Our findings here demonstrate that SMN deficiency causes profound changes in cellular RNA metabolism. It alters the repertoire of snRNAs and perturbs pre-mRNA splicing, leading to numerous splicing defects. The defects are widespread and cell-type specific, affecting mRNAs of functionally diverse genes. These surprising discoveries place the SMN complex, the essential machinery for the biogenesis of snRNPs, as a major factor in splicing regulation. Furthermore, they provide an example of how a deficiency in a ubiquitous protein can result in tissue-specific changes, and cast a new light on the pathophysiology of SMA.
A large degree of SMN decrease (> 80%) is required to cause a significant change in the levels of snRNAs, or cause cell death in cultured cells, suggesting that cells normally contain a large excess capacity of SMN complex to maintain their normal inventory of snRNAs. While a reduction in the amount of snRNAs upon decrease in SMN was not unexpected, its non-uniformity across different snRNAs and its cell type-specificity were unanticipated. Immunoprecipitation of snRNPs from total extract, which could be reliably performed from tissue cultured cells but not from mouse tissues, showed a similar stoichiometry for snRNAs in total RNA preparations and in snRNPs, consistent with previous observations that the pool of unassembled snRNAs is very small, and suggest that the cells have a similarly altered repertoire of snRNPs. The biogenesis of snRNPs is a highly regulated and intricate stepwise process, and the SMN complex is essential for the key step of the Sm core assembly on which all subsequent steps depend (Battle et al., 2006a
; Massenet et al., 2002
; Narayanan et al., 2002
; Will and Luhrmann, 2001
). The steady state level of each snRNA is determined by the gene copy number of that snRNA and by many other factors, including its rate of transcription, processing, transport, assembly, modifications and turnover, as well as availability of both the common and snRNA-specific proteins. Many of these factors could be regulated differently in different cell types and at various stages of development. It is also possible that the SMN complex, in addition to its direct function in Sm core assembly, also plays a role in other steps of snRNP biogenesis and snRNA metabolism. Little is known about the regulation of the biogenesis and turnover of snRNPs in different tissues, and it will be of interest to study these processes under both normal and SMN deficient conditions.
The splicing changes we observe are widespread, affecting hundreds of genes in different tissues of SMN-deficient mice. Different splicing changes occur in different tissues, but the same splicing changes are observed independently in the same tissue in each of the individual SMN-deficient mice. The very high confirmation rate of changes identified by the exon array and the validation of numerous specific cases by RT-PCR, attest to the high reliability of the data set we obtained. Using the stringent criteria we applied to selection of the affected transcripts, only a fraction of genes for which robust signals were obtained showed splicing perturbations (< 2% of genes). However, the number of affected transcripts is likely to be much higher, because the signals for any given exon represent the average for all transcripts in the tissue. To be detected, a significant change in the signal of an exon would have to occur in a large fraction of the transcripts that contain it. Thus, an aberrant splicing of an exon could be severe in some or even all of the transcripts that contain it in a specific cell population and may not be detectable in the total tissue sample. It is possible that a specific splicing defect that is detrimental to motor neurons occurs but was not detected in the total RNA sample from spinal cord. The lack of an effect on the level of the majority of the transcripts nevertheless indicates that general Pol II transcription and mRNA turnover have not been significantly affected, consistent with cDNA expression microarray experiments using different SMA mouse models with milder phenotypes (Balabanian et al., 2007
; Olaso et al., 2006
). In many cases, the aberrant splicing generates an mRNA that contains a premature termination codon. These transcripts are present only at very low levels probably because they were subject to nonsense-mediated decay.
Importantly, although some of the changes we observe in specific exons may correspond to shifts in alternative splicing patterns, most of them are not; they are abnormal RNA processing events that give rise to aberrant splicing products, which normally are not produced. We conclude this because many of the exons that are significantly changed in the SMA mice, particularly exon skipping events, are not involved in known alternative splicing events, such as Hif3a (), Col5a1 and Uaca (Figure S6
). In addition, it is not possible to distinguish based on the available data whether all copies of an affected transcript where more than one exon is aberrantly spliced have the same defect or whether the defects are distributed among many different transcripts.
