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Protein 61K is required for the formation of the U4/U6 ́U5 tri-snRNP. ( A ) Anti-Sm ( a -Sm), anti-116K ( a -116K) and anti-60K ( a -60K) antibodies were used to immunoprecipitate snRNPs from mock-depleted (M), 61K-depleted ( D ) or 61K-depleted extract complemented with native protein 61K (Figure 4B) that was eluted
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In each round of nuclear pre-mRNA splicing, the U4/U6*U5 tri-snRNP must be assembled from U4/U6 and U5 snRNPs, a reaction that is at present poorly understood. We have characterized a 61 kDa protein (61K) found in human U4/U6*U5 tri-snRNPs, which is homologous to yeast Prp31p, and show that it is required for this step. Immunodepletion of protein 6...
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... to restore conditions that allow tri-snRNP formation. As expected, anti-116K antibodies ef®ciently precipitated re- formed U4/U6 ́U5 tri-snRNP from mock-depleted extracts (Figure 5A, lane 4). In contrast, exclusively U5 snRNPs were precipitated from 61K-depleted nuclear extracts (lane 5), indicating that the absence of protein 61K prevented tri-snRNPs from forming. Signi®cantly, addition of protein 61K to the depleted extract fully restored tri-snRNP complex formation (lane 6). Thus, protein 61K is essential for the formation of the tri-snRNP complex in vitro . The lack of stable tri-snRNP formation in the absence of protein 61K was further demonstrated by glycerol-gradient centrifugation of mock versus depleted nuclear extract (Figure 5B and C). In contrast to the mock-depleted extract where the majority of U4 and U6 snRNAs migrated as part of intact 25S tri-snRNPs (Figure 5B, fractions 16±19), most of U4/U6 sedimented as a 13S particle in the 61K- depleted extract (Figure 5C, fractions 7±10). Several ®ndings indicate that the removal of protein 61K from nuclear extract does not affect the stability of the accumulating U4/U6 or U5 snRNP particles. First, the Sm- speci®c monoclonal antibody Y12 precipitated equivalent amounts of the U4, U6 and U5 snRNPs, in addition to U1 and U2 snRNPs, irrespective of whether 61K-depleted, mock-depleted or depleted/complemented nuclear extract was used (Figure 5A, lanes 1±3). Secondly, antibodies against the U4/U6-speci®c 60K protein (Lauber et al ., 1997) precipitated U4/U6 snRNPs from 61K-depleted extract (Figure 5A, lane 7), indicating that the U4/U6 snRNA interaction remains intact and that the binding of protein 60K and thus of the 20K/60K/90K heterotrimer (Horowitz et al ., 1997; Lauber et al ., 1997), is not dependent on protein 61K. Finally, glycerol-gradient centrifugation of 61K-depleted nuclear extract led to the accumulation of 20S U5 and 13S U4/U6 snRNP particles (Figure 4C). No increase in the amount of free U4 or U6 snRNP was observed after 61K-depletion. Taken together, these results demonstrate that protein 61K plays a critical and speci®c part in the formation of the tri-snRNP from U4/U6 and U5 snRNPs. This is a prerequisite for the integration of the tri-snRNP into spliceosomes and thus also for subsequent splicing catalysis. The 61K protein could promote tri-snRNP formation in at least two ways. First, the binding of 61K to the U4/U6 snRNP could promote a conformation of this particle compatible with docking to U5 snRNPs but without contacting directly U5 snRNP. Alternatively, protein 61K could bridge U4/U6 and U5 snRNPs by interacting with components of both U4/U6 and U5 particles. This model would predict a speci®c interaction of 61K with one or more U5 snRNP component. Consistent with this idea, in vitro translated 35 S-labeled 61K protein binds to puri®ed 20S U5 snRNPs, as demonstrated by co-immunoprecipita- tion of 61K with U5 snRNPs at low salt concentration using anti-Sm (Y12) antibodies (Figure 6A, lane 4). The interaction of 61K with the U5 snRNP is speci®c and required the presence of U5-speci®c proteins since no binding was observed with 10S U5 or U1 snRNP core particles (lanes 5 and 6, respectively) or with 12S U1 and U2 snRNPs (lane 3). We next searched for proteins that physically interact with the 61K protein by performing two-hybrid interaction screens with the U5-speci®c 15K, 40K, 52K, 100K, 102K, 116K, 200K and 220K proteins. In addition, the tri-snRNP proteins 65K and 110K, as well as the U4/U6-speci®c proteins 15.5K, 60K and 90K, were also included in the screen; in each case 61K was always used as a bait. Interestingly, only one out of the 13 tested proteins interacted with protein 61K, namely the U5 snRNP- speci®c protein 102K (Figure 6B; data not shown). This interaction was also observed in the reciprocal two-hybrid screen (with 102K as bait), indicating a tight interaction between these two proteins (Figure 6B). Physical and speci®c interaction between the 61K and 102K proteins was independently con®rmed using a biochemical assay. In glutathione S -transferase (GST)-pulldown experiments in vitro translated, 35 S-labeled 102K protein ef®ciently interacts with a puri®ed recombinant fusion protein of 61K with GST (Figure 6C). This interaction is speci®c, as only a low level of 102K protein was precipitated with beads containing GST alone (Figure 6C). In summary, our results indicate that protein 61K functions as a bridge in the tri-snRNP, physically interacting with its U4/U6 and U5 snRNP subunits. A crucial step in the assembly pathway of the spliceosomal U4/U6 ́U5 tri-snRNP is the interaction between the U4/U6 and U5 snRNPs. The results presented here demonstrate that the 61K protein present in tri-snRNPs is required for this step. This conclusion is corroborated by the following observations: (i) immunodepletion of 61K protein from HeLa cell nuclear extract inhibited tri-snRNP formation while U4/U6 and U5 snRNPs accumulated (Figure 5); (ii) the removal of protein 61K did not affect the stability of the U4/U6 or the U5 snRNPs as indicated by the ®ndings that both particles retain their particle-speci®c proteins and that there was no increase in free U4 or U6 snRNPs or 10S U5 core snRNPs (Figure 5); and (iii) the formation of U4/U6 ́U5 tri-snRNPs was restored by complementation of the depleted extract with recombinant 61K protein, con®rming the speci®city of the effect of protein 61K depletion on tri-snRNP stability (Figure 5). Major steps in the splicing process are the formation of spliceosome complex A, which consists of the U1 and U2 snRNPs bound to the pre-mRNA, and its subsequent transformation into complex B upon addition of the tri- snRNP. It is generally believed that the latter takes place in one step, i.e. by the binding of a pre-formed tri-snRNP to complex A (Cheng and Abelson, 1987; Konarska and Sharp, 1987; Utans et al ., 1992), rather than by the sequential and/or independent addition of the U5 and U4/U6 snRNPs. The results described here provide additional direct evidence that it is indeed the pre-formed tri-snRNP that