A major goal in SMA research has been to identify approaches to improve expression of SMN protein from SMN2 for the treatment of the disease. Increasing SMN2 exon 7 splicing has been studied intensely as a means to elevate full-length SMN protein levels in SMA. One key question is how much of an increase in SMN2 exon 7 splicing is required for therapeutic value. Because SMN protein itself functions in the pre-mRNA splicing pathway, it is important to understand how the protein may influence splicing of its own pre-mRNA. We now demonstrate that the abundance of SMN protein determines, in part, the outcome of SMN2 alternative splicing. The discovery of a feedback loop in SMN protein expression has important implications for SMA by suggesting that treatment strategies that lead to modest increases in SMN protein may have significant therapeutic value for the disease.
The SMN feedback loop likely involves the key function of SMN in the snRNP assembly. We (Figs and ) and others (13
) have found that some spliceosomal snRNA species decrease, following a reduction in the SMN protein abundance. This change in snRNAs consequently lowers snRNP abundance and correlates with changes in alternative splicing of a number of gene transcripts, presumably due to a change in the relative concentration of snRNPs (14
). It is not clear how changes in the relative abundance of snRNPs lead to changes in alternative splicing. Using an in vitro
splicing assay, we tested directly the possibility that subtle changes in the relative abundance of the snRNPs can have effects on alternative splicing of SMN2
exon 7 (Fig. ). We found that altering the abundance of individual snRNPs in vitro
had dramatically differing effects on exon 7 splicing, demonstrating that shifts in the balance of snRNPs can regulate alternative splicing (Fig. ). Our results suggest that a change in the relative amount of individual snRNPs alters SMN2
exon 7 splicing, which, in turn, affects SMN protein levels which feeds back to further disrupt snRNP abundance.
exon 7 splicing is particularly sensitive to U1 snRNP levels. We present data showing that U1 snRNA is reduced when SMN protein abundance is low (Figs and ) and that exon 7 splicing decreases following a decrease in U1 snRNP activity or abundance (Figs –). U1 snRNP recognizes the 5' splice site through direct base-pairing interactions between the pre-mRNA and the snRNA (55
). U1 snRNP binding is an early determinant of splice site selection (56
). Thus, the 5' splice site sequence, and likely also the abundance of U1 snRNP, plays an important role in the initial recognition of the site. 5' splice sites with weak base-pairing potential to U1 snRNA are not recognized as efficiently as those with high base-pairing potential (58
). The efficiency of 5' splice site selection is important when 5' splice sites are in competition with each other in alternatively spliced exons. For example, in the case of SMN2
, the 5' splice site of exon 7 is an important determinant of exon inclusion (36
). Exon skipping will occur if exon 6 splicing to exon 8 occurs before exon 7 can splice to exon 8. Improvement of the base-pairing potential of its 5' splice site to U1 improves exon 7 splicing, likely due, in part, to its improved ability to compete with the 5' splice site of exon 6 for splicing to exon 8. We observed that a decrease in U1 snRNP abundance in vitro
results in a decrease in exon 7 inclusion relative to exon skipping (Fig. ). This may be due to the weak exon 7 5' splice site, which is further weakened under conditions of low U1 snRNP. This site may also not be strong enough to promote exon definition across exon 7 for enhancement of the 3' splice site of exon 7, which has also been demonstrated to be a relatively weak splice site (22
). Limiting U1 snRNP may further weaken exon 7 definition, thereby lowering the occurrence of splicing from the 5' splice site of exon 6 to the 3' splice site of exon 7. In this case, splicing from exon 6 to the 3' splice site of exon 8 may be selected over the exon 7 3' splice site, resulting in exon 7 skipping.
