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. 2010 Aug;38(14):4798-806.
doi: 10.1093/nar/gkq185. Epub 2010 Apr 12.

The C-terminus of Utp4, mutated in childhood cirrhosis, is essential for ribosome biogenesis

Emily F Freed  1 , Susan J Baserga
Affiliations

The C-terminus of Utp4, mutated in childhood cirrhosis, is essential for ribosome biogenesis

Emily F Freed et al. Nucleic Acids Res. 2010 Aug.

Abstract

The small subunit (SSU) processome is a large ribonucleoprotein that is required for maturation of the 18S rRNA of the ribosome. Recently, a missense mutation in the C-terminus of an SSU processome protein, Utp4/Cirhin, was reported to cause North American Indian childhood cirrhosis (NAIC). In this study, we use Saccharomyces cerevisiae as a model to investigate the role of the NAIC mutation in ribosome biogenesis. While we find that the homologous NAIC mutation does not cause growth defects or aberrant ribosome biogenesis in yeast, we show that an intact C-terminus of Utp4 is required for cell growth and maturation of the 18S and 25S rRNAs. A protein-protein interaction map of the seven-protein t-Utp subcomplex of which Utp4 is a member shows that Utp8 interacts with the C-terminus of Utp4 and that this interaction is essential for assembly of the SSU processome and for the function of Utp4 in ribosome biogenesis. Furthermore, these results allow us to propose that NAIC may be caused by dysfunctional pre-ribosome assembly due to the loss of an interaction between the C-terminus of Utp4/Cirhin and another SSU processome protein.

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Figures

Figure 1.
Figure 1.
Pre-RNA processing in S. cerevisiae. The pre-rRNA is transcribed as a 35S polycistronic precursor that undergoes multiple cleavage events to give rise to the mature 18S, 5.8S and 25S rRNAs. The first cleavage can occur at either site A0 (left) or site A3 (right). If cleavage occurs at A0 first, the 33S rRNA is generated and then subsequently cleaved at sites A1 and A2 giving rise to the 20S and 27SA2 rRNAs. If cleavage occurs at A3 first, the 27SA3 pre-rRNA is generated in addition to the 23S rRNA. The 27SA2/27SA3 rRNA is further cleaved at multiples sites, before export to the cytoplasm for final maturation to the 5.8S and 25S rRNAs. The 20S rRNA is also exported to the cytoplasm for final cleavage to give the 18S rRNA.
Figure 2.
Figure 2.
Alignment of yeast Utp4 and homologs. (A) Alignment of several yeast and vertebrate Utp4 sequences created by ClustalX (11). (B) Close-up of the alignment in part (A) showing the C-terminus of Utp4. The K616W and K627W point mutations are marked by lightning bolts and the E601X, E691X, D745X and L760X truncations are indicated with arrowheads. (C) Diagram of motifs predicted by the SMART database (12). Mutations indicated as in (B).
Figure 3.
Figure 3.
Utp4 mutations cause growth defects in yeast. (A) Schematic of the yeast strain used for testing Utp4 mutants. Endogenous Utp4 was placed under the control of the inducible GAL4 promoter. HA-tagged wild-type or mutant Utp4 constructs were constitutively expressed from the p415GPD plasmid. (B) Western blot confirming that endogenous Utp4 was not expressed when yeast are grown in glucose and that plasmid-encoded Utp4s were expressed. Blotting with an anti-Mpp10 antibody was used to assess gel loading. (C) Serial dilutions of yeast expressing Utp4 constructs were grown on solid medium at different temperatures for 3 days (30 and 37°C) or 6 days (17°C). (D) Table summarizing the results in (C).
Figure 4.
Figure 4.
Utp4 mutations cause a reduction in mature 18S and 25S rRNA levels in yeast. Total RNA was extracted from yeast expressing Utp4 constructs after endogenous Utp4 was depleted for the indicated amount of time. Left: ethidium bromide staining of total RNA. Right: quantitation of band intensities, each shown as a ratio of the depleted to undepleted band intensities.
Figure 5.
Figure 5.
Yeast two-hybrid analysis of protein–protein interactions in the t-Utp subcomplex. (A) Each t-Utp was cloned into both bait and prey vectors and transformed pair wise into the pJ69-4a yeast two-hybrid strain. Cells were spotted onto medium lacking leucine and tryptophan (permissive) to select for the presence of both bait and prey vectors and onto medium lacking leucine, tryptophan and histidine (selective) to assay for interactions between proteins. Growth on selective medium indicates that the two proteins interact. Bait proteins are listed along the top and prey proteins are listed along the side. (B) Interaction network drawn from results in (A). Arrows are pointing from prey to bait. (C) Comparison of results obtained here to those of Tarassov et al. (27). Green arrows represent interactions found in both studies, yellow arrows represent interactions found in Tarassov et al. only, and blue arrows represent interactions found only here. Note that Tarassov et al. did not include Utp17 in their assay.
Figure 6.
Figure 6.
An intact C-terminus of Utp4 is required for interaction with Utp8. Each Utp4 truncation was used as bait and either Utp5 (A) or Utp8 (B) was used as prey. Yeast was struck out onto medium lacking leucine, tryptophan and histidine (selective) to assay for interactions between proteins.
Figure 7.
Figure 7.
An intact C-terminus of Utp4 is required for association of the t-Utp subcomplex and formation of the SSU processome. (A) Total protein was extracted from yeast expressing p415GPD 3HA-UTP4 constructs after endogenous Utp4 was depleted for 16 h. Utp4 was immunoprecipitated with an anti-HA antibody. Total (5%) and immunoprecipitated proteins were separated by SDS–PAGE and transferred to Immobilon PVDF membranes. Association with the t-Utp subcomplex was assayed by western blotting for Utp17, another t-Utp subcomplex member. Association with the SSU processome was assayed by western blotting for Mpp10 with an anti-Mpp10 antibody. (B) Table summarizing the results from (A) and showing the correlation between cell growth (from Figure 3), the ability of Utp4 and Utp8 to interact (from Figure 6) and the assembly of the t-Utp subcomplex and SSU processome.
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