Mammalian and yeast U3 snoRNPs are matured in specific and related nuclear compartments

Abstract

Nucleolar localization of vertebrate box C/D snoRNA involves transit through Cajal bodies, but the significance of this event is unknown. To define better the function of this compartment, we analyzed here the maturation pathway of mammalian U3. We show that 3'-extended U3 precursors possess a mono-methylated cap, and are not associated with fibrillarin and hNop58. Importantly, these precursors are detected at both their transcription sites and in Cajal bodies. In addition, mature U3, the core box C/D proteins and the human homolog of the methyltransferase responsible for U3 cap tri-methylation, hTgs1, are all present in Cajal bodies. In yeast, U3 follows a similar maturation pathway, and equivalent 3'-extended precursors are enriched in the nucleolus and in the nucleolar body, a nucleolar domain that concentrates Tgs1p under certain growth conditions. Thus, spatial organization of U3 maturation appears to be conserved across evolution, and involves specialized and related nuclear compartments, the nucleolus/nucleolar body in yeast and Cajal bodies in higher eukaryotes. These are likely places for snoRNP assembly, 3' end maturation and cap modification.

Fig. 1. Tgs1p accumulates in the nucleolar body in yeast cells grown on solid medium. TGS1/tgs1::KAN diploid cells expressing a Tgs1–GFP fusion protein under its natural promoter were grown on solid medium for 24–48 h and imaged live (A) or following fixation (B and C). DNA of living and fixed cells was stained with Hoechst and DAPI, respectively. (A) Living cells expressing a DsRed2–Snu13 fusion protein. Tgs1–GFP co-localizes with DsRed2–Snu13 (red) in the majority of the cells (a), but not in ∼25% of cases (c). DsRed2–Snu13p always co-localizes with Gar1p (b). (B) Fixed cells stained for Nop1p (red). (C) Fixed cells overexpressing U14/MS2x2 and hybridized in situ with a probe specific for the artificial snoRNA (red). GFP signal (green) and DNA (blue). Each field is 16 µm².

Fig. 2. Yeast U3 precursors are present in the nucleolus and enriched in the nucleolar body. (A) Schematic of the maturation of yeast U3 with the position of the probes indicated in red (adapted from Kufel et al., 2000). (B) Localization of the indicated U3 species (red) by in situ hybridization in cells expressing U3 from a 2 µm plasmid, and grown in liquid medium. The U3-intron probe was used in a strain also lacking the exosomal component RRP6. The nucleolus was labeled with Gar1–GFP fusion protein (green) and the DNA was stained with DAPI (blue). (C) The same as in (B), but with cells expressing the U3del construct. (D) Localization of the indicated U3 species in cells expressing Tgs1–GFP and U3del, and grown on solid medium. Each field is 25 µm².

Fig. 3. Yeast and mammalian U3 precursors are not associated with all the core box C/D proteins. (A) RNA co-immunoprecipitation with yeast cells expressing a chromosomal ZZ-Nop58 protein alone or together with U3del. Upper panel: wild-type endogeneous U3. Lower panels: U3del. Left and right: two exposures of the same northern blot. The same amount of material was loaded in each lane. (B) RNA co- immunoprecipitation in mammalian cells. HeLa (a and b) or 293T (c) cells were transfected with the rU3B.7 gene alone or together with the indicated construct, and the RNA was co-immunoprecipitated with the relevant antibody and analyzed by RNase protection assays. I, input; S, supernatant; P, pellet (five times the input). Non-transfected cells (NT) showed no signal, while a U3 gene lacking box C′ (U3ΔC′) allowed identification of both U3 precursors, denoted U3-I and U3-II, and mature U3. For (b) and (c), P+ corresponds to the pellet lane after longer exposure of the gel.

Fig. 4. Mammalian 3′-extended U3 precursors are localized at their transcription sites and in Cajal bodies. (A) HeLa cells were transfected with the rU3B.7 gene and hybridized in situ with the indicated probe. (a) U3 3′-extended precursors (red) are present in Cajal bodies (green) and in bright foci often adjacent to them. Inset: enlargement of the boxed area. (b) U3 3′-extended precursors (red) are present at their transcription sites, which are visualized with a probe against the plasmid (green). (c) Mature U3B.7 RNA (red) is present in nucleoli and Cajal bodies (green). The image shown in (a) was deconvolved. (B) Localization of 15.5 kDa–GFP (a), hNop56–GFP (b) and hNop58–GFP (c) in nucleoli and Cajal bodies (red) of HeLa cells. DNA (blue) is stained with DAPI and GFP is green. Cajal bodies are visualized by indirect immunofluorescence with an antibody against coilin. Each field is 24 × 30 µm.

Fig. 5. The cap of mammalian U3 is hypermethylated in Cajal bodies. (A) 3′-extended U3 precursors have an m⁷G cap. RNAs purified from HeLa cells transfected with the rU3B.7 gene were immunoprecipitated with K121, specific for the m₃G cap, or H20, which recognize m⁷G and m₃G cap structures. Details are as in the legend for Figure 3B. (B) hTgs1 is localized in Cajal bodies. HeLa cells were stained by immunofluorescence with anti-hTgs1 (left) and anti-fibrillarin antibodies (middle). DNA is stained with DAPI (right). Cajal bodies are indicated by arrows. In this image, hTgs1 is not detected in the cytoplasm. Indeed, optimal detection in the cytoplasm required permeabilization conditions different from those used to detect it in the nucleus. Each field is 30 × 36 µm.

Fig. 6. Schematic of box C/D snoRNA biogenesis in yeast and mammalian cells. In yeast, U3 becomes associated with the complete set of the core box C/D proteins in the nucleolus and/or the nucleolar body, and this triggers 3′ end maturation and cap hypermethylation. In mammals, these events take place in Cajal bodies.