The survival of motor neuron (SMN) protein plays an important role in the biogenesis of spliceosomal snRNPs and is one factor required for the integrity of nuclear Cajal bodies (CBs). CBs are enriched in small CB-specific (sca) RNAs, which guide the formation of pseudouridylated and 29-O-methylated residues in the snRNAs. Because SMN-deficient cells lack typical CBs, we asked whether the modification of internal residues of major and minor snRNAs is defective in these cells. We mapped modified nucleotides in the major U2 and the minor U4atac and U12 snRNAs. Using both radioactive and fluorescent primer extension approaches, we found that modification of major and minor spliceosomal snRNAs is normal in SMN-deficient cells. Our experiments also revealed a previously undetected pseudouridine at position 60 in human U2 and 29-O-methylation of A1, A2, and G19 in human U4atac. These results confirm, and extend to minor snRNAs, previous experiments showing that scaRNPs can function in the absence of typical CBs. Furthermore, they show that the differential splicing defects in SMN-deficient cells are not due to failure of post-transcriptional modification of either major or minor snRNAs.

During the early steps of snRNP biogenesis, the survival motor neuron (SMN) complex acts together with the methylosome, an entity formed by the pICln protein, WD45, and the PRMT5 methyltransferase. To expand our understanding of the functional relationship between pICln and SMN in vivo, we performed a genetic analysis of an uncharacterized Schizosaccharomyces pombe pICln homolog. Although not essential, the S. pombe ICln (SpICln) protein is important for optimal yeast cell growth. The human ICLN gene complements the ⌬icln slow-growth phenotype, demonstrating that the identified SpICln sequence is the bona fide human homolog. Consistent with the role of human pICln inferred from in vitro experiments, we found that the SpICln protein is required for optimal production of the spliceosomal snRNPs and for efficient splicing in vivo. Genetic interaction approaches further demonstrate that modulation of ICln activity is unable to compensate for growth defects of SMN-deficient cells. Using a genome-wide approach and reverse transcription (RT)-PCR validation tests, we also show that splicing is differentially altered in ⌬icln cells. Our data are consistent with the notion that splice site selection and spliceosome kinetics are highly dependent on the concentration of core spliceosomal components. In eukaryotes, an essential step in the production of functional mRNAs is the spliceosome-mediated removal of introns from pre-mRNAs. This machinery is composed of 5 spliceosomal snRNPs and additional non-snRNP-associated factors (1, 2). The biogenesis of these snRNPs is an ordered multistep process. After transcription, the m 7 G-capped snRNAs are exported to the cytoplasm, where they bind to the seven Sm proteins SmB/B=, SmD1, SmD2, SmD3, SmE, SmF, and SmG. Accurate assembly of the Sm core domain is required for subsequent m 3 G cap formation, which is followed by the active transport of snRNPs to the nucleus (3).Although formation of the snRNP core can occur spontaneously in vitro, the process is highly regulated in vivo, and the survival motor neuron (SMN) protein, encoded by the survival motor neuron (SMN1) gene, is a major player in these preliminary assembly steps (4-6). Mutations in SMN1 cause the autosomal recessive disease spinal muscular atrophy (SMA) (7). The SMN protein forms a stable complex with a group of proteins called gemins and is found in the cytoplasm, as well as the nuclei, of cells, where it is enriched within discrete bodies called Cajal bodies (8,9). During the cytoplasmic step of snRNP biogenesis, the SMN complex interacts with the methylosome, a complex formed by the pICln and WD45 proteins and the PRMT5 methyltransferase (10-12). The methylosome recruits Sm proteins via the pICln subunit, and PRMT5 allows the symmetric dimethylation of arginines within the C tails of SmB, SmD1, and SmD3 (13, 14). The SMN complex further facilitates the loading of Sm proteins onto the snRNA, resulting in the formation of a basic snRNP particle (15,16). In this process, pICln acts as an assembly chaperone and SMN acts ...

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