A cytoplasmic signal corresponding to a 3′-extended 5.8S pre-rRNA can be detected. (A) Fluorescent in situ hybridization (FISH) was performed using the Cy3-labeled ITS2-1 LNA probe (red in merge panels) in various WT strains. Cells were fixed in paraformaldehyde and spheroplasted with Zymolyase. FISH was performed as described in Materials and Methods. The nucleoplasm was visualized by DAPI (blue in merge panels). (B) Wild-type (CRM1) and crm1(T₅₃₉C) strains were grown at 25°C and, where indicated (+), treated with LMB (Lep B; 100 ng ml⁻¹) for 1 h. Cells were fixed in paraformaldehyde and spheroplasted. FISH was performed using the ITS2-1 LNA Cy3-labeled probes (red in merge panels). The nucleoplasm was stained with DAPI. These figures show a single optical section from a deconvolved stack. All images shown in panels A and B, respectively, were captured using identical exposure times. Following acquisition, data were processed in an identical fashion for each image.

6S pre-rRNA levels decrease when nuclear-cytoplasmic export is blocked. (A) Wild-type (CRM1) and crm1(T₅₃₉C) strains were treated with LMB (LepB; 100 ng/ml), at the 0-min time point (shown by *). Low-molecular-weight RNA was separated on an 8% polyacrylamide-8.3 M urea gel. Following transfer, membranes were probed with ³²P-labeled oligonucleotide probes. ³²P signal for 7S, 6S, and 5S was imaged using a Fuji imager system and quantified using AIDA software. (B) Quantification of the 6S/7S ratio from experiments performed in triplicate. The 6S/7S ratio was standardized by normalizing the first time point (0 min) to 1. Error bars represent ±1 standard error. For clarity, error bars have been excluded for the WT and the trf4Δ single mutant.

Nuclear localization of 5.8S precursors in rrp6Δ mutants. (A) Pulse-labeling in wild-type and rrp6Δ strains. Cells were grown in SD-Ura media and pulse-labeled with [8-³H]uracil for 2 min followed by a chase with a large excess of cold uracil for the times indicated. Low-molecular-weight RNA was separated on an 8% polyacrylamide-8.3 M urea gel. (B) FISH performed in an rrp6Δ strain. The LNA modified oligonucleotide ITS2-1 is shown as red in merge panels, and the nucleoplasm was visualized by DAPI (blue in merge panels). The cell periphery is shown by a dashed line.

Model for the final steps in 25S and 5.8S processing. Cleavage of the 27SB pre-rRNA at the C₂ site, by an as yet unidentified enzyme, generates the 7S and 26S pre-rRNA species. Following this cleavage, the processing and export factor Arx1 associates with the preribosomal particle. The 7S pre-rRNA is trimmed to a 5.8S+30 species by the 3′→5′ exonuclease activity of Rrp44, a component of the nuclear exosome. The 26S species is processed by the 5′→3′ exonuclease Rat1 to the 25S′ pre-rRNA, which is extended at the 5′ end by some 8 nucleotides compared to the mature 25S. This species is trimmed back to the mature form, probably through the activity of Rat1. The 5.8S+30 form is processed to 6S pre-rRNA by the nuclear exonuclease Rrp6. The 25S- and 6S-containing preribosomes are competent for export to the cytoplasm and associate with export adapter molecule Nmd3, which recruits the export receptor Crm1. Following export through the nuclear pore complexes (NPC), the recycling factor Lsg1 associates with the preribosome. 6S pre-rRNA processing to the mature 5.8S is completed by Ngl2. Finally, the remaining nonribosomal factors dissociate from the subunit, generating functional 60S subunits.

Pre-rRNA processing pathway in Saccharomyces cerevisiae. (A) The structure of the 35S rRNA precursor and locations of processing sites. The pre-rRNA encodes the 18S, 5.8S, and 25S rRNAs, which are flanked by the 5′ and 3′ external transcribed spacers (5′-ETS and 3′-ETS) and separated by internal transcribed spacers 1 and 2 (ITS1 and ITS2). The positions of oligonucleotide probes used for Northern hybridization and FISH analysis are shown in blue and red boxes, respectively. (B) The pre-rRNA processing pathway. RNA polymerase I (Pol I) transcripts undergo one of two alternate fates. The 35S precursor, contained within the 90S preribosome, is generated by cleavage at site B₀ within the 3′-ETS. This is followed by posttranscriptional endonucleolytic cleavages (PTC) within the 5′-ETS, at A₀ and A₁ at the 5′ end of the mature 18S rRNA and within ITS1 at A₂. Cleavage at A₂ separates the precursors to the 40S and 60S subunits and generates the 20S and 27SA₂ pre-rRNAs. Alternately, Pol I transcripts can undergo cotranscriptional cleavage (Co-TC) at the A₀, A₁, and A₂ sites, within the small subunit (SSU) processome. Co-TC cleavage is followed by the assembly of the large subunit (LSU) processome on the nascent RNA transcript. Following either posttranscriptional or cotranscriptional cleavage, the 20S pre-rRNA, contained within a pre-40S particle, is exported from the nucleus to the cytoplasm, where maturation to 18S is completed. The pathway of 5.8S and 25S synthesis occurs within a series of pre-60S particles. The 27SA₂ pre-rRNA follows one of two alternate pathways: around 85% is cleaved at the A₃ site within ITS2, followed by 5′→3′ exonucleolytic processing gener-ating the 27SB_s pre-rRNA. The remaining 15% is processed at site B_1L, which is located 8 nt 5′ to B_1S, yielding the 27SB_L pre-rRNA. These two alternate forms of 27SB are cleaved within ITS2 at site C₂, yielding 26S pre-rRNA and the long and short forms of 7S. The 7S pre-rRNAs are converted to 6S_L and 6S_S by the nuclear exosome and Rrp6. Maturation of 26S to 25S rRNA proceeds by a two-step 5′-exonuclease pathway. Subsequently, pre-60S particles are exported to the cytoplasm, where final maturation to 5.8S is completed.

