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. 2008 May;28(10):3089-100.
doi: 10.1128/MCB.01574-07. Epub 2008 Mar 10.

Different Mechanisms for Pseudouridine Formation in Yeast 5S and 5.8S rRNAs

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Free PMC article

Different Mechanisms for Pseudouridine Formation in Yeast 5S and 5.8S rRNAs

Wayne A Decatur et al. Mol Cell Biol. .
Free PMC article

Abstract

The selection of sites for pseudouridylation in eukaryotic cytoplasmic rRNA occurs by the base pairing of the rRNA with specific guide sequences within the RNA components of box H/ACA small nucleolar ribonucleoproteins (snoRNPs). Forty-four of the 46 pseudouridines (Psis) in the cytoplasmic rRNA of Saccharomyces cerevisiae have been assigned to guide snoRNAs. Here, we examine the mechanism of Psi formation in 5S and 5.8S rRNA in which the unassigned Psis occur. We show that while the formation of the Psi in 5.8S rRNA is associated with snoRNP activity, the pseudouridylation of 5S rRNA is not. The position of the Psi in 5.8S rRNA is guided by snoRNA snR43 by using conserved sequence elements that also function to guide pseudouridylation elsewhere in the large-subunit rRNA; an internal stem-loop that is not part of typical yeast snoRNAs also is conserved in snR43. The multisubstrate synthase Pus7 catalyzes the formation of the Psi in 5S rRNA at a site that conforms to the 7-nucleotide consensus sequence present in other substrates of Pus7. The different mechanisms involved in 5S and 5.8S rRNA pseudouridylation, as well as the multiple specificities of the individual trans factors concerned, suggest possible roles in linking ribosome production to other processes, such as splicing and tRNA synthesis.

Figures

FIG. 1.
FIG. 1.
Pseudouridylated nucleotides of the small rRNAs of the yeast cytoplasmic large ribosomal subunit. (A) S. cerevisiae 5.8S rRNA has a Ψ at position 73. The secondary structure includes portions of 25S rRNA (light gray) that interact with 5.8S rRNA (15). (B) Alignment of the yeast 5.8S rRNA sequence with those of human (Homo sapiens) (50, 78) and wheat (Triticum aestivum) (64) shows that the Ψ in yeast occurs in a commonly modified stretch of 5.8S rRNA (59, 72, 83). The number of nucleotides upstream of each 5.8S portion is shown in parentheses; the numbering for yeast is for the short (major) version (28, 39). S. cer., S. cerevisiae; T. aes., T. aestivum; H. sap., H. sapiens. (C) The S. cerevisiae cytoplasmic 5S rRNA secondary structure (15, 53) showing the position of the Ψ.
FIG. 2.
FIG. 2.
Formation of the Ψ in 5.8S rRNA, but not the Ψ in 5S RNA, is lost in the strain severely compromised for the activity of the snoRNP-associated Ψ synthase Cbf5. Primer extension reactions were performed using appropriate primers (complementary to 5S rRNA in lanes 1 to 4 and to 5.8S rRNA in lanes 5 to 8) with CMC-treated (+) and mock-treated RNA (−) from the haploid control laboratory strain YS602 (WT) and a strain in which the Cbf5 protein harbors a point mutation in the active site (cbf5-D95A). The site of the CMC-dependent pause in the ladder of extension products is indicated for the respective Ψs.
FIG. 3.
FIG. 3.
snR43 is responsible for the formation of both the Ψ in 5.8S rRNA and Ψ966 in 25S rRNA. (A) The predicted secondary structure of snR43 is shown paired with a segment of pre-rRNA corresponding to 5.8S rRNA to guide the appropriate U into the pseudouridylation pocket. (B) The same domain functions in the pseudouridylation of 25S rRNA. The putative base pairing between the 5′ domain of snR43 and the 966 region of 25S rRNA is illustrated in a condensed manner. Primer extension reactions detecting Ψs in 5.8S rRNA (C) and the 960 to 990 region of 25S rRNA (D) show that the formation of Ψ in 5.8S rRNA and Ψ966 in 25S rRNA is abolished in an snR43 deletion strain (snr43Δ). (D) Primer extension sequencing ladder reactions (lanes 1 to 4) were performed with the same primer as that for the adjacent lanes using total RNA. Each panel is labeled in the same manner as that described in the legend to Fig. 2, with the RNA in this case being isolated from the haploid control strain (WT) and the snR43 deletion strain; the numbering scheme for rRNA positions matches that for the yeast genome (43, 46).
FIG.4.
FIG.4.
snR43 is predicted to maintain both guide functions in the hemiascomycetes. (A) The DNA encoding the S. cerevisiae snR43 RNA is shown aligned with the sequences encoding putative snR43 in other hemiascomycetes. Conserved nucleotides are emphasized in boldface; asterisks along the bottom indicate positions identical in all sequences. D. hansenii, Debaryomyces hansenii. In the bottom right, S. cerevisiae snR43 is shown with the secondary structure labeled according to the annotation along the top of the alignment; the guides are illustrated paired with the 25S rRNA target. (B) Potential pairings between the putative guides of yeast snR43 orthologs and the respective rRNA regions are shown, with differences from the nucleotides present in S. cerevisiae highlighted (shaded boxes). As drawn, the last base pair in the 5.8S diagrams would compete with pairing in the P1:P1′ helix; several other yeast snoRNAs (e.g., snR5, snR9, snR35, snR36, and snR46) display this feature.
FIG.4.
FIG.4.
snR43 is predicted to maintain both guide functions in the hemiascomycetes. (A) The DNA encoding the S. cerevisiae snR43 RNA is shown aligned with the sequences encoding putative snR43 in other hemiascomycetes. Conserved nucleotides are emphasized in boldface; asterisks along the bottom indicate positions identical in all sequences. D. hansenii, Debaryomyces hansenii. In the bottom right, S. cerevisiae snR43 is shown with the secondary structure labeled according to the annotation along the top of the alignment; the guides are illustrated paired with the 25S rRNA target. (B) Potential pairings between the putative guides of yeast snR43 orthologs and the respective rRNA regions are shown, with differences from the nucleotides present in S. cerevisiae highlighted (shaded boxes). As drawn, the last base pair in the 5.8S diagrams would compete with pairing in the P1:P1′ helix; several other yeast snoRNAs (e.g., snR5, snR9, snR35, snR36, and snR46) display this feature.
FIG. 5.
FIG. 5.
Formation of the 5S rRNA Ψ is dependent on Pus7. (A) RNA from several strains was screened for the presence of the 5S rRNA Ψ as described in the legend to Fig. 2 and with the panel labeled in a similar manner. The primer extension sequencing ladder reactions (lanes 1 to 4) were performed with the same primer as that used for the adjacent lanes using total RNA. The following deletion strains were examined: pus1Δ (Open Biosystems yeast clone no. 11080), pus4Δ (clone no. 11152), and pus7Δ (clone no. 12499) strains. (B) Restoring the expression of Pus7 to the deletion strain rescues the formation of the 5S rRNA Ψ. Primer extension reactions to detect the 5S rRNA Ψ were performed with RNA from a pus7Δ strain harboring either an empty yeast shuttle plasmid pRS415 (+ empty vector) or the plasmid bearing the PUS7 gene [+ pPUS7(RS415)]. Two independent isolates are shown for each set.
FIG. 6.
FIG. 6.
Alignment of the seven nucleotides encompassing the sites of characterized S. cerevisiae Pus7 activity (9, 104). The consensus was established previously (9). R, purine; S, G/C.

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