Splicing Defects of the Profilin Gene Alter Actin Dynamics in an S. pombe SMN Mutant

Summary

Spinal muscular atrophy (SMA) is a devastating motor neuron disorder caused by mutations in the survival motor neuron (SMN) gene. It remains unclear how SMN deficiency leads to the loss of motor neurons. By screening Schizosaccharomyces pombe, we found that the growth defect of an SMN mutant can be alleviated by deletion of the actin-capping protein subunit gene acp1⁺. We show that SMN mutated cells have splicing defects in the profilin gene, which thus directly hinder actin cytoskeleton homeostasis including endocytosis and cytokinesis. We conclude that deletion of acp1⁺ in an SMN mutant background compensates for actin cytoskeleton alterations by restoring redistribution of actin monomers between different types of cellular actin networks. Our data reveal a direct correlation between an impaired function of SMN in snRNP assembly and defects in actin dynamics. They also point to important common features in the pathogenic mechanism of SMA and ALS.

Graphical Abstract

Highlights

• •Splicing defects in the profilin gene in an S. pombe SMN mutant
• •SMN mutant contains excessively polymerized actin
• •Altered actin dynamics in the SMN mutant hinders endocytosis and cytokinesis
• •Deletion of the acp1 subunit restores actin dynamics in the SMN mutant

Biological Sciences; Molecular Biology; Molecular Genetics; Cell Biology

Introduction

Spinal muscular atrophy (SMA) is a common recessive genetic disease and is the leading genetic cause of infant mortality. For 98% of patients, SMA is caused by deletions or mutations in the survival motor neuron gene 1 (SMN1) (Lefebvre et al., 1995, Lorson et al., 1999). The SMN protein is ubiquitously expressed and interacts with a large number of factors, indicating its involvement in multiple biological pathways.

The best-characterized function of the SMN complex is its role in the biogenesis of spliceosomal small nuclear ribonucleoprotein (snRNP) particles, which are major constituents of the spliceosome, the nuclear pre-mRNAs processing machinery (Liu et al., 1997, Fischer et al., 1997, Pellizzoni et al., 2002, Battle et al., 2006, Chari et al., 2009). Several studies have reported that SMN deficiency alters the stoichiometry of snRNPs in SMN-deficient mouse tissues and causes widespread and tissue-specific pre-mRNA splicing defects in SMA mouse models (Gabanella et al., 2007, Zhang et al., 2008, Baumer et al., 2009). There is also evidence that SMN deficiency gives rise to differential defects and alterations in the splicing of various genes (Boulisfane et al., 2011, Lotti et al., 2012, Doktor et al., 2017). These results support the view that SMA might arise from the inefficient splicing of pre-mRNAs coding for proteins required for motor neurons function and/or survival. However, a non-splicing role is also possible, as axons contain SMN complexes lacking Sm proteins but containing other RNA-binding proteins, suggesting a function for SMN in the assembly of ribonucleoproteins involved in the regulation of mRNA transport, stability, and/or local translation (Burghes and Beattie, 2009, Rossoll and Bassell, 2009, Fallini et al., 2012).

SMN also interacts with proteins associated with cytoskeletal dynamics (Bowerman et al., 2009, Fallini et al., 2012). Integrity of the cytoskeleton is essential for correct function of the synaptic terminal as well as for the axons, and several reports indicate that cytoskeletal dynamics might be impaired in SMA. Indeed, SMN was shown to interact and to co-localize in motor neurons with the small actin-binding proteins profilin I and II, and SMN knockdown induced a stronger interaction between profilin-2 and its upstream kinase ROCK (Giesemann et al., 1999, Bowerman et al., 2007, Bowerman et al., 2009). Involvement of the actin cytoskeleton in SMA is also based on studies showing that the actin-bundling protein Plastin 3 acts as a protective modifier of SMA in humans (Oprea et al., 2008). Accordingly, PLS3 overexpression rescues axon outgrowth defects in SMN-deficient zebrafish embryos, in cultured motor neurons, and in mouse models of SMA (Oprea et al., 2008, Hao le et al., 2012, Ackermann et al., 2013, Kaifer et al., 2017). SMN deficiency resulted also in impaired endocytosis in various models of SMA including human, mice, zebrafish, C. elegans, or Drosophila (Dimitriadi et al., 2016, Hosseinibarkooie et al., 2016, Riessland et al., 2017, Janzen et al., 2018). Importantly, endocytosis defects can also be rescued by genetic modifiers such as PLS3 (Hosseinibarkooie et al., 2016), NCALD (Riessland et al., 2017), and CHP1 (Janzen et al., 2018).