binds to complex A; in the absence of protein 61K the tri-snRNP fails to form and neither of its snRNP components becomes stably bound to complex A, as investigated by native gel electrophoresis (Figure 4D). The function of protein 61K bears a certain resemblance to that of the U4/U6 ́U5 tri-snRNP 65K and 110K proteins (Makarova et al ., 2001), but differs in several important aspects. As is the case for 61K, the removal of either the 65K or 110K protein prevents binding of the tri-snRNP to complex A. However, in the absence of these two proteins the stability of the tri-snRNP is unaffected. Thus, while a major function of the 61K protein is to promote U4/U6 ́U5 tri-snRNP formation, the 65K and 110K proteins play a major part in recruiting the pre-formed tri-snRNP to the pre-spliceosome. The human protein 61K clearly exhibits signi®cant similarity in sequence to the yeast Prp31p protein (Figure 1), which is also classi®ed as a U4/U6-speci®c protein in yeast (Weidenhammer et al ., 1997). There appears to be a mechanistic difference in the function of the two proteins, however. Evidence was presented that Prp31p mediates the binding of the pre-formed tri-snRNP to complex A (Weidenhammer et al ., 1997), while our data clearly point to a crucial role for protein 61K at the earlier step of tri-snRNP formation. The conclusions drawn by Weidenhammer et al . (1997) rest to a large part on the observation that heat inactivation of extracts from a temperature-sensitive strain inhibits the assembly of mature spliceosomes while signi®cant amounts of U4/U6 ́U5 tri-snRNPs were maintained. These experiments do not exclude the possibility, however, that at the non-permissive temperature the inactivated mutant Prp31p protein may induce a conformation of the tri-snRNP incompatible with docking to the pre-spliceosome, while it is still able to tether U4/U6 to U5 snRNPs. It will therefore be interesting to test whether, similar to the situation in HeLa extracts, physical depletion of Prp31p from yeast extracts prevents stable formation of the U4/U6 ́U5 tri-snRNP. The 61K protein could facilitate the formation of the U4/U6 ́U5 tri-snRNP either indirectly, by promoting a conformation of the U4/U6 snRNP compatible with docking to U5 snRNP or, more directly, by physically tethering U4/U6 to U5 snRNP. The observations described here that the 61K protein speci®cally interacts with both U4/U6 and U5 snRNPs strongly favor a bridging role for this protein in tri-snRNP formation. A strong interaction of 61K with U4/U6 snRNPs was initially documented with anti-61K antibodies, which precipitated U4/U6 ́U5 tri- snRNP at low salt concentrations (where the tri-snRNP is stable), but only U4/U6 snRNPs at higher salt concentrations (where the tri-snRNP dissociates into U5 and U4/U6 snRNPs, see Figure 2B). Several lines of evidence indicate that there is also a speci®c binding site for protein 61K on the U5 snRNP. At low salt concentrations in vitro translated 61K protein also binds to puri®ed 20S U5 snRNPs and this binding requires U5-speci®c proteins (Figure 6A). Most importantly, using two-hybrid screens and biochemical pulldown experiments we could demonstrate that 61K speci®cally interacts with the 102K protein of the 20S U5 snRNP in vivo and in vitro (Figure 6B and C). These data indicate that the U5±102K protein is the major interaction partner of protein 61K on the U5 snRNP subunit of the tri-snRNP. Interestingly, independent evidence for a role of the 61K's interaction partner U5±102K and its yeast ortholog Prp6p in bridging U5 and U4/U6 snRNPs in the tri-snRNP has been provided previously. For example, antibodies directed against the C-terminal region of the 102K protein speci®cally immunoprecipitated free U5 snRNPs, and not U4/U6 ́U5 tri-snRNPs, from HeLa nuclear extract, sug- gesting that in the tri-snRNP, the C-terminal ...
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... extracts (lane 5), indicating that the absence of protein 61K prevented tri-snRNPs from forming. Signi®cantly, addition of protein 61K to the depleted extract fully restored tri-snRNP complex formation (lane 6). Thus, protein 61K is essential for the formation of the tri-snRNP complex in vitro . The lack of stable tri-snRNP formation in the absence of protein 61K was further demonstrated by glycerol-gradient centrifugation of mock versus depleted nuclear extract (Figure 5B and C). In contrast to the mock-depleted extract where the majority of U4 and U6 snRNAs migrated as part of intact 25S tri-snRNPs (Figure 5B, fractions 16±19), most of U4/U6 sedimented as a 13S particle in the 61K- depleted extract (Figure 5C, fractions 7±10). Several ®ndings indicate that the removal of protein 61K from nuclear extract does not affect the stability of the accumulating U4/U6 or U5 snRNP particles. First, the Sm- speci®c monoclonal antibody Y12 precipitated equivalent amounts of the U4, U6 and U5 snRNPs, in addition to U1 and U2 snRNPs, irrespective of whether 61K-depleted, mock-depleted or depleted/complemented nuclear extract was used (Figure 5A, lanes 1±3). Secondly, antibodies against the U4/U6-speci®c 60K protein (Lauber et al ., 1997) precipitated U4/U6 snRNPs from 61K-depleted extract (Figure 5A, lane 7), indicating that the U4/U6 snRNA interaction remains intact and that the binding of protein 60K and thus of the 20K/60K/90K heterotrimer (Horowitz et al ., 1997; Lauber et al ., 1997), is not dependent on protein 61K. Finally, glycerol-gradient centrifugation of 61K-depleted nuclear extract led to the accumulation of 20S U5 and 13S U4/U6 snRNP particles (Figure 4C). No increase in the amount of free U4 or U6 snRNP was observed after 61K-depletion. Taken together, these results demonstrate that protein 61K plays a critical and speci®c part in the formation of the tri-snRNP from U4/U6 and U5 snRNPs. This is a prerequisite for the integration of the tri-snRNP into spliceosomes and thus also for subsequent splicing catalysis. The 61K protein could promote tri-snRNP formation in at least two ways. First, the binding of 61K to the U4/U6 snRNP could promote a conformation of this particle compatible with docking to U5 snRNPs but without contacting directly U5 snRNP. Alternatively, protein 61K could bridge U4/U6 and U5 snRNPs by interacting with components of both U4/U6 and U5 particles. This model would predict a speci®c interaction of 61K with one or more U5 snRNP component. Consistent with this idea, in vitro translated 35 S-labeled 61K protein binds to puri®ed 20S U5 snRNPs, as demonstrated by co-immunoprecipita- tion of 61K with U5 snRNPs at low salt concentration using anti-Sm (Y12) antibodies (Figure 6A, lane 4). The interaction of 61K with the U5 snRNP is speci®c and required the presence of U5-speci®c proteins since no binding was observed with 10S U5 or U1 snRNP core particles (lanes 5 and 6, respectively) or with 12S U1 and U2 snRNPs (lane 3). We next searched for proteins that