Specific Sequences of the Sm and Sm-like (Lsm) Proteins Mediate Their Interaction with the Spinal Muscular Atrophy Disease Gene Product (SMN)

Abstract

The spinal muscular atrophy disease gene product (SMN) is crucial for small nuclear ribonuclear protein (snRNP) biogenesis in the cytoplasm and plays a role in pre-mRNA splicing in the nucleus. SMN oligomers interact avidly with the snRNP core proteins SmB, -D1, and -D3. We have delineated the specific sequences in the Sm proteins that mediate their interaction with SMN. We show that unique carboxyl-terminal arginine-and glycine-rich domains comprising the last 29 amino acids of SmD1 and the last 32 amino acids of SmD3 are necessary and sufficient for SMN binding. Interestingly, SMN also interacts with at least two of the U6-associated Sm-like (Lsm) proteins, Lsm4 and Lsm6. Furthermore, the carboxyl-terminal arginine-and glycine-rich domain of Lsm4 directly interacts with SMN. This suggests that SMN also functions in the assembly of the U6 snRNP in the nucleus and in the assembly of other Lsm-containing complexes. These findings demonstrate that arginineand glycine-rich domains are necessary and sufficient for SMN interaction, and they expand further the range of targets of the SMN protein.Spinal muscular atrophy (SMA) 1 is a neuromuscular disease that results in muscular weakness and atrophy due to degeneration of motor neurons of the spinal cord (1-4). Over 98% of SMA patients have mutations or deletions of the Survival Motor Neuron (SMN1) gene, which is present as an inverted repeat on chromosome 5 at 5q13 (5-7). Only deletions or mutations in the telomeric copy of SMN (SMN1) lead to SMA (7)(8)(9)(10)(11)(12)(13)(14). The centromeric copy of the gene (SMN2) produces mostly an alternatively spliced form of SMN deleted of amino acids encoded by exon 7 (SMN⌬Ex7), and SMN2 can only partially compensate for mutations or deletions in SMN1 (7,15,16). Indeed, some patients, instead of complete deletion of SMN1, have shorter deletions of at least exon 7 or single point mutations within the conserved YG domain (17,18).As measured by coimmunoprecipitation from cytoplasmic extracts, SMN exists in a complex with Sm proteins (19), Gemin2 (formerly SIP1) (19), Gemin4 (20), and the DEAD box RNA helicase Gemin3 (21). The amino terminus of SMN tightly associates with Gemin2, whereas the carboxyl-terminal conserved YG domain is necessary for self-association and interaction with the core snRNP Sm proteins (19,22). We have recently demonstrated that oligomerization of SMN through the conserved YG domain is required for efficient interaction with SmB, -D1, and -D3 and suggested that upon oligomerization a high affinity Sm protein binding site is formed (23).SnRNPs are formed in the cytoplasm where the core Sm proteins bind to the Sm site on U snRNA, and following hypermethylation of the m 7 G cap to a 2,2,7-trimethyl-gaunosine (m 3 G) cap the snRNP is transported to the nucleus (24 -30). Using oocyte injections, it was demonstrated that the SMN⅐Gemin2 complex plays a role in spliceosomal snRNP assembly that takes place in the cytoplasm. Injection of Gemin2-specific monoclonal antibodies inhibited, whereas injection of SMN-specifi...

Direct Interaction of the Spinal Muscular Atrophy Disease Protein SMN with the Small Nucleolar RNA-associated Protein Fibrillarin

“…4 and 5), and other studies indicate that SMN also interacts with hnRNP U (34), several Sm and Sm-like (Lsm) proteins (23,36,41,44,62), and RNA helicase A (30) via glycine/arginine-rich domains. On the other hand, the interaction between the Caenorhabditis elegans SMN and fibrillarin homologs in a yeast two-hybrid system was found to be independent of the GAR domain of fibrillarin (63).…”

“…This finding is of potential importance to our understanding of snoRNP biogenesis as well as the etiology of spinal muscular atrophy. SMN interacts with fundamental components of multiple nuclear RNA-protein complexes as follows: spliceosomal snRNPs (23,35,36,41,44), Box C/D snoRNPs (this work), and also Box H/ACA snoRNPs. 3 The interaction of SMN with each of these RNPs occurs by a similar mechanism involving the Tudor domain of SMN (Refs.…”

“…Full-length, in vitro translated fibrillarin was incubated with each of the tagged SMN truncation mutants. It was recently shown that the conserved YG box of SMN (encoded by exons VI and VII) mediates the interaction of SMN with other proteins rich in glycine and arginine (Sm and Lsm proteins) and that the interaction is disrupted by a point mutation found in some SMA patients, Y272C (36,44). However, the C-terminal half of SMN including the YG box (159/294) failed to interact with fibrillarin (as did this same region containing the Y272C point mutation) (Fig.…”

The C-terminal domain of coilin interacts with Sm proteins and U snRNPs

“…Previously, we showed that the C-terminal 214 amino acid (aa) residues of coilin are sufficient for interaction with SmB′, making coilin a candidate (Hebert et al 2001). Since we had not previously defined the region of SmB′ that interacts with coilin, we were specifically interested in assessing if coilin binds SmB′ at a location distinct from SMN, which predominantly binds to the RG-rich tails of Sm proteins (Brahms et al 2001;Friesen and Dreyfuss 2000;Selenko et al 2001). We therefore generated two His-T7-tagged SmB′ truncations; one truncation deletes all of the RG dipeptides found in the C-terminal tail, whereas the other construct leaves approximately half of them.…”

Tudor and its domains: germ cell formation from a Tudor perspective

“…Tudor domains have highly conserved amino acids and predicted structure, which suggests that the structure of the Tudor domain in SMN is likely to be very similar to those found in other Tudor domain containing proteins. SMN binds to two SM proteins, SmD1 and SmD3, at their carboxy-terminal ends, which are arginine-and glycine-rich domains [39]. Methylation of the carboxy-terminal ends of SmD1 and SmD3 enhances the binding ability of SMN by several folds.…”