Human box H/ACA pseudouridylation guide RNA machinery

Abstract

Pseudouridine, the most abundant modified nucleoside in RNA, is synthesized by posttranscriptional isomerization of uridines. In eukaryotic RNAs, site-specific synthesis of pseudouridines is directed primarily by box H/ACA guide RNAs. In this study, we have identified 61 novel putative pseudouridylation guide RNAs by construction and characterization of a cDNA library of human box H/ACA RNAs. The majority of the new box H/ACA RNAs are predicted to direct pseudouridine synthesis in rRNAs and spliceosomal small nuclear RNAs. We can attribute RNA-directed modification to 79 of the 97 pseudouridylation sites present in the human 18S, 5.8S, and 28S rRNAs and to 11 of the 21 pseudouridines reported for the U1, U2, U4, U5, and U6 spliceosomal RNAs. We have also identified 12 novel box H/ACA RNAs which lack apparent target pseudouridines in rRNAs and small nuclear RNAs. These putative guide RNAs likely function in the pseudouridylation of some other types of cellular RNAs, suggesting that RNA-guided pseudouridylation is more general than assumed before. The genomic organization of the new box H/ACA RNA genes indicates that in human cells, all box H/ACA pseudouridylation guide RNAs are processed from introns of pre-mRNA transcripts which either encode a protein product or lack protein-coding capacity.

FIG. 1.

Schematic structure of box H/ACA RNAs and cDNA construction. (A) Selection of pseudouridylation sites by box H/ACA guide RNAs. For details, see the text. (B) Construction of a cDNA library of human box H/ACA RNAs. HeLa cell RNAs immunoprecipitated by an anti-GAR1 antibody were incubated with a phosphorylated oligoribonucleotide in the presence of T4 RNA ligase. RNA sequences tagged at both termini were converted into double-stranded DNA by a reverse transcription-PCR amplification approach. The amplified DNA was cloned into a plasmid vector, and individual clones were characterized by sequence analysis.

FIG. 2.

Potential base-pairing interactions between box H/ACA RNAs and human 18S rRNA. (A) Selection of known pseudouridylation sites. The upper strands represent box H/ACA RNA sequences in a 5′-to-3′ orientation. Solid lines represent the upper parts of the 5′ or 3′ hairpins of guide RNAs. The ACA motifs are in closed boxes. The first three nucleotides of the putative H motifs are in open-ended boxes. The lower strands represent 18S rRNA sequences in a 3′-to-5′ orientation. The positions of pseudouridine residues were reported previously (36). The pseudouridine residues defined by interactions with guide RNAs are indicated (Ψ). The sequence of human 18S rRNA is from GenBank accession number U13369. (B) H/ACA RNAs distinguishing between two or three potential pseudouridylation sites. A bar below rRNA sequences indicates that one of the underlined uridines is pseudouridine. (C) Prediction of new pseudouridylation sites. Question marks indicate novel pseudouridylation sites revealed by identification of the corresponding guide RNAs.

FIG. 3.

Verification of pseudouridine residues in human 18S and 28S rRNAs predicted by guide RNA-rRNA interactions. CMC-alkali-modified (Ψ) or control (N) HeLa cell RNAs were analyzed by primer extension with ³²P-labeled oligonucleotide primers complementary to the appropriate regions of the human 18S and 28S rRNAs. Lanes A, G, C, and U show dideoxy sequencing reactions performed on recombinant plasmids carrying the human 18S or 28S rRNA genes. Brackets and asterisks indicate uridines that were reported to be pseudouridylated.

FIG. 4.

Potential base-pairing interactions between box H/ACA RNAs and human 28S rRNA. The sequence of human 28S rRNA is from GenBank accession number U13369. The positions of pseudouridine residues were reported previously (45). See the legend to Fig. 2 for other details.

FIG. 5.

Putative pseudouridylation guide RNAs directing the modification of spliceosomal snRNAs. (A) Proposed base-pairing interactions between box H/ACA guide RNAs and human spliceosomal snRNAs. (B) Fluorescence in situ localization of guide RNAs transiently overexpressed in HeLa cells. The schematic structure of the pCMV-globin expression construct is shown. The promoter region of cytomegalovirus (CMV), the exons in the human β-globin gene (E1 to E3), the polyadenylation region in the bovine growth hormone gene (PA), and the SP6 promoter are shown. The relevant restriction sites are indicated (H, HindIII; C, ClaI; X, XhoI). Fluorescence in situ hybridization with oligonucleotide probes specific for the ACA26, ACA35, and ACA57 scaRNAs was combined with indirect immunofluorescence with an antibody against the Cajal body marker protein, p80-coilin. The nuclear DNA was stained with 4′,6′-diamidino-2-phenylindole (blue). Bar, 10 μm.