Identification of genes that function in the biogenesis and localization of small nucleolar RNAs in Saccharomyces cerevisiae

Abstract

Small nucleolar RNAs (snoRNAs) orchestrate the modification and cleavage of pre-rRNA and are essential for ribosome biogenesis. Recent data suggest that after nucleoplasmic synthesis, snoRNAs transiently localize to the Cajal body (in plant and animal cells) or the homologous nucleolar body (in budding yeast) for maturation and assembly into snoRNPs prior to accumulation in their primary functional site, the nucleolus. However, little is known about the trans-acting factors important for the intranuclear trafficking and nucleolar localization of snoRNAs. Here, we describe a large-scale genetic screen to identify proteins important for snoRNA transport in Saccharomyces cerevisiae. We performed fluorescence in situ hybridization analysis to visualize U3 snoRNA localization in a collection of temperature-sensitive yeast mutants. We have identified Nop4, Prp21, Tao3, Sec14, and Htl1 as proteins important for the proper localization of U3 snoRNA. Mutations in genes encoding these proteins lead to specific defects in the targeting or retention of the snoRNA to either the nucleolar body or the nucleolus. Additional characterization of the mutants revealed impairment in specific steps of U3 snoRNA processing, demonstrating that snoRNA maturation and trafficking are linked processes.

FIG. 1.

Aberrant patterns of U3 snoRNA localization detected in temperature-sensitive mutants at the restrictive temperature. Yeast cells were fixed and hybridized with Cy3-labeled anti-U3 probe (green). The nucleolar protein Nop1 was detected by immunofluorescence with an anti-Nop1 antibody (blue). DAPI was used to stain DNA and indicate the location of the nucleoplasm (red). Corresponding DIC (differential interference contrast) images are shown. (A) U3 snoRNA is normally detected in the nucleoli of S. cerevisiae by FISH. (B) FISH analysis was performed on yeast temperature-sensitive mutants following a 4-h shift from 25°C to 37°C (37°C panels). Representative phenotypes are shown. The 25°C panels show the nucleolar localization of U3 in the yeast temperature-sensitive mutants at the permissive temperature (25°C). The illustrations on the left show the relationship of the patterns to the nucleolus (crescent) and nucleolar body (small circle) based on the subsequent analysis presented in this work. wt, wild type; NP, nucleoplasm.

FIG. 2.

mRNA export is not affected in temperature-sensitive mutants. FISH analysis was performed on temperature-sensitive mutants maintained at the permissive temperature (25°C) or following a 4-h shift to the nonpermissive temperature (37°C). Poly(A) RNA was detected with a Cy3-labeled oligo(dT) probe (green). Poly(A) RNA accumulates within the nuclei of Ran cycle mutant cells (gsp1) but not in wild-type (wt) control cells or any of the mutants identified in the U3 screen. DAPI was used to stain DNA and indicate the location of the nucleoplasm (red).

FIG. 3.

Levels and processing of U3 snoRNA in temperature-sensitive mutants. Northern analysis was performed on wild-type (wt) and mutant cells maintained at the permissive temperature (25°C) (0-h lanes) or following a 4-h shift to the nonpermissive temperature (37°C) (4-h lanes). The prp21 mutant showed a partial growth defect at the semipermissive temperature of 30°C and was further analyzed at this temperature (*, lane 6). Precursor forms of U3 containing an intron (U3 intron) or a 3′-extended species (U3-3′ ext.), as well as mature U3 snoRNA (U3), were detected with oligonucleotides A and B, which are specific to different regions of U3, as shown in the schematic (see Materials and Methods for details). 5S rRNA was probed as an internal RNA loading control.

FIG. 4.

rRNA levels and processing in temperature-sensitive mutants. (A) Northern analysis was performed with total RNA isolated from the temperature-sensitive mutants and the wild-type (wt) strain at the permissive temperature (25°C) or after a 4-h shift to the nonpermissive temperature (37°C) (0-h and 4-h lanes, respectively). Lane 6 shows analysis of the prp21 mutant at the semipermissive temperature of 30°C (*). The relative positions of the oligonucleotide probes (a to f) used for each panel are diagrammed in panel B, and the sequences are given in Materials and Methods. We hybridized panels I and VI with probe e, panel II with probe c, panel III with probe b, panel IV with probe f, panel V with probe a, panel VII with probe d, and panel VIII with a probe against the 5S rRNA. (B) Primary Pol I transcript (35S) and simplified pre-rRNA processing pathway. The sequences of the mature 18S, 5.8S, and 25S rRNAs are embedded within external (5′ and 3′-ETS) and internal (ITS1 and ITS2) transcribed spacers. Cleavage sites A₀ to E are indicated.

FIG. 5.

Localization of a box H/ACA snoRNA (snR30) in temperature-sensitive mutants. FISH analysis was performed on wild-type (wt) and mutant strains at the permissive temperature (25°C) or after a 4-h shift to the nonpermissive temperature (37°C). U3 snoRNA and snR30 snoRNA were detected simultaneously with a Cy3-labeled U3-specific probe (green) and an Oregon green-labeled snR30-specific probe (blue).

FIG. 6.

Localization of nucleolar protein Nop1 in temperature-sensitive mutants. FISH analysis was performed on the wild-type (wt) and mutant strains at the permissive temperature (25°C) or after a 4-h shift to the nonpermissive temperature (37°C). U3 snoRNA (green) was detected by FISH with a Cy3-labeled U3-specific probe (60). The subcellular localization of Nop1 (blue) was detected in the same cells by immunofluorescence with antibodies specific for Nop1. DAPI was used to stain DNA (red).

FIG. 7.

Temperature-sensitive mutants do not exhibit major alterations in nucleolar structure. Cells grown for 4 h at the nonpermissive temperature (37°C) were collected, processed by acetylation, and inspected by EM. Representative nuclei are presented. wt, wild type; F, fibrillar strands; G, granular areas. Scale bar is 0.2 μm.

FIG. 8.

Spatial relationship of the nucleolar body to U3 in temperature-sensitive mutants. Temperature-sensitive mutants were transformed with a plasmid overexpressing a box C/D snoRNA variant (pG14-U14/MS2X2) that is used as a marker for the nucleolar body (91). FISH analysis of endogenous U3 snoRNA (green) and the ectopically expressed variant snoRNA (red) was performed following a 4-h shift to the nonpermissive temperature (37°C). wt, wild type.

FIG. 9.

Proposed roles of the newly identified trans-acting factors in the snoRNA trafficking pathway. After synthesis, intron removal, and core protein assembly in the nucleoplasm, yeast U3 snoRNA accumulates within the nucleolar body for cap hypermethylation, 3′-end trimming, and final assembly and then localizes to the nucleolus for function in rRNA biogenesis. The novel trans-acting factors identified in our study that are important for nucleolar body accumulation (NOP4, PRP21, and TAO3) and nucleolar targeting (SEC14 and HTL1) of U3 snoRNA are indicated.