Sof1p is a trans-acting protein that is essential for biogenesis of the 40S ribosomal subunits in the yeast Saccharomyces cerevisiae. Because of its involvement in the early cleavage steps of precursor rRNA, its interaction with Nop1p and its ability to coprecipitate U3 snoRNA, Sof1p has so far been regarded as a protein that is specific to the U3 snoRNP. To determine whether a site exists within U3 snoRNA with which Sof1p directly or indirectly associates, we studied the ability of ProtA-tagged Sof1p to coimmunoprecipitate mutant versions of U3 snoRNA. None of the tested mutations had a significant effect on the recovery of mutant U3 from cell extracts. Further coimmunoprecipitation experiments, using cells that could be genetically depleted for either Sof1p or U3 snoRNA demonstrated that the two factors associate independently of each other with the 35S precursor RNA. Indeed, association between Sof1p and U3 snoRNA was abolished in cells in which 35S pre-rRNA transcription was blocked. Finally, we found that an overall reduction in the levels of box C/D snoRNPs by genetic depletion of the common Nop58p protein did not affect coprecipitation of 35S pre-rRNA by Sof1p. From these data, we conclude that Sof1p does not assemble into the 90S preribosome as part of the U3, or any other box C/D, snoRNP. The early and independently assembling trans-acting factor Rrp5p also proved to be dispensable for assembly of Sof1p. Copyright © 2006, American Society for Microbiology. All Rights Reserved.