Article
- The EMBO Journal (2005) 24, 2403 - 2413
- doi:10.1038/sj.emboj.7600718
Published online: 16 June 2005
Subject Category:
Pseudouridylation of yeast U2 snRNA is catalyzed by either an RNA-guided or RNA-independent mechanism
Xiaoju Ma1,a, Chunxing Yang1,a, Andrei Alexandrov1, Elizabeth J Grayhack1, Isabelle Behm-Ansmant2 and Yi-Tao Yu1
- Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, USA
- Laboratoire de Maturation des ARN et Enzymologie Moleculaire, UMR 7567 CNRS-UHP Nancy I, Faculte des Sciences, Vandoeuvre-les-Nancy, France
Correspondence to:
Yi-Tao Yu, Department of Biochemistry and Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA. Tel.: +1 585 275 1271; Fax: +1 585 275 6007; E-mail: yitao_yu@urmc.rochester.edu
aThese authors contributed equally to this work
Received 25 April 2005; Accepted 25 May 2005
Abstract
Yeast U2 small nuclear RNA (snRNA) contains three pseudouridines (
35, 42, and 44). Pus7p and Pus1p catalyze the formation of 35 and 44, respectively, but the mechanism of 42 formation remains unclear. Using a U2 substrate containing a single 32P radiolabel at position 42, we screened a GST-ORF library for pseudouridylase activity. Surprisingly, we found a 42-specific pseudouridylase activity that coincided with Nhp2p, a protein component of a Box H/ACA sno/scaRNP (small nucleolar/Cajal body-specific ribonucleoprotein). When isolated by tandem affinity purification (TAP), the other protein components of the H/ACA sno/scaRNP also copurified with the pseudouridylase activity. Micrococcal nuclease-treated TAP preparations were devoid of pseudouridylase activity; however, activity was restored upon addition of RNAs from TAP preparations. Pseudouridylation reconstitution using RNAs from a Box H/ACA RNA library identified snR81, a snoRNA known to guide rRNA pseudouridylation, as the 42-specific guide RNA. Using the snR81-deletion strain, Nhp2p- or Cbf5p-conditional depletion strain, and a cbf5 mutation strain, we further demonstrated that the pseudouridylase activity is dependent on snR81 snoRNP in vivo. Our data indicate that snRNA pseudouridylation can be catalyzed by both RNA-dependent and RNA-independent mechanisms.
Keywords:
- pseudouridylation reconstitution,
- RNA pseudouridylation,
- S. cerevisiae,
- snR81 snoRNP,
- U2 snRNA
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