Sequences of the branch site recognition region of vertebrate U2 and S. cerevisiae U2. The three pseudouridines numbered 34, 41, and 43 in vertebrate U2 are equivalent to the three pseudouridines numbered 35, 42, and 44 in yeast U2 (three arrows). Cm and Um in vertebrate U2 indicate 2′-O-methylated residues. The branch site recognition sequences are boxed.

Nhp2p-associated pseudouridylase activity is specific for the formation of Ψ42 in yeast U2 snRNA. Yeast U2 snRNA containing a ³²P label at the 5′ side of either U42 (lanes 1–4), U35 (lanes 5–8), or U44 (lanes 9–12) was used in pseudouridylation assays (see Materials and methods, and Figure 2 legend). The final concentration of the GST-Nhp2p preparation (purified from strain 2A) in the reactions was ∼15 ng/ml (lanes 2, 6, and 10), ∼75 ng/ml (lanes 3, 7, and 11), or ∼375 ng/ml (lanes 4, 8, and 12). As controls, the wild-type yeast cell extract (lanes 5 and 9) or water (lane 1) was used in place of the GST-Nhp2p preparation.

The formation of Ψ42 in yeast U2 requires an RNA component. (A) Pseudouridylation assay was performed using yeast U2 containing a single ³²P label at the 5′ side of U42 (see Materials and methods, and previous figure legends). (B) Pseudouridylation assay was performed using a fragment of yeast 25S rRNA containing a single ³²P label at the 5′ side of U989. In lane 2 (in panels A and B), the Gar1p-TAP preparation (∼75 ng/ml) was present in the reaction. In lane 3 (A, B), micrococcal nuclease (MN)-treated Gar1p-TAP preparation was used. The reaction in lane 4 (A, B) was identical to that in lane 3 except that it contained total RNA prepared from the Gar1p-TAP preparation (not treated with micrococcal nuclease). Lane 1 in each case shows a control in which no TAP preparation was added.

Depletion of either Nhp2p or Cbf5p specifically abolishes Ψ42 formation in yeast U2 snRNA in vivo. (A) Growth curves for three yeast strains after the switch from galactose medium to glucose medium (YPD). Closed circles represent the nhp2-depletion strain in which chromosomal nhp2 was replaced by a plasmid containing a wild-type NHP2 gene under the control of the Gal promoter. Closed triangles represent the strain in which chromosomal cbf5 was replaced by a plasmid containing a wild-type CBF5 gene under the control of the Gal promoter. Closed squares represent the wild-type strain. (B) The U2 pseudouridylation assay (CMC modification followed by primer extension) was performed using total RNA isolated either from the wild-type strain that had been incubated in YPD for 6 h (lanes 1 and 2), from the nhp2-depletion strain that had been incubated in YPD for 6 h (lanes 3 and 4), 16 h (lane 5), or 48 h (lane 6), or from the cbf5-depletion strain that had been incubated in YPD for 6 h (lanes 7 and 8), 16 h (lane 9), or 48 h (lane 10). In lanes 1, 3, and 7, CMC modification was omitted. The lanes labeled U, C, G, and A represent a yeast primer-extension sequencing ladder. The lane labeled H₂O contained no ddNTP. The three primer-extension stops/pauses representing the three pseudouridines (Ψ35, Ψ42, and Ψ44) in U2 snRNA are indicated. (C) A pseudouridylation assay for yeast 25S rRNA was performed exactly as in panel B except that the primer-extension primer was complementary to yeast 25S rRNA (see Materials and methods). The naturally occurring pseudouridylation sites are indicated.

The formation of Ψ42 in U2 and Ψ1051 in 25S rRNA is dependent on snR81 RNA. The pseudouridylation assay for both U2 snRNA and 25S rRNA was carried out exactly as in Figure 7B and C except that total RNA was isolated from different yeast strains grown in different media (see Materials and methods, and Figure 7B and C legends). Specifically, the wild-type yeast strain (lanes 5 and 6) and snR81Δ strain (lanes 7 and 8) were grown in YPD medium. The snR81Δ strain that had been transformed with a plasmid containing a wild-type snR81 gene under the control of P_GAL1 promoter (lanes 9–12) was grown either in YPD (lanes 11 and 12) or YPGal (lanes 9 and 10) medium. Total RNA recovered from each of these strains was subjected to pseudouridylation assay (see Materials and methods, and Figure 7B and C legends).