As there is no SMN homolog in the budding yeast Saccharomyces cerevisiae, a detailed analysis of the SMN functions has never been possible using genetic and biochemical approaches in this model system. In contrast, the fission yeast Schizosaccharomyces pombe contains an SMN gene, which is essential for growth. In this work, we used a genetic approach to find genes able to modulate growth of fission yeast cells carrying a hypomorphic temperature-degron SMN (tdSMN) mutant, which has differential snRNP assembly and splicing defects (Campion et al., 2010). We report that deletion of the acp1⁺ gene encoding a subunit of the heterodimeric actin-capping protein has a protective effect on this tdSMN mutant. We found also that tdSMN cells contain lower levels of profilin and have excessively polymerized and stable actin networks leading to delays in endocytosis, cytokinesis, and cellular growth. Our work provides a framework for understanding how actin dynamics might become altered in SMN-deficient cells.

Results

The acp1⁺ Gene Is a Protective Modifier for SMN-deficient S. pombe Cells

To characterize biological pathways connected to SMN, we focused on a hypomorphic fission yeast tdSMN mutant displaying a growth defect even at the permissive temperature (Campion et al., 2010). We took an Epistatic MiniArray Profiles (E-MAP) approach (Collins et al., 2010) to screen for deletion strains that either enhance or suppress the tdSMN growth defect. As shown in Table S1, we identified 10 hits with significant scores, which include four suppressors and six enhancers. Remarkably, the vast majority of the encoded proteins have human homologs (Table S1). As expected and based on known links between splicing, chromatin structure, and transcription (Naftelberg et al., 2015), several identified genes have functions in chromatin remodeling, transcription, protein transport, and dephosphorylation. Further validation of the E-MAP screen was provided by identification of the deletion of the fission yeast icln⁺ gene, which encodes a subunit of the PRMT5-complex known to act with the SMN complex in early steps of snRNP biogenesis (Meister et al., 2001, Chari et al., 2008, Barbarossa et al., 2014), as an enhancer of tdSMN growth defect.

To decipher the molecular bases of the protective effects of modifier genes and due to potential links between deregulation of actin dynamics and SMA pathogenesis (Oprea et al., 2008, Bowerman et al., 2009), we focused on the protective/modifier gene acp1⁺ (actin-capping protein of muscle Z-line subunit alpha 1, CAPZA1 in human), which together with acp2⁺, encodes the heterodimeric actin-capping protein (CP) binding to the fast growing barbed ends of actin filaments. Neither acp1⁺ nor acp2⁺ are required for cell viability, and S. pombe cells lacking either capping protein subunits have normal morphology at 25°C (Nakano et al., 2001, Kovar et al., 2005). Throughout this work, we examined the effects of tdSMN and acp1Δ on cell growth, protein levels, and actin assembly at the permissive temperature (25°C) because tdSMN cells already display snRNP assembly, splicing, and growth defects at 25°C (Campion et al., 2010). The suppressive phenotype of acp1Δ was confirmed by a growth assay using serial dilutions of wild-type, tdSMN, and tdSMN acp1Δ strains (Figure S1A), which showed that the double mutant is slightly healthier than the single tdSMN strain. Growth curves also showed a slight improvement in growth upon deletion of acp1⁺ in the tdSMN background (Figure S1B).

tdSMN Cells Contain Higher Levels of Filamentous Actin

To characterize the molecular basis explaining the protective effect of acp1⁺ deletion on the tdSMN mutant, we first characterized the filamentous/globular (F/G)-actin ratio in wild-type, tdSMN, and tdSMN acp1Δ strains. As shown in Figure 1A, when actin is prepared using NaOH/TCA treated extracts, the total amount of actin is similar in all three strains. However, when actin is prepared by differential centrifugation following the protocol of the cytoskeleton in vivo F/G-actin assay kit, we found that monomeric G-actin is barely detectable in all strains, whereas F-actin is easily detected and migrates similarly to control rabbit skeletal muscle actin as a 42 kDa protein (Figure 1A). Interestingly, quantification of the blot showed that F-actin is found at lower levels in the wild-type and tdSMN acp1Δ cells compared with tdSMN cells (Figures 1B and S2).

