The pathway taken by 5S RNA in oocytes of Xenopus laevis.
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In Xenopus oocytes, 5S ribosomal RNA (5S RNA) is synthesised prior to other ribosomal components, and stored in the cytoplasm in ribonucleoprotein particles (RNPs). At vitellogenesis, when ribosome assembly begins, 5S RNA is imported into the nucleus and targeted to the amplified nucleoli for assembly into the 60S ribosomal subunit. In this thesis, I have investigated some of the steps of this pathway taken by 5S RNA. In vivo assays using a series of mutant 5S RNAs revealed that only one mutant, with base substitutions in loop A, was defective for binding the 7S RNP storage protein, TFIIIA. All mutants were capable of binding to ribosomal protein L5, a precursor to ribosome assembly. Four of the mutants tested were defective for incorporation into 60S subunits, possibly due to a loss of recognition sites for interactions with other ribosomal proteins. Nucleolar localisation studies showed that the defective ribosome incorporation of these mutants was not due to defective nucleolar targeting. Taken together, these results reveal that different structural features of 5S RNA and different oocyte factors are required for different steps in the pathway taken by 5S RNA. Nucleolar localisation studies also revealed that a large proportion of oocyte-type 5S RNA and L5 in the nucleus are not associated with nucleoli. In contrast, somatic-type 5S RNA was predominantly asssociated with nucleoli, suggesting that nuclear factors directly recognise the sequence differences between the two types of 5S RNA. These could be nucleolar components, which have a higher affinity for somatic-type, or nucleoplasmic factors which have a higher affinity for oocyte-type. Finally, the mechanism by which 7S RNPs are sequestered in the cytoplasm of previtellogenic oocytes was investigated. The results show that neither cytoskeletal or membrane structures are responsible for cytoplasmic retention. Other possibilities for cytoplasmic retention are discussed.