physically interact with the 61K protein by performing two-hybrid interaction screens with the U5-speci®c 15K, 40K, 52K, 100K, 102K, 116K, 200K and 220K proteins. In addition, the tri-snRNP proteins 65K and 110K, as well as the U4/U6-speci®c proteins 15.5K, 60K and 90K, were also included in the screen; in each case 61K was always used as a bait. Interestingly, only one out of the 13 tested proteins interacted with protein 61K, namely the U5 snRNP- speci®c protein 102K (Figure 6B; data not shown). This interaction was also observed in the reciprocal two-hybrid screen (with 102K as bait), indicating a tight interaction between these two proteins (Figure 6B). Physical and speci®c interaction between the 61K and 102K proteins was independently con®rmed using a biochemical assay. In glutathione S -transferase (GST)-pulldown experiments in vitro translated, 35 S-labeled 102K protein ef®ciently interacts with a puri®ed recombinant fusion protein of 61K with GST (Figure 6C). This interaction is speci®c, as only a low level of 102K protein was precipitated with beads containing GST alone (Figure 6C). In summary, our results indicate that protein 61K functions as a bridge in the tri-snRNP, physically interacting with its U4/U6 and U5 snRNP subunits. A crucial step in the assembly pathway of the spliceosomal U4/U6 ́U5 tri-snRNP is the interaction between the U4/U6 and U5 snRNPs. The results presented here demonstrate that the 61K protein present in tri-snRNPs is required for this step. This conclusion is corroborated by the following observations: (i) immunodepletion of 61K protein from HeLa cell nuclear extract inhibited tri-snRNP formation while U4/U6 and U5 snRNPs accumulated (Figure 5); (ii) the removal of protein 61K did not affect the stability of the U4/U6 or the U5 snRNPs as indicated by the ®ndings that both particles retain their particle-speci®c proteins and that there was no increase in free U4 or U6 snRNPs or 10S U5 core snRNPs (Figure 5); and (iii) the formation of U4/U6 ́U5 tri-snRNPs was restored by complementation of the depleted extract with recombinant 61K protein, con®rming the speci®city of the effect of protein 61K depletion on tri-snRNP stability (Figure 5). Major steps in the splicing process are the formation of spliceosome complex A, which consists of the U1 and U2 snRNPs bound to the pre-mRNA, and its subsequent transformation into complex B upon addition of the tri- snRNP. It is generally believed that the latter takes place in one step, i.e. by the binding of a pre-formed tri-snRNP to complex A (Cheng and Abelson, 1987; Konarska and Sharp, 1987; Utans et al ., 1992), rather than by the sequential and/or independent addition of the U5 and U4/U6 snRNPs. The results described here provide additional direct evidence that it is indeed the pre-formed tri-snRNP that binds to complex A; in the absence of protein 61K the tri-snRNP fails to form and neither of its snRNP components becomes stably bound to complex A, as investigated by native gel electrophoresis (Figure 4D). The function of protein 61K bears a certain resemblance to that of the U4/U6 ́U5 tri-snRNP 65K and 110K proteins (Makarova et al ., 2001), but differs in several important aspects. As is the case for 61K, the removal of either the 65K or 110K protein prevents binding of the tri-snRNP to complex A. However, in the absence of these two proteins the stability of the tri-snRNP is unaffected. Thus, while a major function of the 61K protein is to promote U4/U6 ́U5 tri-snRNP formation, the 65K and 110K proteins play a major part in recruiting the pre-formed tri-snRNP to the pre-spliceosome. The human protein 61K clearly exhibits signi®cant similarity in sequence to the yeast Prp31p protein (Figure 1), which is also classi®ed as a U4/U6-speci®c protein in yeast (Weidenhammer et al ., 1997). There appears to be a mechanistic difference in the function of the two proteins, however. Evidence was presented that Prp31p mediates the binding of the pre-formed tri-snRNP to complex A (Weidenhammer et al ., 1997), while our data clearly point to a crucial role for protein 61K at the earlier step of tri-snRNP formation. The conclusions drawn by Weidenhammer et al . (1997) rest to a large part on the observation that heat inactivation of extracts from a temperature-sensitive strain inhibits the assembly of mature spliceosomes while signi®cant amounts of U4/U6 ́U5 tri-snRNPs were maintained. These experiments do not exclude the possibility, however, that at the non-permissive temperature the inactivated mutant Prp31p protein may induce a conformation of the tri-snRNP incompatible with docking to the pre-spliceosome, while it is still able to tether U4/U6 to U5 snRNPs. It will therefore be interesting to test whether, similar to the situation in HeLa extracts, physical depletion of Prp31p from yeast extracts prevents stable formation of the U4/U6 ́U5 tri-snRNP. The 61K protein could facilitate the formation of the U4/U6 ́U5 tri-snRNP either indirectly, by promoting a conformation of the U4/U6 snRNP compatible with docking to U5 snRNP or, more directly, by physically tethering U4/U6 to U5 snRNP. The observations described here that the 61K protein speci®cally interacts with both U4/U6 and U5 snRNPs strongly favor a bridging role for this protein in tri-snRNP formation. A strong interaction of 61K with U4/U6 snRNPs was initially documented with anti-61K antibodies, which precipitated U4/U6 ́U5 tri- snRNP at low salt concentrations (where the tri-snRNP is stable), but only U4/U6 snRNPs at higher salt concentrations (where the tri-snRNP dissociates into U5 and U4/U6 snRNPs, see Figure 2B). Several lines of evidence indicate that there is also a speci®c binding site for protein 61K on the U5 snRNP. At low salt concentrations in vitro translated 61K protein also binds to puri®ed 20S U5 snRNPs and this binding requires U5-speci®c proteins (Figure 6A). Most importantly, using two-hybrid screens and biochemical pulldown experiments we could demonstrate that 61K speci®cally interacts with the 102K protein of the 20S U5 snRNP in vivo and in vitro (Figure 6B and C). These data indicate that the U5±102K protein is the major interaction partner of protein 61K on the U5 snRNP subunit of the tri-snRNP. Interestingly, independent evidence for a role of the 61K's interaction partner U5±102K and its yeast ortholog Prp6p in bridging U5 and U4/U6 snRNPs in the tri-snRNP has been provided previously. For example, antibodies directed against the C-terminal region of the 102K protein speci®cally immunoprecipitated free U5 snRNPs, and not U4/U6 ́U5 tri-snRNPs, from HeLa nuclear extract, sug- gesting that in the tri-snRNP, the C-terminal region of the 102K protein is covered by U4/U6-speci®c proteins (Makarov et al ., 2000). Consistent with a bridging function for the 102K protein, it could be shown that in vitro translated U5±102K binds to puri®ed 13S U4/U6 snRNPs, which contained all U4/U6-speci®c ...