The mechanistic basis for the widespread splicing defects caused by changes in the levels of SMN, hitherto known to function in the biogenesis of snRNPs, remains to be determined. Splicing is dependent both on snRNPs and RNA-binding proteins (hnRNPs and SR proteins). Knockdowns of mRNA processing factors, including spliceosomal protein components, have been shown to have differential effects on the splicing of a subset of pre-mRNAs in yeast (Clark et al., 2002
; Pleiss et al., 2007
) and on alternative splicing of a few pre-mRNAs in Drosophila (Park et al., 2004
). Rather than depletion of an individual splicing factor, deficiency in SMN, which is not itself a component of the splicing machinery, causes changes in multiple snRNAs, leading to an altered snRNP repertoire. Despite this, there is no reason to believe that the composition of snRNPs within each spliceosome is altered in SMN-deficient cells. Importantly, while changes in the stoichiometry of snRNPs have not been previously associated with global regulation or defects in splicing, our observations merit consideration of such a link. Like the major spliceosomal snRNPs, many of the hnRNP and SR proteins that play a role in splicing are extremely abundant and present in vast excess over their high-affinity binding sites on pre-mRNAs (Dreyfuss et al., 2002
; Dreyfuss et al., 1993
), yet even moderate changes in their relative stoichiometry have significant effects on alternative splicing patterns (Black, 2003
; Hou et al., 2002
; Kashima and Manley, 2003
; Licatalosi and Darnell, 2006
; Martinez-Contreras et al., 2006
; Paradis et al., 2007
; Wang and Manley, 1995
; Zhu et al., 2001
). The complexity of the network of interactions among the snRNPs and between snRNPs and the enormous assortment of splicing factors precludes meaningful prediction of specific splicing outcomes based on a particular snRNA repertoire, at this time. However, we suggest that a change in the stoichiometry of snRNPs perturbs this network and affects the efficiency, rate and fidelity of spliceosome assembly on different introns. Because each cell type has a unique assortment of splicing factors and SMN deficiency causes its snRNP repertoire to change in a unique way, the resulting perturbations are distinct and give rise to cell-type specific effects on splicing. We note, however, that alternative mechanisms cannot be ruled out. For example, the SMN complex could have an snRNA-independent effect on splicing, as experiments with an SMN mutant in vitro
have suggested (Pellizzoni et al., 1998
), or in other aspects of RNA metabolism. Another possible snRNA-dependent scenario for the splicing perturbations is that some unassembled snRNAs accumulate in SMN-deficient cells, which might then sequester snRNP proteins or other proteins required for splicing. The important conclusion nevertheless remains, that the stoichiometry of the major constituents of the splicing machinery (the “snRNPertoire”) is altered and that transcripts reflecting a faulty splicing process accumulate in SMN-deficient cells.
Further studies will be required to uncover the specific mechanism at fault for particular transcripts out of the hundreds or thousands that are affected. The splicing changes we observe in SMN deficiency appear to reflect a general defect in splicing and do not resemble the splicing changes that result from modulation of specific splicing factors (Black, 2003
; Caceres et al., 1994
; Licatalosi and Darnell, 2006
; Ule et al., 2005
; Wang and Manley, 1995
). However, examination of the affected transcripts reveals several striking features. Pre-mRNAs containing a large number of introns are much more likely to suffer aberrant splicing, suggesting that the likelihood of splicing defects increases with the number of splicing events the transcript undergoes. We note, in addition, that a considerable number of the aberrantly spliced transcripts have more than one splicing abnormality (see ). This appears to exceed the probability of random events and suggests that the affected transcripts have a specific susceptibility to the suboptimal splicing machinery in SMN-deficient cells and that a splicing defect in one intron may be propagated to other introns in the same transcript. We suggest that having a large number of introns makes a transcript more vulnerable to splicing defects upon decreased SMN complex activity, presenting a challenge to evolutionarily more advanced organisms possessing many multi-intron genes. Classification of the aberrantly spliced transcripts by gene ontology indicates that proteins of diverse functions are affected, and reveals a very high preponderance of membrane transporters and extracellular matrix proteins (). Further analysis of the affected transcripts will be required to determine if there are sequence motifs common to these RNAs that cause their splicing defects. Nevertheless, it is apparent that many of the affected transcripts contain a large number of introns which likely explain their susceptibility.
A new view of SMA emerges from these findings. The selective degeneration of motor neurons is a characteristic feature of SMA, and several potential explanations for this pathogenesis have been suggested, including the possibility of a specific function for SMN in these cells (Monani, 2005
). However, our findings that SMN deficiency causes cell type-specific perturbations in the composition of the major components of the spliceosome and a defective transcriptome throughout all cells and tissues, not only motor neurons, indicate that SMA is a general splicing disease. An unanticipated alternative explanation for the motor-neuron selective phenotype in SMA may therefore be that cell-specific factors that influence the relative abundance of individual snRNPs, rather than a cell-specific function of SMN itself, account for the cell-specific phenotype, but their effect is manifested only when SMN, the common factor in the general pathway, becomes limiting. The attrition of motor neurons may be caused by one or more aberrant transcripts or by the cumulative effect of many splicing defects. However, it now seems possible, that splicing defects in surrounding cells, such as those that make up the matrix on which motor neurons depend, may be the cause or a contributing factor to the demise of the motor neurons. As the severity of SMA is directly correlated with the degree of SMN deficiency, it is likely that even moderate reductions in SMN levels alter the snRNP repertoire and perturb splicing, albeit more subtly than those we have studied here. Together, the observations we describe here establish a key role for the SMN complex in gene regulation, particularly in the maintenance of splicing machinery.