and sequence analysis were used to investigate the presence of pseudouridine in oligonucleotides from HeLa 1 8S rRNA.. Pseudouridine occurs only at specific sites in the rRNA. sequences. A. large RNA fragment was prepared by digestion of HeLa 28S rRNA with T1 ribonuclease under mild conditions, followed by separation of the products on sucrose gradients. Base composition analysis showed that the fragment had a very high G + C content (ca, 80%). Electrophoretic separation on neutral polyacrylamide gels suggested that preparations contained several fragments of comparable, but not identical size, Denaturation of fragments on 6M urea gels suggested that these various fragments have at least part of their primary sequence in common. Both pancreatic RNase fingerprints and electrophoresis under denaturing conditions demonstrated that the large fragments contain internal nicks. The integrity of the large fragment must, therefore, be maintained after partial digestion by secondary structural interactions. Low reactivity of the fragment to sodium bisulphite suggested that a high proportion of G-C base pairs is present. Electron microscopy in 80% formamide showed that the fragment contains a high degree of secondary structure which is resistant to denaturation. The presence of modified nucleotides in the fragment was investigated. Measurement of 14 C-methyl radioactivity showed that few methyl groups are present. This was confirmed by fingerprinting analysis which showed only one or two 2'-0-methylated oligonucleotides. Pseudouridine was also found to be relatively less abundant in G-C rich fragments than in 28S rRNA, as a whole. Not more than three pseudouridines were present in the fragment, which is several hundred nucleotides long.

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: B EH Maden
• Keywords: Biochemistry
• Date of Award: 1976
• Depositing User: Enlighten Team
• Unique ID: glathesis:1976-72797
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 11 Jun 2019 11:06
• Last Modified: 11 Jun 2019 11:06
• URI: https://theses.gla.ac.uk/id/eprint/72797