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... is essential for the formation of the tri-snRNP complex in vitro . The lack of stable tri-snRNP formation in the absence of protein 61K was further demonstrated by glycerol-gradient centrifugation of mock versus depleted nuclear extract (Figure 5B and C). In contrast to the mock-depleted extract where the majority of U4 and U6 snRNAs migrated as part of intact 25S tri-snRNPs (Figure 5B, fractions 16±19), most of U4/U6 sedimented as a 13S particle in the 61K- depleted extract (Figure 5C, fractions 7±10). Several ®ndings indicate that the removal of protein 61K from nuclear extract does not affect the stability of the accumulating U4/U6 or U5 snRNP particles. First, the Sm- speci®c monoclonal antibody Y12 precipitated equivalent amounts of the U4, U6 and U5 snRNPs, in addition to U1 and U2 snRNPs, irrespective of whether 61K-depleted, mock-depleted or depleted/complemented nuclear extract was used (Figure 5A, lanes 1±3). Secondly, antibodies against the U4/U6-speci®c 60K protein (Lauber et al ., 1997) precipitated U4/U6 snRNPs from 61K-depleted extract (Figure 5A, lane 7), indicating that the U4/U6 snRNA interaction remains intact and that the binding of protein 60K and thus of the 20K/60K/90K heterotrimer (Horowitz et al ., 1997; Lauber et al ., 1997), is not dependent on protein 61K. Finally, glycerol-gradient centrifugation of 61K-depleted nuclear extract led to the accumulation of 20S U5 and 13S U4/U6 snRNP particles (Figure 4C). No increase in the amount of free U4 or U6 snRNP was observed after 61K-depletion. Taken together, these results demonstrate that protein 61K plays a critical and speci®c part in the formation of the tri-snRNP from U4/U6 and U5 snRNPs. This is a prerequisite for the integration of the tri-snRNP into spliceosomes and thus also for subsequent splicing catalysis. The 61K protein could promote tri-snRNP formation in at least two ways. First, the binding of 61K to the U4/U6 snRNP could promote a conformation of this particle compatible with docking to U5 snRNPs but without contacting directly U5 snRNP. Alternatively, protein 61K could bridge U4/U6 and U5 snRNPs by interacting with components of both U4/U6 and U5 particles. This model would predict a speci®c interaction of 61K with one or more U5 snRNP component. Consistent with this idea, in vitro translated 35 S-labeled 61K protein binds to puri®ed 20S U5 snRNPs, as demonstrated by co-immunoprecipita- tion of 61K with U5 snRNPs at low salt concentration using anti-Sm (Y12) antibodies (Figure 6A, lane 4). The interaction of 61K with the U5 snRNP is speci®c and required the presence of U5-speci®c proteins since no binding was observed with 10S U5 or U1 snRNP core particles (lanes 5 and 6, respectively) or with 12S U1 and U2 snRNPs (lane 3). We next searched for proteins that physically interact with the 61K protein by performing two-hybrid interaction screens with the U5-speci®c 15K, 40K, 52K, 100K, 102K, 116K, 200K and 220K proteins. In addition, the tri-snRNP proteins 65K and 110K, as well as the U4/U6-speci®c proteins 15.5K, 60K and 90K, were also included in the screen; in each case 61K was always used as a bait. Interestingly, only one out of the 13 tested proteins interacted with protein 61K, namely the U5 snRNP- speci®c protein 102K (Figure 6B; data not shown). This interaction was also observed in the reciprocal two-hybrid screen (with 102K as bait), indicating a tight interaction between these two proteins (Figure 6B). Physical and speci®c interaction between the 61K and 102K proteins was independently con®rmed using a biochemical assay. In glutathione S -transferase (GST)-pulldown experiments in vitro translated, 35 S-labeled 102K protein ef®ciently interacts with a puri®ed recombinant fusion protein of 61K with GST (Figure 6C). This interaction is speci®c, as only a low level of 102K protein was precipitated with beads containing GST alone (Figure 6C). In summary, our results indicate that protein 61K functions as a bridge in the tri-snRNP, physically interacting with its U4/U6 and U5 snRNP subunits. A crucial step in the assembly pathway of the spliceosomal U4/U6 ́U5 tri-snRNP is the interaction between the U4/U6 and U5 snRNPs. The results presented here demonstrate that the 61K protein present in tri-snRNPs is required for this step. This conclusion is corroborated by the following observations: (i) immunodepletion of 61K protein from HeLa cell nuclear extract inhibited tri-snRNP formation while U4/U6 and U5 snRNPs accumulated (Figure 5); (ii) the removal of protein 61K did not affect the stability of the U4/U6 or the U5 snRNPs as indicated by the ®ndings that both particles retain their particle-speci®c proteins and that there was no increase in free U4 or U6 snRNPs or 10S U5 core snRNPs (Figure 5); and (iii) the formation of U4/U6 ́U5 tri-snRNPs was restored by complementation of the depleted extract with recombinant 61K protein, con®rming the speci®city of the effect of protein 61K depletion on tri-snRNP stability (Figure 5). Major steps in the splicing process are the formation of spliceosome complex A, which consists of the U1 and U2 snRNPs bound to the pre-mRNA, and its subsequent transformation into complex B upon addition of the tri- snRNP. It is generally believed that the latter takes place in one step, i.e. by the binding of a pre-formed tri-snRNP to complex A (Cheng and Abelson, 1987; Konarska and Sharp, 1987; Utans et al ., 1992), rather than by the sequential and/or independent addition of the U5 and U4/U6 snRNPs. The results described here provide additional direct evidence that it is indeed the pre-formed