Identification of 13 novel human modification guide RNAs

Abstract

Members of the two expanding RNA subclasses termed C/D and H/ACA RNAs guide the 2'-O-methylations and pseudouridylations, respectively, of rRNA and spliceosomal RNAs (snRNAs). Here, we report on the identification of 13 novel human intron-encoded small RNAs (U94-U106) belonging to the two subclasses of modification guides. Seven of them are predicted to direct 2'-O-methylations in rRNA or snRNAs, while the remainder represent novel orphan RNA modification guides. From these, U100, which is exclusively detected in Cajal bodies (CBs), is predicted to direct modification of a U6 snRNA uridine, U(9), which to date has not been found to be pseudouridylated. Hence, within CBs, U100 might function in the folding pathway or other aspects of U6 snRNA metabolism rather than acting as a pseudouridylation guide. U106 C/D snoRNA might also possess an RNA chaperone activity only since its two conserved antisense elements match two rRNA sequences devoid of methylated nucleotides and located remarkably close to each other within the 18S rRNA secondary structure. Finally, we have identified a retrogene for U99 snoRNA located within an intron of the Siat5 gene, supporting the notion that retro-transposition events might have played a substantial role in the mobility and diversification of snoRNA genes during evolution.

Figure 1

Differences in the genomic organisation of the three novel snoRNAs between human and mouse. Each snoRNA sequence (small arrow) is located within an intron of the indicated genes. Exons are represented by boxes and splicing events by dotted lines. Note that AK009175, a spliced EST, and LOC85028, a poorly characterised gene, both located 15–21 kb downstream from Taf12 in mouse and human, are not related to each other. The cartoon is not drawn to scale.

Figure 2

Interaction between C/D snoRNA U106 and 18S rRNA. (A) Predicted base pairing between U106 and 18S rRNA. The sequences shown are for human (nucleotide differences in rodents are indicated in parentheses). Note that G1536 and U1602 do not appear to be 2′-O-methylated (30). (B) Location of the sites of complementarity to snoRNA U106 within the 18S rRNA secondary structure. The 3′ and 5′ antisense elements of U106 are denoted by 3AE and 5AE, respectively, and the rRNA nucleotides potentially involved in base pairing with the snoRNA are boxed. (C) Mapping of ribose methylated nucleotides in 18S rRNA. Primer extension at low concentrations of dNTP was performed with a 5′-³²P-labelled 18S rRNA-specific oligonucleotide, either at 0.04 mM (lane 1) or at 1 mM (control, lane 2). The significance of a pause at C1544 is unclear since 2′-O-methylation at this position has never been reported so far.

Figure 3

The U99 snoRNA gene is transcribed in the opposite orientation to its host intron. (A) Schematic representation of a part of the mouse (top) and human (bottom) genes hosting U99 snoRNA. Exons 3 and 4 are represented by white boxes, while splicing events are denoted by dotted lines. The U99 snoRNA gene is schematised by a white arrow indicating transcription orientation. Thin black arrows denote the transcription orientation of the indicated genes. Several spliced ESTs (i.e. BU588934, BF219096, BU588653 and BI461867 in human, and a single one, AK011444, in the mouse) overlapping and in the same orientation as the U99 snoRNA gene are also depicted (filled boxes denote exons). Not drawn to scale. (B) U99 does not immunoprecipitate with R1131 antibody. Total RNA from HeLa cells was subjected to immunoprecipitation with R1131 antibody (specific for the trimethyl cap structure), and RNAs recovered from either the pellet or supernatant were assayed for U99 by northern blot hybridisation with a U99-specific oligonucleotide probe. 1, input RNA (1:10); 2, pellet; 3, supernatant (1:10). The 5′-trimethyl-capped snoRNA U3 and the intron-encoded U98 and U102 snoRNAs were used as positive and negative controls, respectively.

Figure 4

U100, a novel RNA guide targeting U6 snRNA. (A) Predicted base pairing of U100 with U6 snRNA. The U6 nucleotide predicted to be targeted for modification by U100 is indicated by an arrow. Note that only the the 3′ hairpin domain from U100 is shown. The sequences shown are for human. (B) Mapping of pseudouridines at the 5′ end of the spliceosomal U6 snRNA. Total RNA extracted from HeLa cells, treated (+) or not treated (–) with CMC, was subjected to primer extension analysis with a U6-specific ³²P-labelled oligonucleotide. (C) U100 sequence alignment between H.sapiens and the fish F.rubripes. The fish U100 gene is located in an intron of the Huntington’s disease gene homologue (accession no. X82939). The conserved ACA motif and potential bipartite antisense element are denoted (boxed and overlined, respectively). Below the alignment, conserved nucleotides are denoted by asterisks.

Figure 5

U100 is a novel member of the scaRNA family. (A) Subfractionation of HeLa cells. RNA isolated either from total HeLa cells (Tot) or from cytoplasmic (Cyt), nuclei (Nu), nucleoplasmic (Np) or nucleolar (No) fractions was analysed in a 6% acrylamide/7 M urea gel and the various snoRNAs detected by northern blot analysis using specific oligonucleotide probes. Hybridisations with U3- and U6-specific probes have been used as controls of the cell fractionation procedure. (B) In situ hybridisation showing the localisation of transfected human U100. HeLa cells co-transfected with pCMV-hU100 and pGFP-coilin were hybridised with a specific U100 fluorescent oligonucleotide. The Cajal bodies are visualised by co-expressing GFP–coilin fluorescent protein. The merged picture shows that U100 co-localises with coilin. (C) Schematic representation of the terminal loops of the 5′ and 3′ hairpins of U100. The predicted Cajal body-specific localisation signals (27) are indicated in bold.

Figure 6

Identification of an intronic U99 retrogene. Sequence alignment of a U99 mouse snoRNA retrogene with its functional counterpart. Boundaries of the mature U99 sequence are denoted by vertical arrowheads. Direct repeats flanking the snoRNA retrogene are overlined (arrow), while the poly(A) stretch is underlined and the H and ACA motifs boxed. Bottom: intronic location of the U99 retrogene within the mouse Siat5 gene, with indication of a spliced EST (AK034863) connecting the U99 retrogene-containing intron to the rest of the Siat5 gene. Exons are represented by white boxes, splicing events by dotted lines, and the U99 retrogene by an open arrow (the flanking direct repeats are depicted by arrowheads). Nucleotides conserved between gene and retrogene are indicated by asterisks.