The PINc domain protein Utp24, a putative nuclease, is required for the early cleavage steps in 18S rRNA maturation

Abstract

Ribosome biogenesis is a complex process that requires >150 transacting factors, many of which form macromolecular assemblies as big and complex as the ribosome itself. One of those complexes, the SSU processome, is required for pre-18S rRNA maturation. Although many of its components have been identified, the endonucleases that cleave the pre-18S rRNA have remained mysterious. Here we examine the role of four previously uncharacterized PINc domain proteins, which are predicted to function as nucleases, in yeast ribosome biogenesis. We also included Utp23, a protein homologous to the PINc domain protein Utp24, in our analysis. Our results demonstrate that Utp23 and Utp24 are essential nucleolar proteins and previously undescribed components of the SSU processome. In that sense, both Utp23 and Utp24 are required for the first three cleavage steps in 18S rRNA maturation. In addition, single-point mutations in the conserved putative active site of Utp24 but not Utp23 abrogate its function in ribosome biogenesis. Our results suggest that Utp24 might be the elusive endonuclease that cleaves the pre-rRNA at sites A(1) and/or A(2.).

Fig. 1.

Pre-rRNA processing in S. cerevisiae. The 35S pre-rRNA encoding the 18S, 5.8S, and 25S rRNAs is processed at sites A₀, A₁, A₂, and A₃ via several intermediates to yield the 20S pre-rRNA. The 20S pre-rRNA cleavage at site D matures the 18S rRNA, whereas numerous endo- and exonucleolytic cleavages in the nucleolus mature the 5.8S and 25S rRNAs from the 27SA₂ or 27SA₃ precursor. Small letters above the primary transcripts indicate the oligonucleotides used. The maturation of the large subunit rRNAs downstream of the 27SA₃ pre-rRNA is the same for each pathway.

Fig. 2.

Schematic of the classical PINc domain with conserved putative active-site residues. Residue numbers below the scheme represent corresponding amino acids in Utp23 and Utp24 based on the detailed alignment shown in Fig. 7.

Fig. 3.

Utp23 and Utp24 are essential for SSU pre-rRNA processing. (A) Depletion of Utp23 and Utp24 impairs cell growth. Growth of wild-type cells (squares), tetO7::Utp23 cells (circles), and tetO7::Utp24 cells (triangles) was compared in the presence (open symbols) or absence (filled symbols) of doxycycline. The OD₆₀₀ was measured up to 40 h after the addition of doxycycline. (B) Depletion of Utp23 and Utp24 affects SSU biogenesis. Twenty micrograms of total RNA from cells depleted and undepleted of either Utp23 or Utp24, respectively, was separated on 1.25% formaldehyde/agarose gels. RNA from depleted and undepleted cells was run on the same gel and subjected to the same exposure. Pre-rRNAs and mature rRNAs were detected by Northern blotting with oligonucleotides C (detects 35S, 33/32S, 27SA₂, and 23S pre-rRNAs), B and E (also label the 27SA₃, 27SB, and 20S pre-rRNAs), or A and Y (label mature 18S and 25S rRNAs). (C) Depletion of Utp23 or Utp24 impairs pre-rRNA processing but does not affect rRNA stability. Yeast cells depleted of Utp23 or Utp24 for 10 h with doxycycline were labeled metabolically with ³H-uracil for 2 min and incubated with excess of unlabeled uracil for 0, 3, 12, 30, and 60 min. Twenty thousand counts per million of RNA were separated as in B, transferred to Hybond N⁺ membrane, and detected by autoradiography. The parental strain YPH499 also was grown in 2 μg/ml doxycycline for 10 h.

Fig. 4.

Utp23 and Utp24 are nucleolar proteins. HA-tagged Utp23, Utp24, and the positive control protein Utp8 were localized with α-HA antibodies and rhodamine-conjugated α-mouse IgG. Mpp10, a nucleolar marker protein, was detected with polyclonal antibodies against Mpp10 and fluorescein-conjugated α-rabbit IgG. Nuclei were stained with DAPI. The parental strain YPH499 was used as a negative control.

Fig. 5.

Utp23 and Utp24 are associated with the SSU processome. (A) Utp23 and Utp24 coimmunoprecipitate Mpp10, an SSU processome protein. α-HA antibodies were used to immunoprecipitate HA-tagged Utp23, Utp24, and Utp8. Immunoprecipitates (IP) and 5% of total input (T) were separated by SDS/PAGE, and Mpp10 was detected by Western blotting. (B) Utp23 and Utp24 coimmunoprecipitate the U3 snoRNA. RNA was extracted from IPs (performed as in A) and 5% T, separated in 8% urea/acrylamide gels, and detected by Northern blotting. (C) Depletion of either Utp23 or Utp24 results in failure to form SSU processomes on nascent rRNA transcripts. Shown are representative rRNA genes from chromatin spreads of cells undepleted or 18 h depleted of Utp23 or Utp24. Small arrows indicate typical SSU processome formation on nascent transcripts, brackets indicate shorter transcripts after cotranscriptional cleavage in ITS1, and open arrows indicate the lack of SSU processome formation.

Fig. 6.

Mutations in the putative active site of Utp24 disrupt protein function. (A) Acidic residues in Utp24 but not Utp23 are essential for in vivo protein function. Ten-fold serial dilutions of yeast cells containing empty vector (−), wild-type (WT), or indicated mutant Utp23 or Utp24 proteins were analyzed for growth in the absence (−Dox) and presence (+Dox) of doxycycline at 17°C, 23°C, 30°C, and 35°C. The pictures were modified so that the dots align. (B) Expression of Utp24 mutants results in accumulation of the 22S pre-rRNA. Cells transformed with empty vector or expressing WT Utp24 or the Utp24 mutants D68N or D138N from p415GPD were harvested 0, 10, and 20 h after growth in media with (depleted) or without (undepleted) doxycycline. Total RNA was extracted and equal amounts were separated on 1.25% formaldehyde/agarose gels. Pre-rRNAs were detected by Northern blotting with oligos B and E.