tri-snRNP that binds to complex A; in the absence of protein 61K the tri-snRNP fails to form and neither of its snRNP components becomes stably bound to complex A, as investigated by native gel electrophoresis (Figure 4D). The function of protein 61K bears a certain resemblance to that of the U4/U6 ́U5 tri-snRNP 65K and 110K proteins (Makarova et al ., 2001), but differs in several important aspects. As is the case for 61K, the removal of either the 65K or 110K protein prevents binding of the tri-snRNP to complex A. However, in the absence of these two proteins the stability of the tri-snRNP is unaffected. Thus, while a major function of the 61K protein is to promote U4/U6 ́U5 tri-snRNP formation, the 65K and 110K proteins play a major part in recruiting the pre-formed tri-snRNP to the pre-spliceosome. The human protein 61K clearly exhibits signi®cant similarity in sequence to the yeast Prp31p protein (Figure 1), which is also classi®ed as a U4/U6-speci®c protein in yeast (Weidenhammer et al ., 1997). There appears to be a mechanistic difference in the function of the two proteins, however. Evidence was presented that Prp31p mediates the binding of the pre-formed tri-snRNP to complex A (Weidenhammer et al ., 1997), while our data clearly point to a crucial role for protein 61K at the earlier step of tri-snRNP formation. The conclusions drawn by Weidenhammer et al . (1997) rest to a large part on the observation that heat inactivation of extracts from a temperature-sensitive strain inhibits the assembly of mature spliceosomes while signi®cant amounts of U4/U6 ́U5 tri-snRNPs were maintained. These experiments do not exclude the possibility, however, that at the non-permissive temperature the inactivated mutant Prp31p protein may induce a conformation of the tri-snRNP incompatible with docking to the pre-spliceosome, while it is still able to tether U4/U6 to U5 snRNPs. It will therefore be interesting to test whether, similar to the situation in HeLa extracts, physical depletion of Prp31p from yeast extracts prevents stable formation of the U4/U6 ́U5 tri-snRNP. The 61K protein could facilitate the formation of the U4/U6 ́U5 tri-snRNP either indirectly, by promoting a conformation of the U4/U6 snRNP compatible with docking to U5 snRNP or, more directly, by physically tethering U4/U6 to U5 snRNP. The observations described here that the 61K protein speci®cally interacts with both U4/U6 and U5 snRNPs strongly favor a bridging role for this protein in tri-snRNP formation. A strong interaction of 61K with U4/U6 snRNPs was initially documented with anti-61K antibodies, which precipitated U4/U6 ́U5 tri- snRNP at low salt concentrations (where the tri-snRNP is stable), but only U4/U6 snRNPs at higher salt concentrations (where the tri-snRNP dissociates into U5 and U4/U6 snRNPs, see Figure 2B). Several lines of evidence indicate that there is also a speci®c binding site for protein 61K on the U5 snRNP. At low salt concentrations in vitro translated 61K protein also binds to puri®ed 20S U5 snRNPs and this binding requires U5-speci®c proteins (Figure 6A). Most importantly, using two-hybrid screens and biochemical pulldown experiments we could demonstrate that 61K speci®cally interacts with the 102K protein of the 20S U5 snRNP in vivo and in vitro (Figure 6B and C). These data indicate that the U5±102K protein is the major interaction partner of protein 61K on the U5 snRNP subunit of the tri-snRNP. Interestingly, independent evidence for a role of the 61K's interaction partner U5±102K and its yeast ortholog Prp6p in bridging U5 and U4/U6 snRNPs in the tri-snRNP has been provided previously. For example, antibodies directed against the C-terminal region of the 102K protein speci®cally immunoprecipitated free U5 snRNPs, and not U4/U6 ́U5 tri-snRNPs, from HeLa nuclear extract, sug- gesting that in the tri-snRNP, the C-terminal region of the 102K protein is covered by U4/U6-speci®c proteins (Makarov et al ., 2000). Consistent with a bridging function for the 102K protein, it could be shown that in vitro translated U5±102K binds to puri®ed 13S U4/U6 snRNPs, which contained all U4/U6-speci®c proteins, including protein 61K (Makarov et al ., 2000). In yeast, mutation of the PRP6 gene inhibits tri-snRNP accumulation, while accumulation of the individual U4/U6 and U5 snRNPs is not affected (Galisson and Legrain, 1993). This ...
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Citations
... The PRPF31 transcript comprises 14 exons that, when processed, encode 499 amino acids essential for the stabilization of the U4/U6-U5 tri-snRNP complex during pre-mRNA splicing [33]. Functional domains of RP11 include the NOSIC domain (named after the central domain of 'Nop56/SIK1-like protein'), nucleolar protein (Nop) domain, and nuclear localization signal (NLS) encoded by exons 4-6, 7-10, and 10-11, respectively ( Figure 2a). ...
... The exclusion of exon 12 from the transcript is expected to direct the synthesis of an internally truncated PRPF31 protein, missing 43 amino acids, that may retain function, since exon 12 does not encode any known functional domain(s), in effect generating a hypomorphic protein product. The majority of previous studies on exon skipping were applied to large genes, with the removal of less than 10% of the amino acids without detrimental effects on protein conformation [33,34]. However, the effect of excluding 43 amino acids out of the 499 amino acids on PRPF31 protein conformation is unknown, considering that the neighboring exon 11 encodes the critically important nuclear localization signal for translocation of the PRPF31 protein into the nucleus. ...