Interestingly, targeted reduction of individual snRNAs had differential effects on exon 7 splicing. Unlike U1 snRNA depletion, exon 7 inclusion was much more resistant to the depletion of U2, U4 and U5 snRNA species in vitro
compared with skipping (Fig. ). These results suggest that exon 7 skipping and inclusion are differentially affected by alterations in the relative abundance of individual snRNPs. This phenomenon, in which the relative abundance of individual snRNPs dictates exon 7 splicing, could result in differences in the relative amount of SMN
2 exon 7 inclusion in different cell types and at different stages of development. In the case of SMA, in which SMN1
is lost, this feedback regulation of SMN2
splicing could be crucial for cell survival. It is possible that SMN2
exon 7 splicing in motor neurons is especially sensitive to alteration in snRNPs, further depleting SMN protein and contributing to motor neuron degeneration in SMA. Indeed, snRNP levels and the abundance and activity of SMN protein fluctuate in the spinal cord throughout early development (60
Modulation of individual snRNAs did not always have the expected outcome on SMN2
exon 7 splicing. The relative resistance of exon 7 inclusion to U2 snRNA depletion in vitro
(Fig. ), for example, was surprising. U2 snRNP binds to the 3'ss and is a determinant of 3'ss selection. U2 snRNP binding to the 3' splice site of exon 7 is impaired in SMN2
compared with SMN1
). Based on these results, it might be predicted that the depletion of U2 snRNP would impair SMN2
exon 7 3' splice site recognition and lead to an increase in skipping. Our results, however, are consistent with our previous finding that depletion of U2AF65 or PUF60, which recruit U2 snRNP to the 3' splice site, results in an increase in exon 7 inclusion (28
). Modulation of 3' splice site recognition may weaken the use of the distal exon 8 3' splice site and thereby improve the competitiveness of the exon 7 3' splice site for splicing. Contrary to this interpretation, depletion of the U2 snRNP protein SNRP B" resulted in an increase in exon 7 skipping (Fig. ). This difference may be due to secondary effects of U2 snRNP depletion in cells or may indicate a functional difference in U2 snRNP when the B" protein is limiting.
The effect of snRNP abundance on splicing outcome is more complex in the context of alterations of multiple snRNP species, as we observed in the iPS cells (Fig. ) and SMN knockdown experiments (Fig. ). Our in vitro results (Fig. ) indicate that a decrease in U2, U4 or U5 snRNP abundance causes an increase in exon 7 inclusion. However, in cells, a decrease in SMN protein always correlates with a decrease in exon 7 inclusion (Figs and ). It is possible that early recognition of the 5'ss by U1 snRNP is the determining step in splice site selection, and thus U1 snRNP abundance has a dominant role in exon 7 splicing.
The SMN feedback loop is likely an important regulator of SMN expression in SMA where SMN1 is mutated or deleted and thus the equilibrium of the feedback loop is disrupted and splicing of SMN2 exon 7 is reduced. The ability of a feedback loop to potentially impact endogenous SMN1 and SMN2 splicing and SMN protein production is demonstrated using the in vitro splicing assay and in experiments with the U1 snRNA decoy where both SMN1 and SMN2 exon 7 splicing were affected by alterations in the snRNP abundance (Figs and ). However, in cells, splicing of endogenous or minigene SMN1 exon 7 was not affected by changes in the SMN protein abundance. This insensitivity may be due to the high efficiency of SMN1 exon 7 splicing in combination with the relatively modest reduction in SMN protein and snRNAs. Our results suggest that the feedback loop could play a regulatory role when SMN1 is present in a non-diseased state in situations.
Regulation of exon 7 splicing could reflect an indirect effect of SMN protein on SMN2
exon 7 splicing. Reduction in SMN protein levels has been reported to result in changes in alternative splicing of a large number of transcripts (14
). Alternative splicing changes are likely to have an impact on the abundance or activity of the resulting proteins. A number of protein factors have been described that alter the splicing of SMN2
exon 7. The reduction in SMN protein may cause a change in the splicing of one of these regulators and thus indirectly alter SMN2
exon 7 splicing. Microarray experiments examining global changes in splicing in SMA mice have not revealed changes in alternative splicing of any known regulators of splicing (14
). To address this possibility more directly, we tested whether depletion of SMN protein in cells causes a change in known regulators of SMN2
exon 7 splicing and did not observe quantitative changes in the abundance of a number of these regulatory proteins including SF2/ASF, hnRNPA1, hnRNP Q/R or Tra2β1 (Figs D and B) (19
). However, we cannot rule out the possibility that the effect of lowering the SMN protein levels on exon 7 inclusion results in part from alterations in the abundance or activity of other effectors of exon 7 splicing. Nonetheless, our demonstration that alterations in snRNP levels can alter exon 7 splicing in vitro
suggests that the decrease in exon 7 splicing upon reduction of SMN protein levels is due, at least in part, to change in snRNP levels in the cell. We also provide evidence that changes in the relative abundance of core spliceosomal snRNPs can regulate alternative splicing.
Our results are a first demonstration of feedback regulation whereby an alteration in SMN protein levels controls expression of the protein itself by affecting alternative splicing of its pre-mRNA transcripts. These finding lay the groundwork for future studies to understand the degree to which an initially small increase in exon 7 splicing can result in a disproportionately larger increase in SMN protein levels. From a more broad perspective, our results indicate that the relative abundance of individual snRNPs can regulate alternative splicing.