Some studies on the low molecular weight RNA components of mammalian cells

Clason, Alexander E (1971) Some studies on the low molecular weight RNA components of mammalian cells. PhD thesis, University of Glasgow.

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Abstract

Until recently, RNA was considered to belong to one of three categories; transfer RNA(tRNA), ribosomal RNA or messenger RNA. In addition however, the development of more sophisticated analytical techniques such as polyacrylamide gel electrophoresis, coupled with the improved cell fractionation procedures, has enabled the detection of hitherto unrecognised low molecular weight RNA species in both the nuclear and cytoplasmic fractions of eukaryete cells. The aim of this work was to investigate the identity and molecular characteristics of these various low molecular weight RNA components in the nuclei and cytoplasm of normal, malignant and virus transformed animal cells and to define the molecular processes involved in their biogenesis. (A) Low Molecular Weight RNAs of the Nucleus. Polyacrylamide gel electrophoresis of extracts of isolated mammalian nuclei indicate the existence of approximately fourteen distinct low molecular weight RNA species with a size range of 80-350 nucleotides in length. These RNA components are stable and appear each to be present to the extent of approximately 105 molecules per cell. Many of these RNA molecules are methylated either in the base or ribose moeities and they are distributed between the nuclealar and nucleoplasmic fractions of the nucleus. Analysis of their patterns of synthesis in synchronised cell populations indicate two distinct patterns of synthesis, for some appear to be synthesised only at, or just after, the time of maximum DNA synthesis during the S phase of the cell cycle whilst others appear to be synthesised continually. In addition these low molecular weight methylated RNA components appear to be confined to the nucleus for no methylated RNA components of similar size and base composition can be found in the cytoplasm, (B) Low Molecular Weight RNAs of the Cytoplasm, Electrophoresis of cytoplasmic preparations from mammalian tissue culture cell lines reveals the presence of approximately thirteen low molecular weight RNA components with, a size range of 80-350 nucleotides in length. The bulk of these RNA components possess lower electrophoretic mobilities than the 5s RNA component from the cytoplasmic ribosomes. Kinetics of labelling studies indicate them to be synthesised in a fairly linear fashion and to show no precursor-product relationship one to another. They appear to be fairly stable, being less metabolically stable than tRNA or the RNA of the cytoplasmic ribosomes, but possessing total life spans of the order of approximately 24-30 hours. Unlike the low molecular weight RNA components of the nucleus they appear to be devoid of methylated nucleosides, the only methylated low molecular weight cytoplasmic RNA component being transfer RNA. Studies on the intracellular location of these RNA spedies indicate that they are absent from the cell sap, are not associated with the mitochondrial fraction but are associated principally with the microsomal fraction and appear in fact to be found on the polyribosomes. They are released from the polyribosomes subsequent to EDTA treatment and appear to be released in the form of free RNA strands rather than as ribonucleoprotein associates. Nucleotide composition analysis indicates these RNA species to possess an average' (G+C) content of 54% and experiments employing ethidium bromide, which specifically inhibits mitochondrial RNA synthesis, indicate that they are not derived from the transcription of mitochondrial DNA. Inhibitor studies using low levels of actinomycin D suggest them to be of nucleoplasmic origin whilst experiments employing puromycin and cordycepin suggest that their function is not that of mRNA. Exporiments utilising the adenosine analogue, toyocamycin, raise the possibility that they could represent the cleavage products of some larger precursor molecule(s). Analyses of the synthesis of these low molecular weight RNA components of the cytoplasm in synchronised cell populations indicate that they are not synthesised coordinately with the DNA during the S phase of the cell cycle but are synthesised throughout the S and phases with a rate which apparently increases through these periods of the cell cycle. Additional experiments investigating the effects of the drug amanitin on RNA synthesis in mammalian cells indicate that the in vitro effects of the drug are different from the in vivo effects and suggest, that an extranucleolar control of nucleolar function exists in mammalian cells.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: R H Burdon
Keywords: Biochemistry
Date of Award: 1971
Depositing User: Enlighten Team
Unique ID: glathesis:1971-73793
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 14 Jun 2019 08:56
Last Modified: 14 Jun 2019 08:56
URI: http://theses.gla.ac.uk/id/eprint/73793

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