Retinitis pigmentosa 11 is an untreatable, dominantly inherited retinal disease caused by heterozygous mutations in pre-mRNA processing factor 31 PRPF31. The expression level of PRPF31 is linked to incomplete penetrance in affected families; mutation carriers with higher PRPF31 expression can remain asymptomatic. The current study explores an antisense oligonucleotide exon skipping strategy to treat RP11 caused by truncating mutations within PRPF31 exon 12 since it does not appear to encode any domains essential for PRPF31 protein function. Cells derived from a patient carrying a PRPF31 1205C>A nonsense mutation were investigated; PRPF31 transcripts encoded by the 1205C>A allele were undetectable due to nonsense-mediated mRNA decay, resulting in a 46% reduction in PRPF31 mRNA, relative to healthy donor cells. Antisense oligonucleotide-induced skipping of exon 12 rescued the open reading frame with consequent 1.7-fold PRPF31 mRNA upregulation in the RP11 patient fibroblasts. The level of PRPF31 upregulation met the predicted therapeutic threshold of expression inferred in a non-penetrant carrier family member harbouring the same mutation. This study demonstrated increased PRPF31 expression and retention of the nuclear translocation capability for the induced PRPF31 isoform. Future studies should evaluate the function of the induced PRPF31 protein on pre-mRNA splicing in retinal cells to validate the therapeutic approach for amenable RP11-causing mutations.
... PRPF31 is a highly conserved gene composed of 14 exons, 1 non-coding and 13 coding, located at chromosome 19q13.4. PRPF31 encodes a ubiquitous splicing factor, which facilitates the formation and stabilization of the U4/U6-U5 tri-snRNP (small nucleolar ribonucleoprotein) complex and plays essential roles in the mRNA splicing process 23,24 . Homozygous Prpf31 knockout mice and zebrafish are embryonic lethal, indicating the essentiality of PRPF31 25,26 . ...
Mutations in PRPF31 cause autosomal dominant retinitis pigmentosa, an untreatable form of blindness. Gene therapy is a promising treatment for PRPF31-retinitis pigmentosa, however, there are currently no suitable animal models in which to develop AAV-mediated gene augmentation. Here we establish Prpf31 mutant mouse models using AAV-mediated CRISPR/Cas9 knockout, and characterize the resulting retinal degeneration phenotype. Mouse models with early-onset morphological and functional impairments like those in patients were established, providing new platforms in which to investigate pathogenetic mechanisms and develop therapeutic methods. AAV-mediated PRPF31 gene augmentation restored the retinal structure and function in a rapidly degenerating mouse model, demonstrating the first in vivo proof-of-concept for AAV-mediated gene therapy to treat PRPF31-retinitis pigmentosa. AAV-CRISPR/Cas9-PRPF31 knockout constructs also mediated efficient PRPF31 knockout in human and non-human primate retinal explants, laying a foundation for establishing non-human primate models using the method developed here. PRPF31-RP is a blinding disease, caused by insufficient levels of a pre-mRNA splicing factor. Here, the authors show that CRISPR-Cas9 editing of the Prpf31 gene in mice leads to retinal degeneration similar to human patients, and, in the same model, demonstrate benefits from PRPF31 gene therapy.
... accessed on 18 September 2021), in 2020, liver cancer was ranked sixth in incidence with 905,677 new cases (4.7% of new cases) but third regarding mortality with 830,180 deaths (8.3% of all cancer-related deaths). Liver cancer includes several forms of cancers, but in most adult patients, these tumors are the splicing of the pre-mRNA as components of the U4/U6-U5 tri-snRNP complex and the spliceosome B complexes [27]. ...
Simple Summary
Among the top ten deadly solid tumors are the two most frequent liver cancers, hepatocellular carcinoma, and intrahepatic cholangiocarcinoma, whose development and malignancy are favored by multifactorial conditions, which include aberrant maturation of pre-mRNA due to abnormalities in either the machinery involved in the splicing, i.e., the spliceosome and associated factors, or the nucleotide sequences of essential sites for the exon recognition process. As a consequence of cancer-associated aberrant splicing in hepatocytes- and cholangiocytes-derived cancer cells, abnormal proteins are synthesized. They contribute to the dysregulated proliferation and eventually transformation of these cells to phenotypes with enhanced invasiveness, migration, and multidrug resistance, which contributes to the poor prognosis that characterizes these liver cancers.
Abstract
The two most frequent primary cancers affecting the liver, whose incidence is growing worldwide, are hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), which are among the five most lethal solid tumors with meager 5-year survival rates. The common difficulty in most cases to reach an early diagnosis, the aggressive invasiveness of both tumors, and the lack of favorable response to pharmacotherapy, either classical chemotherapy or modern targeted therapy, account for the poor outcome of these patients. Alternative splicing (AS) during pre-mRNA maturation results in changes that might affect proteins involved in different aspects of cancer biology, such as cell cycle dysregulation, cytoskeleton disorganization, migration, and adhesion, which favors carcinogenesis, tumor promotion, and progression, allowing cancer cells to escape from pharmacological treatments. Reasons accounting for cancer-associated aberrant splicing include mutations that create or disrupt splicing sites or splicing enhancers or silencers, abnormal expression of splicing factors, and impaired signaling pathways affecting the activity of the splicing machinery. Here we have reviewed the available information regarding the impact of AS on liver carcinogenesis and the development of malignant characteristics of HCC and iCCA, whose understanding is required to develop novel therapeutical approaches aimed at manipulating the phenotype of cancer cells.
... Le splicéosome devient actif après la liaison du complexe protéique, formé par l'association des snRNP U5 et U4 / U6, 5' de la séquence d'épissage. Chacune des snRNP est liée spécifiquement avec un facteur d'épissage de pré-ARNm, respectivement PRPF3, PRPF31 et PRPF8 (Makarova et al., 2002). Divers réarrangements ont lieu entre les snRNP, les facteurs d'épissage et la séquence intronique : la snRNP U5 se lie en 5', libérant ainsi le snRNP U1. ...
Notre projet consiste à modéliser une forme spécifique de rétinite pigmentaire (RP) en utilisant des cellules iPS de patients. Nous avons d'abord optimisé un protocole de différenciation pour obtenir à partir de cellules iPS des organoïdes rétiniens avec une organisation structurelle plus proche de la rétine in vivo, permettant une maturation avancée des photorécepteurs. Cet outil nous a permis de récapituler entièrement le phénotype RP (dégénérescence des bâtonnets et des cônes), observé chez les patients présentant une mutation du gène RHODOPSINE, codant pour le pigment visuel. Nous avons ensuite utilisé la même approche pour comprendre la pathogénicité des RP liées à des mutations du gène PRPF31, codant pour un facteur d'épissage. Les organoïdes rétiniens ont résumé la dégénérescence des bâtonnets et la perte secondaire des cônes, observées chez les patients. Les cellules de l'épithélium pigmenté de la rétine présentaient également des défauts organisationnels et fonctionnels. Ces phénotypes dégénératifs rétiniens sont corrélés à un niveau d'expression plus faible de la protéine PRPF31, liant la pathogénicité à un mécanisme d'haploinsuffisance. Nous avons donc développé une stratégie d'augmentation du gène, en apportant une copie fonctionnelle de PRPF31 par CRISPR/Cas9 ou en utilisant un vecteur AAV, qui ont permis le sauvetage de la dégénérescence des cellules rétiniennes.
