A missense mutation in the C-terminus of human Utp4/Cirhin has been reported to cause North American Indian childhood cirrhosis (9
). In this study we show that, unexpectedly, the homologous mutation does not affect ribosome biogenesis in the baker’s yeast, S. cerevisiae
. However, we have also shown that truncation of yeast Utp4 by as few as 31 amino acids leads to reduced growth and reduced levels of mature rRNAs likely due to the loss of an interaction with Utp8. Moreover, truncation of Utp4 prevents the formation of both the t-Utp subcomplex and the SSU processome. These findings indicate that the region including the NAIC mutation is important for the function of Utp4 in ribosome biogenesis, and our results are therefore important for understanding the protein contacts that make the subcomplexes that compose the SSU processome.
Although the NAIC mutation does not result in a mutant phenotype in yeast, S. cerevisiae
still provides a good model for studying eukaryotic ribosome biogenesis. Overall rRNA processing is well conserved from yeast to humans (19
). Furthermore, approximately 90% of ribosome biogenesis genes are conserved from yeast to humans, and recent studies have shown that ribosome biogenesis proteins that are essential in yeast, including Utp4, are also essential in mice (20–22
). Interestingly, Utp8, the protein that interacts with the C-terminus of Utp4, is one of the few proteins in the SSU processome that is not conserved in mammals. The lack of an Utp8 ortholog in humans is one possible reason why human Utp4/Cirhin does not complement yeast Utp4. In humans, Utp8 is likely replaced by a functional analogue that has yet to be discovered, although one candidate, Cirip, was identified in a yeast two-hybrid screen using human Utp4/Cirhin as a bait (23
). Cirip increases the activity of the HIV-1 LTR enhancer element, an NF-κB responsive gene. However, since Cirip has not been detected in the nucleolus, it is unlikely to replace the role of Utp8 in ribosome biogenesis.
In order to more completely understand the assembly and composition of large pre-ribosome complexes, we carried out a directed one-by-one yeast two-hybrid analysis of the seven member t-Utp subcomplex. This enabled us to determine binary protein–protein interactions of this subcomplex and gives us insight into how the individual components come together to function as a single macromolecule. Previous high-throughput yeast two-hybrid studies largely failed to include the t-Utps and have only identified the interaction between Utp5 and Utp15 (24–26
). Recently, however, a new method using a protein-fragment complementation assay (PCA) reported interactions involving six of the seven t-Utp proteins (27
). While many of the detected interactions were the same between that study and this one, Tarassov et al.
found several interactions that we did not and vice versa (summarized in C). While it is difficult to determine exactly why these differences exist, it is clear that the two methods have different physiochemical limitations: yeast two-hybrid is biased toward nuclear interactions while PCA is biased towards proteins with transmembrane domains (28
). Since the t-Utps are nucleolar proteins, the yeast two-hybrid system is an appropriate method for determining interactions between subcomplex members. Furthermore, yeast two-hybrid analysis can detect weak interactions with Kd
values in the range of 10–100 µM (29
). Importantly, both yeast two-hybrid and PCA detect an interaction between Utp4 and Utp8, strengthening the validity of this interaction.
Although we were not able to use S. cerevisiae as a model for NAIC, we were able to determine that an intact C-terminus of Utp4 is required for ribosome biogenesis and that this role is mediated by its interaction with Utp8. Future work should be carried out to determine whether the NAIC mutation abrogates the interaction between human Utp4/Cirhin and the as-yet-to-be discovered human functional analogue of Utp8, disrupting ribosome biogenesis and leading to disease.