Functional characterization of the nucleolar Nopp140 and Srp40 in yeast
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Rat Nopp140 has been implicated to transport box C/D and H/ACA snoRNPs between the nucleolus and the Cajal body. Its yeast homolog Srp40p is localized to the nucleolus, implying that it might also be involved in ribosome biogenesis. To clarify its function, synthetic lethal screens were performed to identify proteins whose mutations were not lethal alone, but were lethal in combination with SRP40 disruption. SHM2 (cytosolic serine hydroxymethyltransferase) and ADE3 (one-carbon tetrahydrofolate synthase) were identified in these screens. Both proteins are enzymes in independent but convergent pathways to produce 5,10-methylene tetrahydrofolate, the primary donor in one carbon metabolism. However, point mutations in the catalytic domain of Shm2p that abolished its enzymatic activity were able to complement the synthetic lethality, indicating that it had an unknown function that might link to SRP40 and ribosome biogenesis. The synthetic lethal screens provided us with strains in which SRP40 was essential. This allowed us to explore the function of Srp40p. Thus, depletion of Srp40p in the synthetic lethal strain resulted in growth arrest and specific depletion of box H/ACA snoRNAs, indicating that Srp40p was genetically associated with H/ACA snoRNAs. Furthermore, this growth arrest and depletion of box H/ACA snoRNAs were rescued by expression of Nopp140, indicating that Nopp140 and Srp40p are indeed functional homologs.;We also performed a two-hybrid screen to identify rat Nopp140-interacting proteins. p80 coilin, the marker protein of Cajal bodies, was identified and the interaction was confirmed by coimmunoprecipitation. This finding provides additional evidence that Nopp140 serves as a molecular basis between the nucleolus and Cajal bodies.;Nopp140-associated protein NAP57 is a putative pseudouridylase in box H/ACA snoRNPs. Since it was highly conserved from yeast to mammals, we tested if it was able to replace its yeast homolog Cbf5p. Expression of NAP57 in yeast complemented the growth defect and box H/ACA snoRNA depletion in a cbf5p-depleted strain, but failed to do so in a CBF5 null strain or a Cbf5p temperature-sensitive strain. Therefore, NAP57 could only partially replace Cbf5p in yeast.
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