... The PRPF31 gene encodes the pre-mRNA processing factor-31 protein, which forms part of the U4/U6/U5 small nuclear ribonucleoprotein (snRNP) subunits of the spliceosome, a large RNP complex central to the process of pre-mRNA splicing [9][10][11][12][13]. Pre-mRNA splicing is an essential step in gene expression whereby non-coding intronic sequences are removed from the transcribed Pre-mRNA molecule to produce a mature mRNA transcript carrying the protein coding sequence. ...
Retinitis pigmentosa 11 (RP11) is caused by dominant mutations in PRPF31, however a significant proportion of mutation carriers do not develop retinopathy. Here, we investigated the relationship between CNOT3 polymorphism, MSR1 repeat copy number and disease penetrance in RP11 patients and non-penetrant carriers (NPCs). We further characterized PRPF31 and CNOT3 expression in fibroblasts from eight RP11 patients and one NPC from a family carrying the c.1205C>T variant. Retinal organoids (ROs) and retinal pigment epithelium (RPE) were differentiated from induced pluripotent stem cells derived from RP11 patients, an NPC and a control subject. All RP11 patients were homozygous for the 3-copy MSR1 repeat in the PRPF31 promoter, while 3/5 NPCs carried a 4-copy MSR1 repeat. The CNOT3 rs4806718 genotype did not correlate with disease penetrance. PRFP31 expression declined with age in adult cadaveric retina. PRPF31 and CNOT3 expression was reduced in RP11 fibroblasts, RO and RPE compared with controls. Both RP11 and NPC RPE displayed shortened primary cilia compared with controls, however a subpopulation of cells with normal cilia lengths was present in NPC RPE monolayers. Our results indicate that RP11 non-penetrance is associated with the inheritance of a 4-copy MSR1 repeat, but not with CNOT3 polymorphisms.
... Mutations in seven PRPF genes have been identified in adRP, including PRPF3,4,6,8,31,SNRNP200 (Brr2), and RP9 (PAP-1) (Mordes et al., 2006;Chen et al., 2014;Daiger et al., 2014;Ruzickova and Stanek, 2017), all of which except RP9 encode components of tri-snRNP and play important roles in the assembly of spliceosome complex B. During spliceosome assembly, U4 and U6 snRNP strongly bind with each other via snRNA paring to form U4/U6 di-snRNP which subsequently associates with U5 snRNP leading to U4/U6.U5 tri-snRNP conformation . Amongst the seven RP-PRPF proteins, PRPF3, PRPF4, and PRPF31 are U4/U6 snRNP-specific factors, while PRPF6, PRPF8, and SNRNP200 are components of U5 snRNP and RP9 is a non-snRNP splicing factor (Liu et al., 2006;Supplementary The interactions among PRPFs and PRPF-snRNA crosslinks within tri-snRNP have been well described in other reviews (Abovich et al., 1990;Makarov et al., 2000;Gonzalez-Santos et al., 2002;Kuhn et al., 2002;Makarova et al., 2002;Nottrott et al., 2002;Schaffert et al., 2004;Liu et al., 2006). Moreover, recent yeast and human cryo-EM structures of spliceosomes at different stages of splicing have shed light on the RNA and protein structural rearrangements of the spliceosome during splicing cycle. ...
... PRPF31 is a U4/U6 specific protein that binds directly to U4 snRNA (Figure 1; Nottrott et al., 2002). PRPF31 interacts with PRPF6 and tethers U5 snRNP to the U4/U6 snRNP facilitating the formation of tri-snRNPs (Makarova et al., 2002). PRPF31 contains a Nop domain that mediates protein-RNA interactions . ...
Retinitis pigmentosa (RP) is the most common inherited retinal disease characterized by progressive degeneration of photoreceptors and/or retinal pigment epithelium that eventually results in blindness. Mutations in pre-mRNA processing factors ( PRPF3, 4, 6, 8, 31, SNRNP200, and RP9 ) have been linked to 15–20% of autosomal dominant RP (adRP) cases. Current evidence indicates that PRPF mutations cause retinal specific global spliceosome dysregulation, leading to mis-splicing of numerous genes that are involved in a variety of retina-specific functions and/or general biological processes, including phototransduction, retinol metabolism, photoreceptor disk morphogenesis, retinal cell polarity, ciliogenesis, cytoskeleton and tight junction organization, waste disposal, inflammation, and apoptosis. Importantly, additional PRPF functions beyond RNA splicing have been documented recently, suggesting a more complex mechanism underlying PRPF -RPs driven disease pathogenesis. The current review focuses on the key RP- PRPF genes, depicting the current understanding of their roles in RNA splicing, impact of their mutations on retinal cell’s transcriptome and phenome, discussed in the context of model species including yeast, zebrafish, and mice. Importantly, information on PRPF functions beyond RNA splicing are discussed, aiming at a holistic investigation of PRPF -RP pathogenesis. Finally, work performed in human patient-specific lab models and developing gene and cell-based replacement therapies for the treatment of PRPF -RPs are thoroughly discussed to allow the reader to get a deeper understanding of the disease mechanisms, which we believe will facilitate the establishment of novel and better therapeutic strategies for PRPF -RP patients.
... This was a rather unexpected finding because inhibition of tri-snRNP assembly by PRPF31 and PRPF6 knockdown induced Cajal body accumulation of U4 and U6 snRNAs only 24 . We therefore first wanted to confirm that U5 snRNP proteins and specifically PRPF6, which is the U5-specific protein that interacts with U4/U6 di-snRNP and is essential for tri-snRNP assembly 23,46 , are present in U5 snRNP after TSSC4 downregulation. TSSC4 was downregulated in HeLa cells stably expressing PRPF8-GFP 47 , PRPF8-GFP was immunoprecipitated using anti-GFP antibodies and co-precipitation of PRPF6 and other U5-specific proteins assayed by western blotting (Fig. 7a). ...
