We have demonstrated that pICln binds to Sm proteins by several independent methods. First, microsequencing of proteins purified by affinity to pICln identified two pICln-binding proteins as SmB/B′ and SmD3. Second, immunoprecipitation of Sm proteins coimmunoprecipitated pICln. Third, a bait containing pICln extracted SmD3 from a cDNA library in a yeast two-hybrid screen. Fourth, purified GST-ICln bound to in vitro-translated SmB′, SmD1, and SmD3. Finally, proteins which coimmunoprecipitated with pICln from MDCK cell cytoplasmic extracts had electrophoretic mobilities appropriate for SmB/B′ and SmD1 and SmD3. Although these experiments do not define which Sm proteins directly bind to pICln, they clearly demonstrate that Sm proteins and pICln form a complex. Furthermore, this interaction is likely to be functionally important, since immunodepletion experiments showed that in MDCK cells the majority of cytoplasmic Sm protein (or at least SmB/B′) is bound to pICln.
Binding of Sm proteins to pICln inhibited their assembly on U RNA to form the Sm core domain, as determined by immunoprecipitation and mobility shift assays. By inhibiting Sm core domain assembly on U RNA, high concentrations of pICln interfered with snRNP nuclear import in Xenopus
oocytes. At the maximal GST-xICln concentration tested (5.5 μM), there was a 2.5-fold inhibition of nuclear import. Although this concentration of pICln was 25-fold higher than the normal oocyte pICln concentration (0.2 μM [15
]), this was expected given the high concentration of Sm proteins in the oocyte cytoplasm. In previous studies, 1.5 to 15 μM U1 RNA was required to completely bind the endogenous cytoplasmic pool of free Sm proteins (4
). Using this as a minimal estimate of the oocyte cytoplasmic Sm protein concentration, the concentration of pICln necessary for effective inhibition of nuclear import was reasonable if inhibition required stoichiometric binding of pICln to Sm proteins.
Although lack of U RNA, rather than the presence of pICln, is likely the primary factor which regulates snRNP assembly in oocytes (36
), the nuclear import assay demonstrates clearly a functional consequence of pICln binding to Sm proteins. In contrast to oocytes, in the majority of mammalian cells the cytoplasmic Sm concentration is low and pICln is relatively abundant, and the majority of cytoplasmic Sm is bound to pICln. Under these conditions, pICln may play an important role in the regulation of snRNP assembly.
Much has been learned recently about the assembly pathway of the Sm core domain. The Sm proteins have been found in three stable RNA-free complexes (D1-D2, E-F-G, and D3-B/B′) (5
). The D1-D2 and E-F-G Sm protein complexes can together form a stable complex with U RNA. This subcore particle then binds the D3-B/B′ complex to form the complete core domain (33
). Our finding that pICln binding to complexes containing SmB/B′, D1, and D3 interfered with the ability of Sm proteins to associate with U RNA is consistent with this model.
We have also found that pICln binding to Sm proteins inhibited their association with SMN protein. This protein forms a complex with the protein SIP1, and the SMN-SIP1 complex binds to SmB/B′, D1-3, and E (20
). Antibodies directed against SIP1 blocked assembly of the Sm core domain on U RNA, while anti-SMN antibodies enhanced this process. These results implicate the SMN-SIP1 complex in Sm core domain assembly and suggest that by binding Sm proteins, the complex may facilitate their binding to U RNA (5
). In combination with our results, these data lead us to propose a model in which pICln inhibits Sm protein assembly on U RNA, at least in part by inhibiting Sm protein interaction with SMN (Fig. ). Consistent with this model, U1 and U5 RNAs efficiently coimmunoprecipitated with SIP1 or SMN and were most inhibited by pICln, while U2 and U4 RNAs were weakly coimmunoprecipitated with SIP1 or SMN (5
) and were relatively insensitive to pICln (Fig. and ). Although this model is compatible with the existing data, alternative models are possible. For instance, we have depicted SMN as an integral component of the assembly pathway, whereas it may actually be a regulatory element. SMN and pICln both influence snRNP biogenesis at the level of Sm core domain assembly, but a role for these proteins in regulating other steps in the biogenesis pathway has not been excluded.
FIG. 8 Model of pICln and SMN regulation of Sm protein assembly on U RNA. pICln is depicted as inhibiting Sm core domain assembly and U RNA nuclear import by preventing Sm interaction with SMN. Hypermethylation and nuclear import of U RNA occur after Sm core (more ...)
In addition to promoting snRNP biogenesis, SMN may also be essential for proper spliceosome function, since a dominant-negative SMN mutant inhibits pre-mRNA splicing in vitro and causes dramatic nuclear snRNP reorganization in vivo (32
). The inhibitory effect of the dominant-negative mutant on in vitro splicing was observed only when the SMN mutant was preincubated with splicing extract prior to addition of pre-mRNA (32
). One interpretation of these results is that splicing results in snRNP rearrangement to an inactive form and that SMN is necessary for the regeneration of functional snRNPs. Since pICln is also present in the nucleus, pICln could potentially regulate snRNP recycling by modulating the interaction of SMN with snRNPs. In keeping with the participation of pICln in a critical cellular function, loss of pICln in mice results in embryonic lethality between 3.5 and 7.5 days postcoitus, and embryonic stem cells lacking both pICln alleles cannot be generated in tissue culture (35
In summary, we have described a novel interaction between pICln and several Sm proteins, and we have demonstrated that this interaction inhibits association of Sm proteins with SMN, Sm protein binding to U RNA, and snRNP biogenesis. We have presented a model that accounts for these observations. Experiments are in progress to test this model and to determine the mechanism by which loss of pICln leads to embryonic lethality.