Cajal body-specific small nuclear RNAs: a novel class of 2'-O-methylation and pseudouridylation guide RNAs

Abstract

Cajal (coiled) bodies are conserved subnuclear organelles that are present in the nucleoplasm of both animal and plant cells. Although Cajal bodies were first described nearly 100 years ago, their function has remained largely speculative. Here, we describe a novel class of human small nuclear RNAs that localize specifically to Cajal bodies. The small Cajal body-specific RNAs (scaRNAs) are predicted or have already been demonstrated to function as guide RNAs in site-specific synthesis of 2'-O-ribose-methylated nucleotides and pseudouridines in the RNA polymerase II-transcribed U1, U2, U4 and U5 spliceosomal small nuclear RNAs (snRNAs). Our results provide strong support for the idea that the Cajal body, this mysterious nuclear organelle, provides the cellular locale for post-transcriptional modification of spliceosomal snRNAs.

Fig. 1. Computer-predicted two-dimensional structures of human box C/D–H/ACA snoRNAs. The evolutionarily conserved box C, D, H, ACA and the putative box C′ and D′ sequence motifs are boxed. The structure of the U85 snoRNA has been adopted from Jády and Kiss (2001). The genomic copies of the U85 and U89 snoRNAs are in the fourth and sixth introns of the structural maintenance of chromatin (D63880) and the B cell-associated protein (U75511) genes, respectively. The U87 and U88 snoRNAs are encoded within the ninth and twelfth introns of a putative protein-coding gene (BC000061).

Fig. 2. Characterization of the novel RNAs. (A) Immunoprecipitation of human snoRNPs. An extract prepared from HeLa cells was incubated with protein A–Sepharose saturated with anti-fibrillarin (α-Fib) or anti-GAR1 (α-Gar) antibodies. Upon collection of Sepharose beads by centrifugation, RNAs were recovered by proteinase K treatment and phenol extraction. Distribution of RNAs was determined by RNase A/T1 mapping using sequence-specific antisense RNA probes as indicated on the right. Control mappings performed with E.coli tRNA (C) or RNAs obtained from the HeLa cell extract (E) are also shown. Mock represents control reaction with protein A–Sepharose alone. Lane M, size markers (terminally labelled HaeIII- and TaqI-digested pBR322). (B) Intracellular localization. RNA isolated either from HeLa cells (T), or from nuclear (Nu), nucleoplasmic (Np), nucleolar (No) or cytoplasmic (Cy) fractions of HeLa cells were analysed by RNase A/T1 mapping using sequence-specific RNA probes. Lane C represents control mapping with E.coli tRNA.

Fig. 3. Potential base-pairing interactions between human box C/D–H/ACA RNAs and spliceosomal snRNAs. Nucleotides predicted to be selected for 2′-O-methylation (closed circle) or pseudouridylation (Ψ) in the human snRNAs are indicated. The 5′-terminal hairpins of the box H/ACA snoRNA-like domains of the U85 and U89 RNAs are schematically represented. Positions of the H, D and D′ boxes are indicated.

Fig. 4. Human U85 box C/D–H/ACA RNA localizes to Cajal bodies. (A) In situ localization of the endogenous U85 RNA. Human HeLa cells were probed with a fluorescent antisense RNA complementary to the human U85 RNA from position 1 to 76 and with an antibody directed against p80 coilin as indicated above the panels. The merged image shows that the U85 RNA co-localizes with p80 coilin. Scale bar, 10 µm. (B) In situ localization of transiently overexpressed U85 and mgU6-53 RNAs in HeLa cells. The schematic structure of the pCMV-globin expression construct is shown. The exons (E1, E2 and E3) and the polyadenylation site (PA) of the human β-globin gene are indicated. The transcription initiation site of the cytomegalovirus promoter (CMV) is indicated. The coding regions of the human U85 and mgU6-53 RNAs in the second intron of the globin gene are represented by an open arrow. Relevant restriction sites are shown (H, HindIII; C, ClaI; X, XhoI). The pCMV-globin-mgU6-53 plasmid was co-transfected with an expression construct producing a GFP-tagged version of the human fibrillarin protein. The overexpressed U85 and mgU6-53 RNAs were visualized by sequence-specific fluorescent RNA probes and Cajal bodies were specifically stained by anti-p80 coilin. Arrows indicate nucleolar stained structures enriched in mgU6-53 and fibrillarin. Arrowheads point to Cajal bodies, which clearly contain mgU6-53, fibrillarin and p80 coilin. The contour of the nucleus of a non-transfected HeLa cell is highlighted.

Fig. 5. In situ localization of human U87, U88 and U89 box C/D–H/ACA RNAs. Human U87, U88 and U89 RNAs were transiently over expressed in HeLa cells using the pCMV-globin expression construct. The intracellular distribution of the overproduced U87, U88 and U89 RNAs as well as the endogenous HeLa U88 RNA (U88endo) was investigated by fluorescent in situ hybridization. Cajal bodies were visualized by staining with anti-p80 coilin antibody. For other details, see the legend to Figure 4.

Fig. 6. Localization of box C/D and H/ACA RNAs predicted to function in modification of pol II-specific spliceosomal snRNAs. (A) Structure and function of human U90, U91 box C/D and U92 box H/ACA RNAs. The conserved box C/D and the potential C′ and D′ motifs of the U90 and U91 RNAs are indicated. Inverted arrows under the sequences of U90 and U91 indicate nucleotides predicted to form terminal and internal helices. The predicted two-dimensional structure of the U92 RNA was obtained by computer folding. The H and ACA sequence motifs are boxed. Predicted base-pairing interactions of the U90, U91 and U92 RNAs with the U1, U4 and U2 snRNAs, respectively, are indicated. The 2′-O-methylated A70 and C8 nucleotides in the selected sequences of the U1 and U4 snRNAs are indicated by dots. The pseudouridine residue (Ψ) at position 44 in the human U2 snRNA is marked. (B) Localization of human U90, U91 and U92 RNAs in HeLa cells. Overexpressed (U90 and U92) and endogenous (U91endo and U92endo) modification guide RNAs were visualized with sequence-specific fluorescent probes. For other details, see legends to Figures 4 and 5.