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Immunopurified Small Nucleolar Ribonucleoprotein Particles Pseudouridylate rRNA Independently of Their Association with Phosphorylated Nopp140
- Source :
- Molecular and Cellular Biology. 22:8457-8466
- Publication Year :
- 2002
- Publisher :
- Informa UK Limited, 2002.
-
Abstract
- The isomerization of up to 100 uridines to pseudouridines (Psis) in eukaryotic rRNA is guided by a similar number of box H/ACA small nucleolar RNAs (snoRNAs), each forming a unique small nucleolar ribonucleoprotein particle (snoRNP) with the same four core proteins, NAP57 (also known as dyskerin or Cbf5p), GAR1, NHP2, and NOP10. Additionally, the nucleolar and Cajal body protein Nopp140 (Srp40p) associates with the snoRNPs. To understand the role of these factors in pseudouridylation, we established an in vitro assay system. Short site-specifically (32)P-labeled rRNA substrates were incubated with subcellular fractions, and the conversion of uridine to Psi was monitored by thin-layer chromatography after digestion to single nucleotides. Immunopurified box H/ACA core particles were sufficient for the reaction. SnoRNPs associated quantitatively and reversibly with Nopp140. However, pseudouridylation activity was independent of Nopp140, consistent with a chaperoning role for this highly phosphorylated protein. Although up to 14 bp between the snoRNA and rRNA were required for the in vitro reaction, rRNA pseudouridylation and release occurred in the absence of ATP and magnesium. These data suggest that substrate release takes place without RNA helicase activity but may be aided by the snoRNP core proteins.
- Subjects :
- Nucleolus
Gene Expression
RNA-binding protein
Biology
Cell Fractionation
Pseudouridine
Dyskerin
chemistry.chemical_compound
Ribonucleoproteins, Small Nucleolar
Animals
Humans
Nucleotide
Phosphorylation
Rats, Inbred BUF
Small nucleolar RNA
Molecular Biology
Cells, Cultured
chemistry.chemical_classification
Nuclear Proteins
Nucleic Acid Hybridization
RNA-Binding Proteins
Cell Biology
Ribosomal RNA
Phosphoproteins
Ribonucleoproteins, Small Nuclear
Rats
Liver
chemistry
Cajal body
Biochemistry
RNA, Ribosomal
Biological Assay
Cell Nucleolus
HeLa Cells
Subjects
Details
- ISSN :
- 10985549
- Volume :
- 22
- Database :
- OpenAIRE
- Journal :
- Molecular and Cellular Biology
- Accession number :
- edsair.doi.dedup.....83b79751e80e7b6635727cfe34393643