Fibrillarin is one of four proteins known to interact selectively with all Box C/D family small nucleolar (sno) 1 RNAs (1-6). In eukaryotes, large numbers of snoRNAs direct cleavages and specific nucleotide modifications of pre-rRNA that are required for ribosome biogenesis (7-11). Most Box C/D family snoRNAs guide the site-specific 2Ј-O-methylation of rRNA (12,13). snoRNAs function as RNA-protein complexes known as small nucleolar ribonucleoprotein particles (snoRNPs) (11,14).Fibrillarin has sequence and structural homology to known methyltransferases (15, 16). This observation, coupled with functional studies in yeast (17), has led to the hypothesis that fibrillarin is the catalytic factor in Box C/D snoRNA-directed 2Ј-O-methylation of ribosomal RNA.The survival motor neuron (SMN) protein is linked with one of the most common inheritable causes of childhood mortality, spinal muscular atrophy (SMA) (18 -21). The SMN1 gene is deleted or mutated in patients with SMA (22) resulting in loss of spinal motor neurons accompanied by progressive muscular atrophy.SMN has been implicated in an array of cellular pathways. Antibody inhibition experiments have demonstrated that SMN is required for the biogenesis of spliceosomal small nuclear RNPs (snRNPs) (23, 24), and studies using in vitro splicing systems and yeast mutants have demonstrated a key role for SMN in pre-mRNA splicing (25-27). SMN has also been implicated in regulation of gene expression at the transcriptional level (28,29), in the assembly of the polymerase II transcription machinery (30), and as a neuron-specific anti-apoptotic factor (31-33).To learn more about snoRNP biogenesis and structure, we performed a yeast two-hybrid screen for proteins that interact with Xenopus fibrillarin. We identified the survival motor neuron (SMN) gene multiple times in screens of both Xenopus and human cDNA libraries. Fibrillarin had been detected previously in a two-hybrid screen using SMN (34). We have now demonstrated an in vivo interaction between SMN and fibrillarin, and we show that the two proteins interact directly in vitro. We have mapped the domains of each protein responsible for the interaction, and we found that SMN interacts with fibrillarin and the SmB snRNP protein via the same domain. Our findings suggest a function for SMN in the biogenesis and/or function of snoRNPs (similar to its established role with snRNPs (23-26, 35, 36)).EXPERIMENTAL PROCEDURES

Deletion or mutation of the SMN1 (survival of motor neurons) gene causes the common, fatal neuromuscular disease spinal muscular atrophy. The SMN protein is important in small nuclear ribonucleoprotein (snRNP) assembly and interacts with snRNP proteins via arginine/glycine-rich domains. Recently, SMN was also found to interact with core protein components of the two major families of small nucleolar RNPs, fibrillarin and GAR1, suggesting that SMN may also function in the assembly of small nucleolar RNPs. Here we present results that indicate that the interaction of SMN with GAR1 is mediated by the Tudor domain of SMN. Single point mutations within the Tudor domain, including a spinal muscular atrophy patient mutation, impair the interaction of SMN with GAR1. Furthermore, we find that either of the two arginine/glycine-rich domains of GAR1 can provide for interaction with SMN, but removal of both results in loss of the interaction. Finally, we have found that unlike the interaction of SMN with the Sm snRNP proteins, interaction with GAR1 and fibrillarin is not enhanced by arginine dimethylation. Our results argue against post-translational arginine dimethylation as a general requirement for SMN recognition of proteins bearing arginine/glycine-rich domains.The best established role of SMN, the protein implicated in spinal muscular atrophy, is in the assembly of small nuclear RNA (snRNA) 1 -protein complexes that function in pre-mRNA splicing. SMN interacts with the common snRNP proteins Sm B/BЈ, D1 and D3, and LSm 4 through arginine/glycine (RG)-rich domains present in these proteins (1-4). Several studies have demonstrated that SMN and the other proteins of the SMN complex are required to facilitate the assembly of snRNPs and the generation of active spliceosomes (5-11).SMN appears to interact with a series of cellular proteins via a common mechanism that depends on the RG-rich domains in the target proteins (reviewed in Ref. 12). In addition to the Sm and LSm snRNP proteins, SMN has been found to interact with RNA helicase A (13), a protein associated with RNA polymerase II; coilin (14), the signature component of nuclear Cajal bodies; heteronuclear RNP binding proteins Q, R, and U (9, 15, 16); and fibrillarin and GAR1 (17, 18), core protein components of the Box C/D and Box H/ACA snoRNPs, respectively. Most of these proteins contain a single RG-rich region that has been shown to be essential for association with SMN. GAR1 has two RG-rich domains, and interestingly, it has been reported that both of these are necessary for interaction (18). Current evidence supports two different models for the basis of the interaction of SMN with partner proteins. One series of studies indicates that sequences near the carboxyl terminus of SMN mediate the interactions (including the interaction with GAR1) (2,11,13,16,18). This domain of SMN is important for the oligomerization of SMN (11,19,20). Other laboratories have implicated the Tudor domain of SMN in its interaction with some of the same RG domain proteins (3,4,7,14,17). ...

U3 small nucleolar RNA (snoRNA) is a member of the Box C/D family of snoRNAs which functions in ribosomal RNA processing. U3-55k is a protein that has been found to interact with U3 but not other members of the Box C/D snoRNA family. We have found that interaction of the U3-55k protein with U3 RNA in vivo is mediated by the conserved Box B/C motif which is unique to U3 snoRNA. Mutation of Box B and Box C, but not of other conserved sequence elements, disrupted interaction of U3-55k with U3 RNA. Furthermore, a fragment of U3 containing only these two conserved elements was bound by U3-55k in vivo. RNA binding assays performed in vitro indicate that Box C may be the primary determinant of the interaction. We have cloned the cDNA encoding the Xenopus laevis U3-55k protein and find strong homology to the human sequence, including six WD repeats. Deletion of WD repeats or sequences near the C-terminus of U3-55k resulted in loss of association with U3 RNA and also loss of localization of U3-55k to the nucleolus, suggesting that protein-protein interactions contribute to the localization and RNA binding of U3-55k in vivo.