Previous experiments have demonstrated that the SMN complex is required for snRNP assembly. Specifically, complete or nearly complete removal or inhibition of the SMN complex results in the inhibition of Sm core assembly in vitro (32
). Here, using an in vitro assay we developed for the quantitative measurement of the Sm core assembly process in cell extracts, we show that there is a linear correlation between the amount of SMN present in the cell extract and the amount of Sm cores that can be formed on specific RNA substrates, suggesting that the amount of SMN determines the capacity for Sm core assembly. SMA results from a reduction in the amount of the full-length SMN protein (6
). Studies on a collection of SMA patient cell lines revealed that SMN expression is more reduced in the severe form (type I) than the mild form (type III) of the disease, demonstrating a direct correlation between the degree of reduction of SMN protein levels in SMA patients and the severity of their clinical phenotypes (7
). However, an understanding of the molecular consequences of the reduced levels of SMN in patients' cells has been lacking.
Our finding that snRNP assembly is impaired in cells of SMA patients provides the first direct evidence for a biochemical deficiency, namely, reduced capacity to assemble Sm cores, in SMA patients. Due to a limited availability of SMA patient cells, our studies focused on the severe type I SMA patients. However, the correlation we find is strong, exhibiting direct proportionality between the amount of the SMN protein and assembly capacity in vitro. Consistent with the reduced activity observed in extracts of cells with low SMN, the rate of production of snRNPs in these cells is profoundly reduced.
Although the overall amount of the major snRNPs in the same cells is not reduced, the strong deficiency in their rate of accumulation could be detrimental to cells in several ways. For dividing cells, biosynthetic capacity, such as the overall translation rate, has been linked to the control of cell growth and division (17
). Similarly, the slower rate of snRNP accumulation could cause a delay in cell cycle progression. This is indeed the case for S5 cells where the growth rate slows down proportionally to the reduction in SMN (45
). In nondividing cells, such as motor neurons, an unmet demand for timely snRNP production at a particular point in the growth and development of the cell could have severe consequences for the cell. It could lead to a deficit in functions that depend on an adequate amount of the major snRNPs (e.g., a general decrease in pre-mRNA splicing or an altered processing pattern of pre-mRNAs that are required by that cell), or to a deficit in a specific snRNP (e.g., an RNA of a lower abundance or one that has a lower affinity for the SMN complex). It is also possible that reduced amounts of the SMN complex result in some loss of the regulation of Sm core assembly, leading to loss of fidelity of Sm core assembly, such that Sm cores assemble on RNAs that are not supposed to receive them. This could be harmful to cells, as it may interfere with the normal functions of these RNAs or cause them to aggregate. We note that the experiments we carried out here examined only the formation of the major snRNPs, and it is therefore possible that the formation of minor snRNPs, motor neuron-specific snRNPs, or other RNAs that require the SMN complex for RNP assembly is strongly affected by the reduction in SMN.
The method we describe here is sensitive, quantitative, easy to set up, and readily suitable for laboratory automation. Unlike the methods commonly used so far, such as RNP gel shift and anti-Sm immunoprecipitation followed by gel electrophoresis, this assay does not require radioactive labeling of the RNAs, nor does it require gel electrophoresis. The biotinylated RNA substrates used for the assay can be prepared ahead of time and stored for many months, allowing off-the-shelf and long-term use. The assay should facilitate detailed studies of the mechanisms of action of the SMN complex and on the process of Sm core assembly. Combined with other approaches, such as RNA interference to systematically reduce protein expression, it should now be possible to determine the roles of the individual Gemins and other components of interest in the assembly reaction. The assay can also be used on extracts from patient cells. The sensitivity of the assay and its colinearity with the amount of SMN make it suitable as an additional measure for the characterization of SMA and as a means to identify potential modifiers, both genetic and pharmacological, of the disease phenotype.