Ψ42-specific pseudouridylase activity coincides with Nhp2p. Yeast U2 snRNA containing a single ³²P label at the 5′ side of U42 was used to screen a GST-ORF library (Phizicky et al, 2002) for pseudouridylase activity. After the pseudouridylation reaction, U2 snRNA was digested with nuclease P1 and analyzed by TLC. (A) Of all 64 pools of GST-ORF fusion proteins, pool #44 was positive. (B) The 96 strains in pool #44 were arranged into 12 columns and eight rows, with each column and row representing a ‘subpool'. The column #2 subpool and row A subpool were positive, implicating strain 2A as the source of the pseudouridylase activity. (C) Individual strains were further screened to confirm that strain 2A, expressing GST-Nhp2p, was indeed positive. The migration of labeled ³²PU and ³²PΨ is indicated. Control reactions (Con.) contained either yeast cell extract (+) or water (−).

TAP-purified Box H/ACA sno/scaRNPs catalyze Ψ42 formation. (A) Yeast U2 snRNA containing a single ³²P label at the 5′ side of U42 was used to assess the pseudouridylase activity of TAP preparations targeting Gar1p (lane 3), Cbf5p (lane 4), Nhp2p (lane 5), or Mak5p (as a control, lane 6). The final protein concentration of the TAP preparations in the pseudouridylation reaction (see Materials and methods, and previous figure legends) was ∼75 ng/ml. As controls, water (lane 1) or the wild-type yeast cell extract (lane 2) was used in place of the TAP preparations. (B) The protein compositions of TAP preparations were analyzed on an SDS–PAGE gel stained with Coomassie blue. Lanes 3, 4, and 5 contain samples of Gar1p-TAP, Cbf5p-TAP, and Nhp2p-TAP preparations, respectively. Lane 6 is a control containing the sample of Mak5p-TAP preparation. The positions of the Box H/ACA sno/scaRNP core proteins, Cbf5p, Gar1p, Nhp2p, and Nop10p (both TAP-tagged and native forms), are indicated on the left. The TAP-tagged proteins migrated more slowly than their native forms. The label 3C indicates protease 3C, which was used during TAP purification. Lane M is a standard protein marker lane.

snR81 snoRNA directs the formation of Ψ42 in yeast U2. (A) Total Box H/ACA RNA isolated from the Gar1p-TAP preparation was fractionated on an 8% polyacrylamide–8 M urea gel. The entire lane (lane 2) was sliced into 11 fractions (A–K). RNAs were eluted from each fraction for reconstitution analysis. Lane 1 is a size marker of MspI-digested pBR322 DNA. (B) Fractionated RNAs (A–K, lanes 5–15) were added to micrococcal nuclease-treated Gar1p-TAP preparation (lanes 3–15) to reconstitute U2 pseudouridylation at position 42. Lanes 3 and 4 are controls in which no RNA (lane 3) or unfractionated total Box H/ACA RNA (lane 4) was added to the reaction. Lane 1 is an unreacted singly radiolabeled U2 snRNA. In lane 2, U2 pseudouridylation was carried out using mock-treated Gar1p-TAP preparation. (C) Reconstitution of Ψ42 formation was carried out using micrococcal nuclease-treated Gar1p-TAP preparation (lanes 2–15) and individual RNAs (lanes 4–15) derived from a Box H/ACA RNA library (see Materials and methods). Lanes 2 and 3 are controls where the reaction was supplemented with no RNA (lane 3) or total RNA isolated from fraction E (see panel B) (lane 2). In lane 1, mock-treated Gar1p-TAP preparation was used. Sequencing data indicated that RNA 8 in lane 11 was snR81 snoRNA. (D) Primary sequence and secondary structure of snR81 snoRNA. As indicated, the 3′ pseudouridylation pocket guides 25S rRNA pseudouridylation at position 1051 (Schattner et al, 2004), whereas the 5′ pseudouridylation pocket directs U2 snRNA pseudouridylation at position 42 (this work). The conserved H and ACA boxes are shaded.

The cbf5D95A mutant abolishes the formation of Ψ42 in yeast U2 snRNA. (A) The yeast 25S rRNA pseudouridylation assay was conducted exactly as in Figure 7C except that total RNA was isolated from the cbf5D95A strain (see Figure 7B and C legends). (B) The yeast U2 snRNA pseudouridylation assay was conducted exactly as in Figure 7B except that total RNA was isolated from the cbf5D95A strain (see Figure 7B legend).