Characterisation of the U83 and U84 small nucleolar RNAs: two novel 2'-O-ribose methylation guide RNAs that lack complementarities to ribosomal RNAs

Abstract

In eukaryotic cells, the site-specific 2'- O -ribose methylation of ribosomal RNAs (rRNAs) and the U6 spliceosomal small nuclear RNA (snRNA) is directed by small nucleolar RNAs (snoRNAs). The C and D box-containing 2'- O -methylation guide snoRNAs select the correct substrate nucleotide through formation of a long 10-21 bp interaction with the target rRNA and U6 snRNA sequences. Here, we report on the characterisation of two novel mammalian C/D box snoRNAs, called U83 and U84, that contain all the elements that are essential for accumulation and function of 2'- O -methylation guide snoRNAs. However, in contrast to all of the known 2'- O -methylation guide RNAs, the human, mouse and pig U83 and U84 snoRNAs feature no antisense elements complementary to rRNA or U6 snRNA sequences. The human U83 and U84 snoRNAs are not associated with maturing nucleolar pre-ribosomal particles, suggesting that they do not function in rRNA biogenesis. Since artificial substrate RNAs complementary to the evolutionarily conserved putative substrate recognition motifs of the U83 and U84 snoRNAs were correctly 2'- O -methylated in the nucleolus of mouse cells, we suggest that the new snoRNAs act as 2'- O -methylation guides for cellular RNAs other then rRNAs and the U6 snRNA.

Figure 1

Selection of 2′-O-methylated nucleotides by box C/D snoRNAs. The consensus sequences of the conserved C, C′, D and D′ boxes are indicated. The upstream (UAE) and/or downstream (DAE) antisense element of the snoRNA forms a double helix with complementary rRNA sequences. A nucleotide in the rRNA that base pairs with the fifth nucleotide 5′ to the D or D′ box of the snoRNA is 2′-O-methylated. The position of an internal hairpin that frequently folds together the D′ and C′ boxes is indicated (13).

Figure 2

Characterisation of human U83 and U84 snoRNAs. (a) Primer extension analyses of the 5′-termini of U83 and U84 RNAs. Terminally labelled oligonucleotides specific for U83 and U84 were annealed to human nuclear RNAs and extended by AMV reverse transcriptase (lanes R). Lanes C, T, A and G represent dideoxy sequencing reactions using the same oligonucleotides as primers and recombinant plasmids carrying the full-length cDNAs of U83 and U84 as templates. The extended products were separated on an 8% sequencing gel. The 5′-terminal sequences of the U83 and U84 RNAs are shown. (b) Determination of the 3′-terminal sequences of human U83 and U84 snoRNAs by the T4 RNA ligase/PCR procedure. The 3′-terminal sequences of the two snoRNAs and the 5′-terminal sequence of the oligoribonucleotide ligated to the U83 and U84 RNAs are shown.

Figure 3

Alignments of human, pig and mouse U83 and U84 snoRNAs. The C and D boxes and the potential C′ and D′ box motifs are indicated. The vertical lines highlight nucleotides conserved in the aligned snoRNAs and dashes stand for gaps. Inverted arrows indicate sequences capable of forming base pairing interactions. The presence of an additional A and C residue in the pig U83 RNA at positions 4 and 61 (circled), as compared to the published sequence (GenBank accession no. Z34846), has been verified by sequence analysis of the appropriate region of the pig BAT1 locus (data not shown). Sequences of the human U83 and U84 snoRNAs have been deposited in the EMBL database (accession nos AJ243200 and AJ243199, respectively).

Figure 4

Human U83 and U84 RNAs are not associated with higher order nucleolar structures. (a) Intracellular localisation of human U83 and U84 snoRNAs. RNAs extracted either from human HeLa cells (T) or from nuclear (N), nucleoplasmic (Np), nucleolar (No) and cytoplasmic (Cy) fractions of HeLa cells were mapped by RNase A/T1 protection using sequence-specific antisense RNA probes as indicated. Lane C, control mapping with Escherichia coli tRNA. Lane M, size markers (a mixture of HaeIII- and TaqI-digested pBR322). (b) Sedimentation analyses of U83, U84 and U14 snoRNP particles. HeLa cell extract was fractionated on a 10–30% glycerol gradient. RNAs were isolated from each fraction and subjected to RNase A/T1 mapping with antisense RNA probes specific for the U83, U84 or U14 snoRNA. Positions of HeLa ribosome markers are indicated. Lanes C and M represent control mappings and size markers, respectively.

Figure 5

Ribose methylation of artificial substrate RNAs in mouse cells. (a) Schematic structure of the pW-U83t and pW-U84t expression constructs used for transfection of mouse cells. The RNA polymerase I promoter and terminator, the terminal regions of the 5′ (hatched box) and 3′ (open box) external transcribed sequences (ETS) and a fragment of the chloramphenicol acetyltransferase (CAT) gene are indicated. To generate pW-U83t and pW-U84t, appropriate synthetic DNA fragments were inserted into the XbaI (Xb) and XhoI (Xh) sites of pW(Xb/Xh). Nucleotides facilitating the cloning are in lower case letters. The potential base pairing interactions formed between the expressed artificial substrate RNAs and the putative antisense elements of the U83 and U84 snoRNA are shown. Nucleotides predicted to be 2′-O-methylated are indicated by closed circles. (b) Primer extension mapping of 2′-O-methylated nucleotides. A 5′-end-labelled deoxyoligonucleotide was annealed with RNAs extracted from mouse cells non-transfected (Nt) or transfected (Tr) with the indicated expression constructs and extended with AMV reverse transcriptase in the presence of 1 or 0.004 mM dNTPs (as indicated above the lanes). Lanes C, T, A and G are dideoxy sequencing reactions performed on the pW-U83t or pW-U84t expression constructs.

Figure 6

Primer extension analysis. RNAs obtained from human HeLa cells (T) or from a nuclear fraction of HeLa cells (N) were annealed with 5′-end-labelled oligo 9 and extended with AMV reverse transcriptase. The extended DNA products were size-fractionated on a 6% sequencing gel. Lane M, size marker.