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PMID: 1563354 Published · ppublish English Comparative Study Journal Article Research Support, Non-U.S. Gov't

Identification and functional analysis of two U3 binding sites on yeast pre-ribosomal RNA.

The EMBO journal ·Vol. 11 ·No. 4 ·1992-04-00 ·Pages 1531-42

Beltrame M, Tollervey D

Abstract

It has long been known that U3 can be isolated hydrogen bonded to pre-ribosomal RNAs, but the sites of interaction are poorly characterized. Here we show that yeast U3 can be cross-linked to 35S pre-rRNA both in deproteinized extracts and in living cells. The sites of cross-linking were localized to the 5' external transcribed spacer (ETS) and then identified at the nucleotide level. Two regions of U3 near the 5' end are cross-linked to pre-rRNA in vivo and in vitro; the evolutionarily conserved box A region and a 10 nucleotide (nt) sequence with perfect complementarity to an ETS sequence. Two in vivo cross-links are detected in the ETS, at +470, within the region complementary to U3, and at +655, close to the cleavage site at the 5' end of 18S rRNA. A tagged rDNA construct was used to follow the effects of mutations in the ETS in vivo. A small deletion around the +470 cross-linking site in the ETS prevents the synthesis of 18S rRNA. This region is homologous to the site of vertebrate ETS cleavage. We propose that this site may be evolutionarily conserved to direct the assembly of a pre-rRNA processing complex required for the cleavages that generate 18S rRNA.

MeSH Terms
Animals Base Sequence Binding Sites Chromosome Deletion Cross-Linking Reagents/pharmacology DNA, Ribosomal/genetics,metabolism Humans Mice Molecular Sequence Data Mutagenesis, Site-Directed Oligodeoxyribonucleotides Polymerase Chain Reaction RNA Precursors/metabolism RNA, Fungal/metabolism RNA, Ribosomal, 18S/biosynthesis RNA, Small Nuclear/metabolism Saccharomyces cerevisiae/genetics,metabolism Sequence Homology, Nucleic Acid Sequence Tagged Sites Transcription, Genetic Trioxsalen/analogs & derivatives,pharmacology Xenopus
Chemicals
Cross-Linking Reagents DNA, Ribosomal Oligodeoxyribonucleotides RNA Precursors RNA, Fungal RNA, Ribosomal, 18S RNA, Small Nuclear aminomethyltrioxsalen Trioxsalen
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Beltrame M
EMBL, Heidelberg, Germany.
Tollervey D
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35 references, click to expand
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
0261-4189
Published
1992-04-00
Pages
1531-42
Language
English
Region
England
NLM ID
8208664
PMCID
PMC556602
Subset
IM
External Links
PubMed source PMC Full Text View journal details
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