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Molecular and Cellular Biology, December 2002, p. 8457-8466, Vol. 22, No. 24
0270-7306/02/$04.00+0     DOI: 10.1128/MCB.22.24.8457-8466.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Immunopurified Small Nucleolar Ribonucleoprotein Particles Pseudouridylate rRNA Independently of Their Association with Phosphorylated Nopp140

Chen Wang,1 Charles C. Query,2 and U. Thomas Meier1*

Department of Anatomy and Structural Biology,1 Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York 104612

Received 29 July 2002/ Returned for modification 6 September 2002/ Accepted 19 September 2002

The isomerization of up to 100 uridines to pseudouridines ({Psi}s) 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 32P-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.

* Corresponding author. Mailing address: Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Phone: (718) 430-3294. Fax: (718) 430-8996. E-mail: meier{at}aecom.yu.edu.

Molecular and Cellular Biology, December 2002, p. 8457-8466, Vol. 22, No. 24
0022-538X/02/$04.00+0     DOI: 10.1128/MCB.22.24.8457-8466.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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