Pseudouridylation of yeast U2 snRNA is catalyzed by either an RNA-guided or RNA-independent mechanism.
Yeast U2 small nuclear RNA (snRNA) contains three pseudouridines (Psi35, Psi42, and Psi44). Pus7p and Pus1p catalyze the formation of Psi35 and Psi44, respectively, but the mechanism of Psi42 formation remains unclear. Using a U2 substrate containing a single (32)P radiolabel at position 42, we screened a GST-ORF library for pseudouridylase activity. Surprisingly, we found a Psi42-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 Psi42-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.
Ma
Xiaoju
X
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY 14642, USA.
Yang
Chunxing
C
Alexandrov
Andrei
A
Grayhack
Elizabeth J
EJ
Behm-Ansmant
Isabelle
I
Yu
Yi-Tao
YT
D002384:Catalysis; D015723:Gene Library; D006836:Hydro-Lyases / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009687:Nuclear Proteins / Q000378:metabolism; D011560:Pseudouridine / Q000378:metabolism*; D012331:RNA, Fungal / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000737:chemistry / Q000378:metabolism*; D020537:RNA, Small Nucleolar / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism; D020718:Ribonucleoproteins, Small Nucleolar / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism
D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D012331:RNA, Fungal; D012342:RNA, Small Nuclear; D020537:RNA, Small Nucleolar; D017411:Ribonucleoproteins, Small Nuclear; D020718:Ribonucleoproteins, Small Nucleolar; D029701:Saccharomyces cerevisiae Proteins; C405484:U2 small nuclear RNA; C069628:NHP2 protein, S cerevisiae; D011560:Pseudouridine; C073180:GAR1 protein, S cerevisiae; D006836:Hydro-Lyases; C082278:CBF5 protein, S cerevisiae