A small nucleolar RNA requirement for site-specific ribose methylation of rRNA in Xenopus

Abstract

Vertebrate cells contain a large number of small nucleolar RNA (snoRNA) species, the vast majority of which bind fibrillarin. Most of the fibrillarin-associated snoRNAs can form 10- to 21-nt duplexes with rRNA and are thought to guide 2'-O-methylation of selected nucleotides in rRNA. These include mammalian UHG (U22 host gene)-encoded U25-U31 snoRNAs. We have characterized two novel human snoRNA species, U62 and U63, which similarly exhibit 15- (with one interruption) and 12-nt complementarities and are therefore predicted to direct 2'-O-methylation of A590 in 18S and A4531 in 28S rRNA, respectively. To establish the function of antisense snoRNAs in vertebrates, we exploited the Xenopus oocyte system. Cloning of the Xenopus U25-U31 snoRNA genes indicated that they are encoded within multiple homologs of mammalian UHG. Depletion of U25 from the Xenopus oocyte abolished 2'-O-methylation of G1448 in 18S rRNA; methylation could be restored by injecting either the Xenopus or human U25 transcript into U25-depleted oocytes. Comparison of Xenopus and human U25 sequences revealed that only boxes C, D, and D', as well as the 18S rRNA complement, were invariant, suggesting that they may be the only elements required for U25 snoRNA stability and function.

Figure 1

Fibrillarin-associated human snoRNAs. (A) Resolution of RNAs isolated from an antifibrillarin precipitate of HeLa cell extract. The 3′ end-labeled RNAs were first fractionated on a standard 15% denaturing polyacrylamide gel. Twenty-three bands were excised, eluted, and further resolved on a 15% gel with a ratio of acrylamide/bis-acrylamide of 9:1. U62 (Upper) and U63 (Lower) snoRNAs are indicated by arrows. Previously identified snoRNAs are indicated by dots. From largest to smallest they are: U16 (27), U14 (28), U45a (11), U45a variant (11), U20 (29), U28 (20), U26 (20), and U45b (11). (B) Sequences of U62 and U63 snoRNAs and their predicted base-pairing interactions with rRNAs. Conserved boxes C, D, and D′ are shown in boldface type. Filled circles indicate 2′-O-methylated residues in rRNA.

Figure 2

(A) Arrangement of the Xenopus U25–U31 and U22 snoRNA genes (schematic, not drawn to scale). The pairs of arrows indicate primers used for either standard or inverted PCR amplification. Organization of the human and mouse U25–U31 and U22 snoRNA genes (20) are shown for comparison. Boxes filled with identical patterns indicate genes for the same snoRNA. The X over the box in Xenopus fragment C indicates that the U26-like sequence is only 76% identical to human U26 snoRNA and contains several nucleotide changes and deletions within box C and the 18S rRNA complementarity, respectively. (B) Xenopus U25–U31 snoRNA sequences and their predicted base pairing interactions with rRNAs. The sequences of all Xenopus U25–U28, U30, and U31 snoRNA variants, as well as the U29D variant, were deduced from genomic sequences by comparison with their mammalian counterparts, whereas the sequences of the other U29 variants were determined by analyzing their cDNAs. Human U25 is shown for comparison. Identical and missing nucleotides are represented by dashes and asterisks, respectively. Conserved boxes C, D, and D′ are shown in bold. A bar and arrows indicate complementary oligonucleotides used in oocyte injections and for primer extension experiments, respectively. Filled circles or arrowheads indicate rRNA residues that are reported or predicted to be 2′-O-methylated, respectively.

Figure 3

Depletion of U25 snoRNA from Xenopus oocytes inhibits 2′-O-methylation of G1448 in 18S rRNA. Oocytes were cytoplasmatically injected with either U25-5′ or a nonspecific oligonucleotide. Sixteen hours later, a Xenopus or human U25 transcript or a Xenopus antisense U25 transcript was injected into germinal vesicles as indicated at the top. After 24 h, the oocytes were dissected, and RNA was isolated from both the nuclear and cytoplasmic compartments. (A) Primer extension analysis of U25 and U28 snoRNAs in the nuclei of uninjected oocytes (lane 1) and oocytes injected either with the nonspecific (lane 2) or U25-5′ (lanes 3–6) oligonucleotide. Oocytes in lanes 4, 5, and 6 were also injected with Xenopus U25, human U25, and Xenopus antisense U25 transcripts, respectively. A mixture of XU25-3′ and XU28-3′ primers was used in lanes 1–4. A combination of XαU25 and XU28-3′ or HU25-3′ and XU28-3′ primers was used in lane 5 or 6, respectively. (B) Mapping ribose methylation at G1448 in 18S rRNA. Nuclear (lanes 1–6) or cytoplasmic (lanes 11–16) RNA was subjected to partial alkaline hydrolysis, and 18S sequences were analyzed by primer extension using 18S-245 primer. Lanes 1 and 11, uninjected oocytes; lanes 2 and 12, oocytes injected with nonspecific oligonucleotide 26; lanes 3–6 and 13–16, oocytes injected with U25-5′ oligonucleotide. Oocytes in lanes 4 and 14, 5 and 15, or 6 and 16 were also injected with Xenopus U25, human U25, or Xenopus antisense U25 transcripts, respectively. Lanes 7–10 show dideoxy sequencing of 18S rRNA performed on the cytoplasmic RNA. An arrow indicates a gap in the ladders of primer extension products caused by 2′-O-methylation of G1448. The primer extension products were resolved on 8% denaturing polyacrylamide gels.