5.8S rRNA synthesis is rapidly inhibited when nuclear-cytoplasmic export is blocked. (A) Wild-type (CRM1) and crm1(T₅₃₉C) strains were transformed with a pURA plasmid and grown in SD-Ura media. CRM1 and crm1(T₅₃₉C) strains were treated with LMB (100 ng/ml) for 15 min prior to labeling with [5,6-3H]uracil (GE) (1 mCi in 25 ml culture). One milliliter of cells was harvested every 30 s. Low-molecular-weight RNA was separated on an 8% polyacrylamide-8.3 M urea gel. (B) CRM1 and crm1(T₅₃₉C) strains were labeled with [5,6-³H]uracil (GE) (1 mCi in 25 ml culture) for 5 min before treatment with LMB (100 ng/ml). Time of LMB addition is shown by *. One milliliter of cells was harvested at each time point indicated. Low-molecular-weight RNA was separated on an 8% polyacrylamide-8.3 M urea gel. (C) Experiments as shown in panel B were performed in quadruplicate, and tritiated 5.8S_L+S was quantified using a Fuji imager system with an FL-5100 screen. Lane background signal (tritium) was subtracted, and loading variations were accounted for by normalizing against the signal gained from a Northern hybridization using a ³²P-labeled probe against 5S rRNA. To compare experiments, each data set was normalized to 5S ³H signal at the time point taken 1 min before LMB treatment. The averages of 4 experiments are shown. Error bars represent ±1 standard error.

The cytoplasmic GTPase Lsg1 precipitates 6S pre-rRNA. (A) Assembly of late pre-60S factors. Arx1 associates in the nucleus and shuttles with the pre-60S particles to the cytoplasm. Nmd3 is the export adapter protein that tethers the 60S preribosome to the export receptor Crm1/Xpo1. Once in the cytoplasm, the cytoplasmic GTPase Lsg1 releases Nmd3 from the 60S subunit. (B, C, and D) Northern and primer extension analysis of RNAs precipitated from the untagged strain (BY4741), Arx1-TAP, Nmd3-TAP, Lsg1-TAP, and Crm1-TAP. Cell lysates from the TAP-tagged strains were incubated with IgG-Sepharose, and the associated RNA was extracted from beads (IP). RNA from total cell extract was also prepared (T). Total RNA loaded corresponds to 1% of the amount used as input for immunoprecipitations. (B) To analyze low-molecular-weight species, RNA was separated on a denaturing 8% polyacrylamide-8.3 M urea gel. Following migration, RNAs were transferred to a nylon membrane and hybridized with the 5′-labeled oligonucleotide probes shown in parentheses on the left of the gel panels. (C) The experiment shown in panel B was performed in triplicate, and the data were quantified. The band intensity for precipitated 7S and 6S was quantified after subtraction of the lane background and expressed as a fraction of the corresponding signal in the total input lane to determine the efficiency of precipitation. The fold over background is the ratio between the efficiency of RNA coprecipitation seen with each protein and the background recovery of 7S/6S in the untagged BY4741 strain. (D) Primer extension analysis of the 5′ end of 25S, using oligonucleotide 007, where total RNA loaded corresponds to 1% of the amount used as input for immunoprecipitations. Reactions were analyzed on a denaturing 6% polyacrylamide-8.3 M urea gel, and images were captured using the Fuji imager system.

Cytoplasmic localization of 5.8S precursors in ngl2Δ mutants. (A) Pulse-labeling in wild-type and ngl2Δ strains, performed as described for Fig. 6A. (B) Northern hybridization of the same filter (10-min time point lanes are shown) using probes hybridizing within 5.8S rRNA (017) or across the 5.8S-ITS boundary (020). The locations of the long (L) and short (S) forms of 6S and 5.8S are indicated. (C) FISH on ngl2Δ, performed as described for Fig. 6B.