In general, transcription of a eukaryotic ribosomal DNA (rDNA) unit yields a 45S precursor ribosomal RNA (rRNA) which undergoes methylations at specific sites and protein additions to form an 80S ribonucleoprotein (RNP) particle. A series of nucleolar processing steps convert the 80S particle into a large (60S) and a small (40S) subunit, which are then transported presumably via the nuclear pores, to the cytoplasm. Relatively little is known about the structural elements (in the nuclear matrix and nuclear envelope), interacting components, machinery, and regulatory elements necessary to transport RNPs in general from the nucleus to the cytoplasm. Whether or not a general mechanism exists for the transport of all RNPs is unknown. Specifically what components of RNPs (RNA and/or protein) interact with the pore complex constituents and/or other nuclear components remains to be determined. The long term research goals of this laboratory are (1) to determine the specific components and interactions involved in transporting rRNPs to the cytoplasm and (2) to determine how rRNP movement to the cytoplasm is regulated in different developmental and disease states. Our main objective outline in this proposal is limited to the identification of sequences in 28S rRNA that impact (either directly or indirectly) the transport of rRNPs form the nucleus to the cytoplasm. We propose to undertake our transport studies using Xenopus oocytes microinjected with engineered rRNAs (made by manipulating specific rDNAs). We have hypothesized that nucleocytoplasmic transport domains exist within rRNA and are defined by conserved rRNA sequences common only to eukaryotes. We will test this hypothesis by (1) determining the competency of E. coli rRNPs to be transported in Xenopus oocytes and (2) determining the ability of heterologous 28S mutant rRNAs (created either by deletions or by using antisense RNAs to block the function of RNA at specific sites) to be transported in Xenopus oocytes. These investigations should enable us to determine the feasibility of a genetic approach to the study of putative RNA interactions involved in rRNP transport in eukaryotes and overall will provide a better understanding of the function of eukaryotic rRNA.
| Khanna-Gupta, A; Ware, V C (1989) Nucleocytoplasmic transport of ribosomes in a eukaryotic system: is there a facilitated transport process? Proc Natl Acad Sci U S A 86:1791-5 |
