The nucleolus is a cytologically prominent organelle of the cell nucleus which is the site of ribosomal RNA transcription and ribosome assembly. As much as 5% of the nuclear DNA can be localized in the nucleoli of mammalian cells, yet only about 2% of this DNA is comprised of the repeating ribosomal gene cluster. Of interest to us is the remaining 98% of the necleolar DNA which may not be directly involved in rRNA synthesis. The small nuclear RNA U3 is specifically localized in the nucleolus, and is currently thought to function in ribosomal RNA synthesis or processing. Our psoralen cross-linking evidence, however, indicated that U3 is associated with poly(A)+ nuclear transcripts, and not with ribosomal RNA. This suggests the possibility that U3 functions in the processing of messenger RNA precursors, and that there may be mRNA precursors specifically located in nucleoli. The goal of this research is to determine whether nucleolus-specific messenger RNA precursors exist, what their relationship is to RNA molecules associated with U3, and whether this classof mRNAs is actually encoded in the nucleolus. Nuclear RNA from psoralen cross-linked HeLa cells will be hybridized to cloned U3 DNA to isolate psoralen cross-linked complexes containing U3 and high molecular weight RNA. An mRNA-derived cDNA library will be constructed and screened directly with these U3-hnRNA complexes to determine whether U3-associated RNAs identify specific cDNA clones. HeLa cell poly(A)+ nucleolar RNA will also be isolated and used to screen the cDNA library. cDNA clones identified by these procedures will then be used as hybridization probes to determine whether certain mRNA precursors are enriched in, and therefore specific to, the nucleolar RNA preparations. To determine whether mRNAs are transcribed in nucleoli, a recombinant nucleolar DNA library will be constructed and screened with HeLa cell mRNA to identify nucleolar DNA sequences complementary to mRNA. The presence of these RNAs in the nucleolus would suggest that there may be a special a class of messenger RNAs whose transcription or processing is coupled with ribosome biosynthesis.
| Maser, R L; Calvet, J P (1989) U3 small nuclear RNA can be psoralen-cross-linked in vivo to the 5' external transcribed spacer of pre-ribosomal-RNA. Proc Natl Acad Sci U S A 86:6523-7 |
| Carley, L R; Raymond, S A (1987) Comparison of the after-effects of impulse conduction on threshold at nodes of Ranvier along single frog sciatic axons. J Physiol 386:503-27 |
