Sfp1 plays a key role in yeast ribosome biogenesis

Abstract

Sfp1, an unusual zinc finger protein, was previously identified as a gene that, when overexpressed, imparted a nuclear localization defect. sfp1 cells have a reduced size and a slow growth phenotype. In this study we show that SFP1 plays a role in ribosome biogenesis. An sfp1 strain is hypersensitive to drugs that inhibit translational machinery. sfp1 strains also have defects in global translation as well as defects in rRNA processing and 60S ribosomal subunit export. Microarray analysis has previously shown that ectopically expressed SFP1 induces the transcription of a large subset of genes involved in ribosome biogenesis. Many of these induced genes contain conserved promoter elements (RRPE and PAC). Our results show that activation of transcription from a reporter construct containing two RRPE sites flanking a single PAC element is SFP1 dependent. However, we have been unable to detect direct binding of the protein to these elements. This suggests that regulation of genes containing RRPEs is dependent upon Sfp1 but that Sfp1 may not directly bind to these conserved promoter elements; rather, activation may occur through an indirect mechanism.

FIG. 1.

The rate of translation is reduced in sfp1 cells. [³⁵S]methionine was added to wild-type and sfp1 cultures grown in SD−Met medium, and total incorporation of the label was measured at the indicated time points by TCA precipitation of the aliquots and counting of the radioactivity in the pellet. The sfp1 mutant shows a ∼2.5-fold decrease in the rate of methionine incorporation.

FIG. 2.

Polyribosome profiles show that sfp1 cells have reduced levels of 80S ribosomes and higher-order polyribosomes. Extracts from wild-type and sfp1 cells were resolved on 7 to 47% sucrose gradients. The OD₂₅₄ was monitored for each sample. Positions of the 40, 60, and 80S ribosome peaks are shown.

FIG. 3.

sfp1 cells are defective in nuclear export of the 60 and 40S ribosomal subunits. (A) DAPI staining and localization of the RPL11b-GFP fusion protein (60S component) are shown for wild-type, kap120, and sfp1 cells. (B) DAPI staining and localization of the Rps2-GFP fusion protein (40S component) are shown for wild-type, noc4-1, and sfp1 cells.

FIG. 4.

sfp1 cells exhibit a delay in 27 and 20S pre-rRNA processing. rRNAs from wild-type and sfp1 cells were pulse-labeled in vivo by using l-[methyl-³H]methionine. Samples were harvested and processed at the indicated time points, electrophoresed on a formaldehyde gel, transferred to a membrane, and visualized by exposure to film after being treated with a fluorescence enhancer. The positions of the 35, 27, 25, 20, and 18S rRNAs are shown.

FIG. 5.

sfp1 cells have reduced levels of rRNA. Equal concentrations of total RNA purified from a wild-type strain and an sfp1 strain were electrophoresed on a formaldehyde agarose gel. The RNA was then transferred to a nylon membrane and probed by a Northern blot procedure with labeled probes specific for 25S rRNA (top), 18S rRNA (center), and ACT1 (bottom) The levels of hybridization of the 25S rRNA, 18S rRNA, and ACT1 probe to the wild-type and sfp1 RNA samples were measured on a phosphorimager.