U5 snRNP is a complex particle essential for RNA splicing. U5 snRNPs undergo intricate biogenesis that ensures that only a fully mature particle assembles into a splicing competent U4/U6•U5 tri-snRNP and enters the splicing reaction. During splicing, U5 snRNP is substantially rearranged and leaves as a U5/PRPF19 post-splicing particle, which requires re-generation before the next round of splicing. Here, we show that a previously uncharacterized protein TSSC4 is a component of U5 snRNP that promotes tri-snRNP formation. We provide evidence that TSSC4 associates with U5 snRNP chaperones, U5 snRNP and the U5/PRPF19 particle. Specifically, TSSC4 interacts with U5-specific proteins PRPF8, EFTUD2 and SNRNP200. We also identified TSSC4 domains critical for the interaction with U5 snRNP and the PRPF19 complex, as well as for TSSC4 function in tri-snRNP assembly. TSSC4 emerges as a specific chaperone that acts in U5 snRNP de novo biogenesis as well as post-splicing recycling.
... In vitro splicing assays were performed as previously described (49). Briefly, nuclear extracts were prepared from HEK293T cells treated with control and SANS siRNAs. ...
... However, SANS deficiency did not change the abundance of U5 snRNP-specific proteins U5-52K and hSNU114 and U5 snRNAs in Cajal bodies. These results may support a role for SANS in the regulation of the tri-snRNP complex assembly which should be evident from the accumulation of the U4/U6 di-snRNP intermediates (47,49) or from the composition of the isolated tri-snRNP complex. However, the abundance of fully maturated tri-snRNP complexes indicated by the presence SART1 and their accumulation in SANS-depleted cells strongly argues against a prominent role of SANS in tri-snRNP complex assembly. ...
Splicing is catalyzed by the spliceosome, a compositionally dynamic complex assembled stepwise on pre-mRNA. We reveal links between splicing machinery components and the intrinsically disordered ciliopathy protein SANS. Pathogenic mutations in SANS/USH1G lead to Usher syndrome—the most common cause of deaf-blindness. Previously, SANS was shown to function only in the cytosol and primary cilia. Here, we have uncovered molecular links between SANS and pre-mRNA splicing catalyzed by the spliceosome in the nucleus. We show that SANS is found in Cajal bodies and nuclear speckles, where it interacts with components of spliceosomal sub-complexes such as SF3B1 and the large splicing cofactor SON but also with PRPFs and snRNAs related to the tri-snRNP complex. SANS is required for the transfer of tri-snRNPs between Cajal bodies and nuclear speckles for spliceosome assembly and may also participate in snRNP recycling back to Cajal bodies. SANS depletion alters the kinetics of spliceosome assembly, leading to accumulation of complex A. SANS deficiency and USH1G pathogenic mutations affects splicing of genes related to cell proliferation and human Usher syndrome. Thus, we provide the first evidence that splicing dysregulation may participate in the pathophysiology of Usher syndrome.
... Pre-mRNAs processing factor 31 (PRPF31) is a constitutive component of spliceosomes, which participates in the assembly and stabilization of U4/U6/U5 tri-snRNP (21)(22)(23). Mutations in PRPF31 have been determined to be lossof-function, resulting in reduced levels of activated snRNPs and decreased splicing efficiency (14,22,24). Remarkably, in patient-derived lymphocytes or siRNA-treated human organotypic retinal cultures, the insufficiency of PRPF31 only impaired the splicing of a subset of genes (22,25). ...
Dysfunction of splicing factors often result in abnormal cell differentiation and apoptosis, especially in neural tissues. Mutations in pre-mRNAs processing factor 31 (PRPF31) cause autosomal dominant retinitis pigmentosa, a progressive retinal degeneration disease. The transcriptome-wide splicing events specifically regulated by PRPF31 and their biological roles in the development and maintenance of retina are still unclear. Here, we showed that the differentiation and viability of retinal progenitor cells (RPCs) are severely perturbed in prpf31 knockout zebrafish when compared with other tissues at an early embryonic stage. At the cellular level, significant mitotic arrest and DNA damage were observed. These defects could be rescued by the wild-type human PRPF31 rather than the disease-associated mutants. Further bioinformatic analysis and experimental verification uncovered that Prpf31 deletion predominantly causes the skipping of exons with a weak 5′ splicing site. Moreover, genes necessary for DNA repair and mitotic progression are most enriched among the differentially spliced events, which may explain the cellular and tissular defects in prpf31 mutant retinas. This is the first time that Prpf31 is demonstrated to be essential for the survival and differentiation of RPCs during retinal neurogenesis by specifically modulating the alternative splicing of genes involved in DNA repair and mitosis.
... Moreover, recent studies have revealed that pre-mRNA splicing depends on ubiquitination and deubiquitination cycles of the spliceosomal component. For example, the tri-snRNP proteins, PRP3 and PRP31, are reported to regulate the spliceosome through ubiquitination and deubiquitination 17,[59][60][61] . First, the ubiquitin ligase, PRP19, containing the U-box spliceosomal protein, ubiquitinates PRP3 and PRP31, and then the ubiquitinated PRP3 and PRP31 complex binds PRP8 and stabilizes the tri-snRNP complex. ...
IK depletion leads to an aberrant mitotic entry because of chromosomal misalignment through the enhancement of Aurora B activity at the interphase. Here, we demonstrate that IK, a spliceosomal component, plays a crucial role in the proper splicing of the ATM pre-mRNA among other genes related with the DNA Damage Response (DDR). Intron 1 in the ATM pre-mRNA, having lengths <200 bp, was not spliced in the IK-depleted cells and led to a deficiency of the ATM protein. Subsequently, the IK depletion-induced ATM protein deficiency impaired the ability to repair the damaged DNA. Because the absence of SMU1 results in IK degradation, the mechanism underlying IK degradation was exploited. IK was ubiquitinated in the absence of SMU1 and then subjected to proteolysis through the 26S proteasome. To prevent the proteolytic degradation of IK, a deubiquitinating enzyme, USP47, directly interacted with IK and stabilized it through deubiquitination. Collectively, our results suggest that IK is required for proper splicing of the ATM pre-mRNA and USP47 contributes toward the stabilization of IK.
