Immunopurified small nucleolar ribonucleoprotein particles pseudouridylate rRNA independently of their association with phosphorylated Nopp140.
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.
Wang
Chen
C
Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Query
Charles C
CC
Meier
U Thomas
UT
D000818:Animals; D001681:Biological Assay / Q000379:methods; D002458:Cell Fractionation; D002466:Cell Nucleolus / Q000737:chemistry / Q000378:metabolism; D002478:Cells, Cultured; D006367:HeLa Cells; D006801:Humans; D008099:Liver / Q000166:cytology / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism*; D009693:Nucleic Acid Hybridization; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011560:Pseudouridine / Q000378:metabolism*; D012335:RNA, Ribosomal / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D051381:Rats; D011915:Rats, Inbred BUF; D017411:Ribonucleoproteins, Small Nuclear; D020718:Ribonucleoproteins, Small Nucleolar / Q000302:isolation & purification / Q000378:metabolism*
C485802:GAR1 protein, human; C407731:GAR1 protein, mammalian; C107170:NAP57; C416929:NHP2 protein, human; C075564:NOLC1 protein, human; C513067:Nolc1 protein, rat; D009687:Nuclear Proteins; D010750:Phosphoproteins; D012335:RNA, Ribosomal; D016601:RNA-Binding Proteins; D017411:Ribonucleoproteins, Small Nuclear; D020718:Ribonucleoproteins, Small Nucleolar; D011560:Pseudouridine