Functional link between ribosome formation and biogenesis of iron-sulfur proteins

Abstract

In genetic screens for ribosomal export mutants, we identified CFD1, NBP35 and NAR1 as factors involved in ribosome biogenesis. Notably, these components were recently reported to function in extramitochondrial iron-sulfur (Fe-S) cluster biosynthesis. In particular, Nar1 was implicated to generate the Fe-S clusters within Rli1, a potential substrate protein of unknown function. We tested whether the Fe-S protein Rli1 functions in ribosome formation. We report that rli1 mutants are impaired in pre-rRNA processing and defective in the export of both ribosomal subunits. In addition, Rli1p is associated with both pre-40S particles and mature 40S subunits, and with the eIF3 translation initiation factor complex. Our data reveal an unexpected link between ribosome biogenesis and the biosynthetic pathway of cytoplasmic Fe-S proteins.

Figure 1

Cloning of RIX19. YIL003w can complement the ts (A) and rix phenotype (B) of the rix19-1 allele. The strain rix19-1 was transformed with pRS315 plasmid either without insert or containing YIL003w. (B, C) Rps2-GFP and Rpl25-GFP accumulation in rix19-1. Cells with the indicated reporter plasmids were examined in the fluorescence microscope 2 h after shift from 23 to 37°C.

Figure 2

NBP35 is genetically linked to RIX19. (A) The NBP35 shuffle strain (nbp35∷KAN-MX4+pRS316-NBP35) was transformed with pRS315 plasmid containing nbp35-1 allele or the wild-type NBP35. After FOA selection, cells were grown on YPD plates for 5 days. (B) Rps2-GFP and Rpl25-GFP accumulation in the nbp35-1 ts mutant, which was transformed with indicated reporter plasmids. Exponentially growing cells were shifted from 30 to 37°C and examined after 4 h in the fluorescence microscope. (C) rix19-1 strain was crossed with the NBP35 shuffle strain. After tetrad analysis, a resulting rix19-1/nb35Δ haploid was transformed with NBP35 or nbp35-1. Two individual transformants were grown on the SDC plate containing 5-FOA for 5 days at 30°C. (D) The levels of ribosomal proteins Rpl10 and Rpl25 are not elevated in nbp35-1 strains. Overnight cultures of indicated stains were diluted to OD₆₀₀ 0.2 and grown at 23 or 37°C for 4 h. Whole cell lysates were separated by SDS–PAGE and transferred to nitrocellulose. Ponceau S staining (upper panel) revealed the protein loading, and the amount of Rpl10 and Rpl25 was determined by Western using specific antibodies (lower panel).

Figure 3

NAR1 is genetically linked to RIX19 and is required for nuclear export of ribosomes. (A) The rix19/nar sl mutant was transformed with pRS315 plasmid without insert or containing NAR1 or RIX19. Cells were grown on 5-FOA containing SDC plates for 5 days at 30°C. (B) The NAR1 shuffle strain was transformed with pRS315 plasmids containing either NAR1 or nar1-2. After plasmid shuffling on 5-FOA containing SDC plates, the cells were grown in serial dilutions on YPD plates at the indicated temperatures for 3 days. (C, D) nar1-2 strain was transformed with Rps2-GFP and Rpl25-GFP plasmids. Cells growing in liquid SDC medium at 30°C were observed in the fluorescence (C) and confocal (D) microscope. (E) Poly(A)⁺ RNA localization in nar1-2 cells, grown at 30°C, was analyzed by in situ hybridization.

Figure 4

Rli1 is a shuttling protein involved in ribosomal export. (A) A yeast strain, in which the endogenous RLI1 promoter was replaced by the Tet^OFF promoter, was transformed with Rpl25-GFP and Rps2-GFP plasmids. The cells were grown in liquid SDC-leu medium at 30°C. At early exponential growth (OD 0.2), doxycycline was added to a final concentration of 5 μg/ml. Cells were examined after 12 h of incubation. (B) The RLI1-GFP/xpo1-1 cells were grown in YPD at 30°C to OD₆₀₀ 0.2 before shift to 37°C. After 1.5 and 4 h of incubation at 37°C, cells were examined in the fluorescence microscope. (C) Rli1 mainly cosediments with 40S subunits and 80S ribosomes. Rli1-TAP was visualized in the fractions derived from the sucrose gradient by Western using anti-protein A antibodies.

Figure 5

Rli1 is involved in pre-rRNA maturation. (A) Schematic of the pre-rRNA showing the positions of the oligonucleotide probes used for hybridization. Total RNA was extracted from strains expressing wild-type or Tet-regulated RLI1 (tet∷rli1) during growth in the absence of doxycycline (0 h samples), and at time points following doxycycline addition. (B) Northern analyses of high molecular weight RNAs separated on a 1.2% agarose/formaldehyde gel. (C) Northern analyses of low molecular weight RNAs separated on an 8% polyacrylamide/urea gel. Pre-rRNAs and rRNAs detected are indicated on the right of the figure. Oligonucleotides used are listed on the left. tRNA^Leu was used as a loading control (oligonucleotide 306). (D) Pulse-chase labeling of high molecular weight RNAs. Wild-type and tet∷rli1 strains were pregrown in minimal medium and treated with doxycycline for 6 h prior to pulse labeling with [5,6-³H]uracil for 2 min. A large excess of unlabeled uracil was added and samples were taken immediately (0 min) and at the time points indicated. RNA was extracted, separated on a 1.2% agarose/formaldehyde gel, transferred to a nylon membrane and visualized by fluorography.

Figure 6

Rli1 is associated with pre-rRNA, rRNA and eIF3. (A) Immunoprecipitation of TAP-tagged Rli1 and Ded1 was performed in the presence of 150 mM NaCl using IgG-Sepharose. RNAs were extracted from the pellets obtained after precipitation (lanes IP) or from an amount of total extract corresponding to 1/30 of that used for the precipitation (lanes T). Co-precipitated RNAs were identified by Northern hybridization. (B) TAP purification of Rli1 was performed in the presence of 150 mM NaCl (lane 2) or 50 mM NaCl (lane 3). Lane 1 depicts a protein standard. Bands marked with asterisks are the commonly found contaminants in TAP purifications. (C) RLI1 and HCR1 are genetically linked. The hcr1Δ strain pretransformed with pRS315-RLI1 plasmid was crossed with RLI1-TAP strain. After subsequent tetrad analysis, two haploid strains from one tetrad carrying both mutations were selected and plated on FOA-containing media together with parental strains. (D) Ribosomal export is impaired in hcr1Δ cells. hcr1Δ strain was transformed with Rpl25-GFP or Rps2-GFP plasmids. The cells were grown in liquid SDC-leu medium at 30°C. Cells were examined in the fluorescence microscope.