cDNA Cloning and Characterization of the Human U3 Small Nucleolar Ribonucleoprotein Complex-Associated 55-Kilodalton Protein

ABSTRACT

The eukaryotic nucleolus contains a large number of small RNA molecules (snoRNAs) which, in the form of small nucleolar ribonucleoprotein complexes (snoRNPs), are involved in the processing and modification of pre-rRNA. The most abundant and one of the best-conserved snoRNAs is the U3 RNA. So far, only one human U3 snoRNA-associated protein, fibrillarin, has been characterized. Previously, the U3 snoRNPwas purified from CHO cells, and three proteins of 15, 50, and 55 kDa were found to copurify with the U3 snoRNA (B. Lübben, C. Marshallsay, N. Rottmann, and R. Lührmann, Nucleic Acids Res. 21:5377–5385, 1993). Here we report the cDNA cloning and characterization of the human U3 snoRNP-associated 55-kDa protein. The isolated cDNA codes for a novel nucleolar protein which is specifically associated with the U3 snoRNA. This protein, referred to as hU3-55k, is the first characterized U3 snoRNP-specific protein from humans. hU3-55k is a new member of the family of WD-40 repeat proteins and is conserved throughout evolution. It appears that the C-terminal end of hU3-55k is required for nucleolar localization and U3 snoRNA binding.

ACKNOWLEDGMENTS

We thank G. J. M. Pruijn for helpful discussions and critical reading of the manuscript and J. M. H. Raats and A. van der Kemp for advice on tissue culture and immunofluorescence techniques. We are grateful to B. Lübben for isolation of the 55-kDa protein from CHO cells, K. M. Pollard and E. M. Tan for providing antifibrillarin (72B9) antibodies, and T. Kiss for providing plasmids of human U8, U13, and U24 snoRNAs.

This work was supported in part by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for Scientific Research (NWO).