Biomedical subjects

B A Peculis

Publications and source records attributed to B A Peculis.

6 recordsLinked to original sources

PubMed1998

The structure of the ITS2-proximal stem is required for pre-rRNA processing in yeast.

Accurate and efficient processing of pre-rRNA is critical to the accumulation of mature functional ribosomal subunits for maintenance of cell growth. Processing requires numerous factors which act in trans as well as RNA sequence/ structural elements which function in cis. To examine the latter, we have used directed mutagenesis and expression of mutated pre-rRNAs in yeast. Specifically, we tested requirements for formation of an ITS2-proximal stem on processing, a structure formed by an interaction between sequences corresponding to the 3' end of 5.8S rRNA and the 5' end of 25S. Pre-rRNA processing is inhibited in templates encoding mutations that prevent the formation of the ITS2-proximal stem. Compensatory, double mutations, which alter the sequence of this region but restore the structure of the stem, also restore processing, although at lower efficiency. This reduction in efficiency is reflected in decreased levels of mature 5.8S and 25S rRNA and increased levels of 35S pre-rRNA and certain processing intermediates. This phenotype is reminiscent of the biochemical depletion of U8 snoRNA in vertebrates for which the ITS2-proximal stem has been proposed as a potential site for interaction with U8 RNP. Thus, formation of the ITS2-proximal stem may be a requirement common to yeast and vertebrate pre-rRNA processing.

Animals↗

PubMed1997

The sequence of the 5' end of the U8 small nucleolar RNA is critical for 5.8S and 28S rRNA maturation.

Ribosome biogenesis in eucaryotes involves many small nucleolar ribonucleoprotein particles (snoRNP), a few of which are essential for processing pre-rRNA. Previously, U8 snoRNA was shown to play a critical role in pre-rRNA processing, being essential for accumulation of mature 28S and 5.8S rRNAs. Here, evidence which identifies a functional site of interaction on the U8 RNA is presented. RNAs with mutations, insertions, or deletions within the 5'-most 15 nucleotides of U8 do not function in pre-rRNA processing. In vivo competitions in Xenopus oocytes with 2'O-methyl oligoribonucleotides have confirmed this region as a functional site of a base-pairing interaction. Cross-species hybrid molecules of U8 RNA show that this region of the U8 snoRNP is necessary for processing of pre-rRNA but not sufficient to direct efficient cleavage of the pre-rRNA substrate; the structure or proteins comprising, or recruited by, the U8 snoRNP modulate the efficiency of cleavage. Intriguingly, these 15 nucleotides have the potential to base pair with the 5' end of 28S rRNA in a region where, in the mature ribosome, the 5' end of 28S interacts with the 3' end of 5.8S. The 28S-5.8S interaction is evolutionarily conserved and critical for pre-rRNA processing in Xenopus laevis. Taken together these data strongly suggest that the 5' end of U8 RNA has the potential to bind pre-rRNA and in so doing, may regulate or alter the pre-rRNA folding pathway. The rest of the U8 particle may then facilitate cleavage or recruitment of other factors which are essential for pre-rRNA processing.

Animals↗

PubMed1996

Ribosomal RNA: small nucleolar RNAs make their mark.

Small nucleolar RNAs direct the location of certain methylations in ribosomal RNA by direct base pairing; although evolutionarily conserved, the physiological significance of these modifications remains unclear.

Animals↗

PubMed1994

Sequence and structural elements critical for U8 snRNP function in Xenopus oocytes are evolutionarily conserved.

We have generated mutants in Xenopus U8 RNA, a nucleolar snRNA required for the maturation of 5.8S and 28S rRNAs, to identify sequences and structural domains essential for RNA stability, particle assembly, and function of the U8 RNP. Activity of the mutants was assayed by microinjection of in vitro-synthesized U8 RNAs into the cytoplasm of Xenopus oocytes. Most of the mutant RNAs were stable, bound fibrillarin, a protein common to several of the nucleolar-specific snRNPs, and became hypermethylated. Although hypermethylation of the 5' cap of U8 RNA and fibrillarin binding can occur in either the cytoplasmic or nuclear compartment of Xenopus oocytes, neither is required for nuclear import. We find that the trimethylguanosine cap, although present on the endogenous U8 RNA, is not essential for stability, particle assembly, or functioning of U8 in the coordinate processing of pre-rRNA at sites 3' of 28S and 5' of 5.8S RNA. Several conserved single- and double-stranded sequences within the 5' domain of U8 RNA are essential for function.

Animals↗

PubMed1993

Disruption of U8 nucleolar snRNA inhibits 5.8S and 28S rRNA processing in the Xenopus oocyte.

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.

Animals↗

PubMed1992

Localization of the nucleolar protein NO38 in amphibian oocytes.

To examine the role of primary amino acid sequence in the localization of proteins within the nucleus, we studied the nucleolar protein NO38 of amphibian oocytes. We synthesized NO38 transcripts in vitro, injected them into the oocyte cytoplasm, and followed the distribution of the translation products. The injected RNA contained a short sequence encoding an epitope derived from the human c-myc protein. We used an mAb against this epitope to detect translation products from injected RNAs by Western blots and by immunofluoresent staining of cytological preparations. When full-length transcripts of NO38 were injected into oocytes, the translation products accumulated efficiently in the germinal vesicle, and a major fraction was localized in the multiple nucleoli. To identify protein domains involved in this nucleolus-specific accumulation, we prepared a series of carboxy-terminal deletions of the cDNA. Oocytes injected with RNA encoding truncated forms of NO38 were examined for altered patterns of protein accumulation. We defined a domain of about 24 amino acids near the carboxy terminus that was essential for nucleolar localization of NO38. This domain is separated by more than 70 amino acids from two putative nuclear localization signals near the middle of the molecule. Hybrid constructs were made which encoded part of Escherichia coli beta-galactosidase or pyruvate kinase fused to a long segment of NO38 containing the essential domain. Injection of RNA from these constructs showed that the essential domain was not sufficient to target the hybrid proteins to the nucleolus. We suggest that nucleolar accumulation of NO38 requires more than a single linear domain.

Amino Acid Sequence↗