A common sequence motif determines the Cajal body-specific localization of box H/ACA scaRNAs

Abstract

Post-transcriptional synthesis of 2'-O-methylated nucleotides and pseudouridines in Sm spliceosomal small nuclear RNAs takes place in the nucleoplasmic Cajal bodies and it is directed by guide RNAs (scaRNAs) that are structurally and functionally indistinguishable from small nucleolar RNAs (snoRNAs) directing rRNA modification in the nucleolus. The scaRNAs are synthesized in the nucleoplasm and specifically targeted to Cajal bodies. Here, mutational analysis of the human U85 box C/D-H/ACA scaRNA, followed by in situ localization, demonstrates that box H/ACA scaRNAs share a common Cajal body-specific localization signal, the CAB box. Two copies of the evolutionarily conserved CAB consensus (UGAG) are located in the terminal loops of the 5' and 3' hairpins of the box H/ACA domains of mammalian, Drosophila and plant scaRNAs. Upon alteration of the CAB boxes, mutant scaRNAs accumulate in the nucleolus. In turn, authentic snoRNAs can be targeted into Cajal bodies by addition of exogenous CAB box motifs. Our results indicate that scaRNAs represent an ancient group of small nuclear RNAs which are localized to Cajal bodies by an evolutionarily conserved mechanism.

Fig. 1. U85 scaRNA is a specific molecular marker for CBs in both human and Drosophila cells. (A) Human U85 scaRNA co-purifies with CBs. Nuclei (Nu) of human HeLa cells were subfractionated into nucleoplasmic (Np), nucleolar (No) and CB fractions (Lam et al., 2002). RNA extracted from each fraction corresponding to 5 × 10⁶ cells was mapped by RNase A/T1 protection by using an excess of sequence-specific antisense RNA probes as indicated on the right. Protected RNAs were separated on a 6% sequencing gel. Lane C, control mapping with E.coli tRNA. Lane M, size markers in nucleotides (HaeIII- and TaqI-digested pBR322). (B) In situ localization of Drosophila U85 scaRNA. Drosophila Schneider II cells were hybridized with fluorescent oligonucleotide probes specific for the U85 scaRNA (dU85), U2 snRNA (dU2) and U3 snoRNA (dU3). CB-like structures are indicated by open arrowheads. Nuclei were visualized by DAPI staining. Scale bar, 5 µm.

Fig. 2. The box H/ACA domain is essential for CB-specific localization of human U85 C/D-H/ACA scaRNA. (A) Computer-predicted two- dimensional structures of the human U85 scaRNA (black) and U64 box H/ACA snoRNA (blue) and schematic structures of the box C/D domain of U85 (U85CD) and the U85CD–U64 chimeric RNAs. Positions of the conserved C, D, H and ACA box elements and the relevant restriction sites are indicated (B, BglII; K, KpnI). (B) Schematic structure of the pCMV/globin expression cassette. The test RNA genes (open arrow) were inserted into the second intron of the human β-globin gene that had been placed under the control of the cytomegalovirus promoter (CMV). The exons (E1, E2 and E3) and the polyadenylation site (PA) of the globin gene as well as the SP6 promoter used to transcribe antisense RNA probes are indicated. Relevant restriction sites are shown (H, HindIII; C, ClaI; X, XhoI). (C) In situ localization of transiently expressed human U85 scaRNA, the box C/D domain of U85 (U85CD) and the U85CD–U64 composite RNA in HeLa cells. The RNAs were detected by sequence-specific fluorescent oligonucleotide probes (see Materials and methods). CBs were detected by an anti-coilin antibody and nucleoli were visualized by co-expression of GFP-tagged fibrillarin (fibrillarin). Nuclei were visualized by DAPI staining. A CB accumulating U85CD is indicated by an open arrow. Scale bar, 10 µm.

Fig. 3. Intranuclear localization of transiently expressed composite U85–U64 RNAs in HeLa cells. Structures of the artificial box H/ACA domains composed of U85- (black) and U64-specific (blue) sequences are shown. The sequence and structure of the common box C/D domain (U85CD) of the expressed chimeric RNAs is shown in Figure 2A. Localization of RNAs was determined by in situ hybridization with sequence-specific fluorescent oligonucleotide probes complementary to the 5′ (U85CD–U64/U85u, U85CD–U64/U85t, U85CD–U64aca/U85t) and 3′ (U85CD–U85/U64) junction sequences of U85 and U64. For other details, see the legend to Figure 2C.

Fig. 4. The terminal stem–loop structures of the H/ACA domain of U85 carry elements essential for localization to CBs. (A) Targeting of human U64 snoRNA into CBs by addition of the terminal stem–loop of the 5′ hairpin of U85. Transiently expressed chimeric U64/U85t and wild-type U64 RNAs were localized by in situ hybridization. (B) Alteration of the terminal stem–loop sequences in the 5′ and/or 3′ hairpin of the box H/ACA domain of human U85. The intranuclear distribution of mutant U85 RNAs overexpressed in HeLa cells was determined by in situ hybridization. For other details, see the legend to Figure 2C.

Fig. 5. Identification of a putative CB localization signal for box H/ACA scaRNAs. (A) Compilation of the terminal stem–loop regions of the 5′ and 3′ hairpins of box H/ACA scaRNAs. Sequences that show significant conservation are boxed. (B) Nucleotide conservation in the proposed cis-acting CB localization elements of box H/ACA scaRNAs.

Fig. 6. Mutational analysis of the minimal stem–loop region of U85 directing localization of the U85CD–U64/U85t chimeric RNA to CBs. Altered nucleotides in the terminal stem–loop region of the U85CD–U64/U85t chimeric RNA are indicated. Subnuclear localization of transiently expressed mutant RNAs was determined by in situ hybridization with a fluorescent oligonucleotide probe complementary to the 5′ junction of U85 and U64 sequences. For other details, see the legend to Figure 2C.