ABSTRACT
The nucleoli of vertebrate cells contain several snRNPs, of which only one, U3, has been assigned a role in rRNA processing. We present the primary sequence of Xenopus U8, a fibrillarin-associated nucleolar snRNA, and examine its expression through oocyte development. Antisense deoxyoligonucleotides were microinjected into Xenopus oocytes to deplete the endogenous pool of U8 RNA. Analysis of the mature rRNAs and rRNA intermediates that accumulate in the U8-depleted oocytes indicate that the U8 snRNP is essential for correct maturation of the 5.8S and 28S rRNAs at both their 5' and 3' ends. U8 is therefore a nucleolar snRNA implicated in a nucleolytic rRNA processing step other than 18S maturation. Evidence for a long-lived 5.8S rRNA intermediate (12S) in Xenopus is also presented.
Subject(s)
RNA, Ribosomal, 28S/drug effects , RNA, Ribosomal, 5.8S/drug effects , RNA, Small Nuclear , Ribonucleoprotein, U4-U6 Small Nuclear/physiology , Animals , Base Sequence , DNA, Antisense/pharmacology , Female , Humans , Molecular Sequence Data , Oocytes , XenopusABSTRACT
Diethyl pyrocarbonate reactivity and thermal denaturation were used to probe potential ribosomal interactions between tRNA and the small 5.8S and 5S rRNAs. Puromycin, an analogue of the terminal aminoacyl-adenosine portion of aminoacyl-tRNA, was observed to increase the accessibility of the 5.8S rRNA, including the highly conserved GAACp sequences. EDTA which releases both tRNA and the 5S rRNA-protein complex resulted in an even greater accessibility in the 5.8S rRNA. The thermal dissociation of whole ribosomes resulted in the release of all three RNAs, with a striking similarity in the denaturation profiles. These results strongly suggest an interdependence in the ribosome-associated structures of the small rRNAs and provide in situ evidence for the various 5S rRNA, 5.8S rRNA, and tRNA containing ribonucleoprotein complexes previously reconstituted through affinity chromatography.
