Box C/D small nucleolar RNA trafficking involves small nucleolar RNP proteins, nucleolar factors and a novel nuclear domain

Abstract

Nucleolar localization of box C/D small nucleolar (sno) RNAs requires the box C/D motif and, in vertebrates, involves transit through Cajal bodies (CB). We report that in yeast, overexpression of a box C/D reporter leads to a block in the localization pathway with snoRNA accumulation in a specific sub-nucleolar structure, the nucleolar body (NB). The human survival of motor neuron protein (SMN), a marker of gems/CB, specifically localizes to the NB when expressed in yeast, supporting similarities between these structures. Box C/D snoRNA accumulation in the NB was decreased by mutation of Srp40 and increased by mutation of Nsr1p, two related nucleolar proteins that are homologous to human Nopp140 and nucleolin, respectively. Box C/D snoRNAs also failed to accumulate in the NB, and became delocalized to the nucleoplasm, upon depletion of any of the core snoRNP proteins, Nop1p/fibrillarin, Snu13p, Nop56p and Nop5p/Nop58p. We conclude that snoRNP assembly occurs either in the nucleoplasm, or during transit of snoRNAs through the NB, followed by routing of the complete snoRNP to functional sites of ribosome synthesis.

Fig. 1. Overexpressed artificial box C/D snoRNA accumulates in the NB. (A) Schematic of the expression vectors used. The snoRNA is represented by a stem–loop in red. Pm, GPD promoter; BP, branch point of the actin intron; RPR1 leader and trailer, promoter and terminator of the RNase P gene. (B) Localization of the snoRNA reporters by light microscopy. Each field is 5 × 5 µm. The snoRNA (red) was detected by fluorescent in situ hybridization, the nucleolus was labelled with a Gar1–GFP fusion protein (green) and the DNA was stained with DAPI (blue). The artificial snoRNA was expressed from either the RNase P gene (RPR1) on a 2 µ plasmid (CD-2m/e), or the actin intron on a 2 µ plasmid (CD-2m/i), or on a centromeric vector (CD-Cen). A similar RNA lacking boxes C and D was expressed from the actin intron on a 2 µ plasmid, and used as a control (ΔCD). (C) Localization of the snoRNA reporter by electron microscopy. The nucleolus (NU) of different yeast strains (upper left panel: CD-Cen; upper right panel: CD-2m/i) was composed of a fibrillar and a granular component. Upon snoRNA overexpression (CD-2m/i), an additional structure of spherical shape was obvious in the nucleolus (the NB). This fibrillar body exhibited strong immunogold labelling after in situ hybridization using a BrdU-labelled MS2 oligonucleotide probe (lower panel). NP, nucleoplasm.

Fig. 2. The NB is a structure hosting several box C/D snoRNAs, and is related to CB. (A) Overexpression of an artificial snoRNA leads to accumulation of endogenous snoRNA in the NB. Yeast cells overexpressing the multicopy, artificial snoRNA were simultaneously labelled for the artificial snoRNA (red), and for endogeneous U14 (green, U14), U3 (green, U3) or Nop1–GFP (green, Nop1). (B) Human SMN localizes in the NB. Human SMN (green) was fused to GFP, and expressed in either wild-type cells (U3), or in cells overexpressing the artificial snoRNA (CD-2m/i). The NB was detected with a probe against the artificial snoRNA (red, CD-2m/i), and the nucleolus with a probe against U3 (red, U3).

Fig. 3. Antagonistic effects of Srp40p and Nsr1p on snoRNA trafficking. (A) Srp40p is required to accumulate box C/D snoRNA in the NB. The multicopy artificial snoRNA (red) was detected in either wild-type cells (Wild-type), or in isogenic cells deficient in SRP40p (srp40-Δ). SRP40p was fused to YFP (green) and detected in the NB (SRP40::YFP). (B) Nsr1p is required to distribute box C/D snoRNA in the entire nucleolus. The centromeric artificial snoRNA (red) was detected in either wild-type cells (Wild-type), in isogenic cells expressing the N-terminal domain of NSR1p (Nsr1-Nter), or following inactivation of RNA polymerase I (Pol I). The nucleolus was visualized with a Gar1–GFP fusion protein (Gar1, green), and the localization of the N-terminal domain of Nsr1p was visualized through a fusion with YFP (Nsr1-Nter::YFP, green). (C) SnoRNA expression levels in Srp40p and Nsr1p-deficient strains. Northern blots were probed with sequences specific for the artificial snoRNA (upper panels), or for SCR1 RNA for normalization (lower panels). The band corresponding to the mature snoRNA is indicated (91 bases, U14/MS2x2) and an arrow points to the precursor (∼250 bases). The molecular weight markers are shown on the left. Nsr1 (left panel) and Srp40 (right panel) strains were expressing U14/MS2x2 from a centromeric or 2 µ plasmid, respectively. Lane 1, wild-type cells; lane 2, Nsr1-Nter; lane 3, Nsr1-Δ; lane 4, Srp40-Δ; lane 5, wild type.

Fig. 4. Nop1p, Nop56p, Nop5/58p and Snu13p are all required for efficient localization of box C/D snoRNAs in the nucleolus. (A) Mislocalization of endogenous U14 in Nop1p- and Nop56p-depleted strains. U14 snoRNA (red) was detected in wild-type cells (U14/wt), or following depletion of Nop56p (U14/Gal::Nop56) or Nop1p (U14/Gal::Nop1). In the right image of each panel, the DNA has been stained with DAPI. The nucleolus is visible as the nuclear region that stains poorly with DAPI. (B) Mislocalization of the artificial snoRNA reporter in strains depleted for Nop1p, Nop56p or Nop58p. The multicopy, intronic, artificial snoRNA (red) was detected in wild-type cells (CD-2m), or following depletion of Nop5p/Nop58p (CD-2m/Gal::Nop58), Nop1p (CD-2m/Gal::Nop1) or Nop56p (CD-2m/Gal::Nop56). For comparison, a similar intronic RNA lacking boxes C and D was stabilized by inactivating the gene for debranching enzyme (ΔCD-2m/dbr). (C) Mislocalization of the artificial snoRNA in strains depleted for Snu13p. The artificial snoRNA (red, CD-2m/e) was expressed from the RPR1 gene on a 2 µ plasmid, and detected in wild-type strains (Wild-type), or following depletion of Snu13p (Gal::Snu13). A similar RNA lacking boxes C and D was used as a control (red, ΔCD-2m/e; Wild-type). The nucleolus was visualized with a Gar1–GFP fusion protein (Gar1, green).

Fig. 5. Nucleolar localization of Nop1p and Nop5/Nop58p requires other box C/D core proteins. (A) Nop1p is mislocalized in strains depleted for Nop5p/Nop58p or Nop56p. Nop1p (green) was detected by immunofluorescence in wild-type cells (Wild-type), or following depletion of Nop5p/Nop58p (Gal::Nop58) and Nop56p (Gal::Nop56). (B) Nop5p/Nop58p is mislocalized in strains depleted for Nop1p or Nop56p. Nop5/Nop58p (green) was detected by immunofluorescence in wild-type cells (Wild-type), or following depletion of either Nop1p (Gal::Nop1) or Nop56p (Gal::Nop56).

Fig. 6. A model for the snoRNA trafficking pathway towards the nucleolus (see text).