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.Eukaryotic cells contain a large number of small nucleolar RNAs (snoRNAs) which, in the form of small nucleolar ribonucleoprotein complexes (snoRNPs), are involved in the various steps of ribosome synthesis (reviewed in references 30 and 46). Several snoRNAs have previously been shown to be required for pre-rRNA processing (12,30,46), and a large set of snoRNAs is involved in ribose methylation and pseudouridylation of rRNA (8,15,24,33,34,49). snoRNAs are heterogeneous in size, structural elements, and protein association. They are produced by two biosynthetic pathways. Most snoRNAs are encoded within the pre-mRNA introns of ribosomal or nucleolar proteins. The processing of such premRNAs via endo-and exonucleolytic cleavages results in the generation of mature noncapped snoRNAs (30). Other snoRNAs, e.g., U3 snoRNA, are transcribed from independent genes and typically possess a modified 5Ј terminus, usually a 5Ј trimethylguanosine (TMG) cap (30).snoRNPs can be divided into four groups, which appear to be functionally distinct (1, 46). Methylation guide snoRNPs direct the site-specific formation of 2Ј-O-methyl groups in mature rRNA. All snoRNAs of this class contain two conserved sequence elements, referred to as box C and box D (30), and contain an extended region (10 to 21 nucleotides) of base complementarity to mature rRNA (8,24,34,49). Members of the second group of snoRNAs, which encompasses U3, U8, U14, and U22 snoRNAs, also contain the conserved box C and D elements and are involved in pre-rRNA processing reactions (reference 46 and references therein). All box C-and D-containing snoRNAs, including methylation guide snoRNAs, are associated with the conserved nucleolar protein fibrillarin, which thus is a common snoRNP component (30). Members of the third class of snoRNAs lack the box C and D elements but share another conserved sequence element, referred to as the ACA box (1). Such snoRNA...

Interaction of the U3-55k protein with U3 snoRNA is mediated by the Box B/C motif of U3 and the WD repeats of U3-55k

“…RNA was resolved on an 8% denaturing polyacrylamide gel and blotted onto a Hybond-N + membrane (Amersham, Buckinghamshire, UK). Northern analysis performed with anti-sense radiolabeled U3 RNA probes was done essentially as previously described (45,66).…”

“…pCI-neo VSV-55k was constructed as described (45). The original hU3-55k cDNA was modified by PCR to introduce an XhoI site before the translational start codon [pGEM3Zf(+)-hU3-55k (45)].…”

“…The RNA is associated with proteins common to all Box C/D snoRNAs, including fibrillarin/Nop1p (34,35), Nop56p (36) and Nop58/Nop5p (37)(38)(39) and proteins specific to the U3 snoRNP, including Sof1p (supressor of fibrillarin; 40), Mpp10 (41,42), Lcp5p (43), Imp3p (44), Imp4p (44) and U3-55k (29,45). Each of these proteins (like U3 RNA) are essential and have been shown to be specifically required for the maturation of 18S rRNA [39][40][41]43,44,46,47 personal communication].…”

“…Lubben et al analyzed deletion mutants of human U3 RNA and determined that a central region of the RNA (nt 97-204) was important for binding of a complex that included CHO U3-55k in vitro (29). The cDNA for the U3-55k protein was subsequently isolated from a human teratocarcinoma library and the protein was characterized in HeLa cells (45). Pluk et al found that human U3-55k (hU3-55k) co-localized with fibrillarin in nucleoli and showed the association of hU3-55k with U3 in vivo (45).…”

“…The cDNA for the U3-55k protein was subsequently isolated from a human teratocarcinoma library and the protein was characterized in HeLa cells (45). Pluk et al found that human U3-55k (hU3-55k) co-localized with fibrillarin in nucleoli and showed the association of hU3-55k with U3 in vivo (45). In addition, characterization of several hU3-55k mutants revealed that replacement of 17 amino acids at the C-terminus disrupted localization of the protein to nucleoli and the ability of U3-55k to associate with U3 snoRNA in vivo (45).…”