Figure 1.

Increased Levels of Filamentous Actin in S. pombe SMN-deficient Cells

(A) Whole-cell extract and F/G-actin fractions were prepared as described in Experimental Procedures, and equal amounts of fractions for each strain were loaded onto SDS-PAGE gels. Immunoblot was done using the AAN01 actin antibody. Representative data from three independent experiments are shown. A lane containing 100 ng of rabbit skeletal muscle actin was included as control.

(B) Changes in the levels of F-actin observed on blots were quantified using ImageJ. Data are from three independent experiments. Data are presented as mean ± SEM for each category. (**) p < 0.01, (*) p < 0.05, (ns) non-significant, Tukey's multiple comparison test.

(C) Actin distribution in wild-type, tdSMN, and tdSMN acp1Δ suppressor cells. The cells were grown at 25°C, fixed with paraformaldehyde and stained for actin with Alexa Fluor 488 phalloidin. DNA was stained with DAPI (blue). Fluorescence was quantified using ImageJ from n > 200 cells for each strain with three trials per genotype. Images were acquired using identical parameters. Data are presented as mean ± SEM for each category. (***) p < 0.001, (ns) non-significant, Tukey's multiple comparison test.

(D) Actin distribution was examined in wild-type and acp1Δ cells in conditions similar to those described in 1C. Quantification was performed using ImageJ from n = 30 cells with three trials per genotype. (***) p < 0.001, two-sided unpaired Student t tests.

(E) Latrunculin A sensitivity of strains. Cells were grown in YES media, and serial dilutions were spotted on YES plates containing no or the indicated concentrations of latrunculin A (LatA).

To confirm levels of F-actin in the strains, quantitative fluorescence microscopy was performed on fixed cells stained with Alexa Fluor 488 phalloidin, which is known to bind tightly and selectively to F-actin polymers. Stacks (z series) of images were acquired at high (63×) magnification, and one section was used to calculate the background-corrected total cell fluorescence (CTCF) from n > 200 cells using ImageJ. Representative images of phalloidin staining are shown in Figures 1C and 1D as well as in Figure S3 for the strains that were grown exponentially under similar conditions. Quantification of the fluorescence intensity showed increased fluorescence in tdSMN, tdSMN acp1Δ, and acp1Δ cells compared with wild type (Figures 1C and 1D), indicating increased levels of F-actin.

We reasoned that higher levels of F-actin in the tdSMN mutant could have an impact on actin dynamics. To test this, we monitored the sensitivity of the strains to latrunculin A (LatA), which is a potent inhibitor of F-actin polymerization and has been shown to cause disruption of the actin cytoskeleton (Fujiwara et al., 2018). As shown in Figure 1E, all strains grew on rich media and were sensitive to 0.5 μM LatA. However, on plates containing lower amounts of LatA (0.1 μM), wild-type and tdSMN cells were resistant, whereas tdSMN acp1Δ and acp1Δ cells were hypersensitive. Remarkably, tdSMN cells were found to be considerably resistant to 0.3 μM LatA, whereas growth of wild-type, tdSMN acp1Δ, and acp1Δ cells was inhibited. Altogether, these experiments demonstrate that actin dynamics is altered in tdSMN cells and that the tdSMN mutant contains excessively polymerized actin compared with wild-type and suppressor cells.

Analysis of Actin Distribution In Vivo

To investigate actin dynamics in live cells, we constructed strains carrying a Pact1-LifeAct-GFP reporter, which allows visualization of the actin cytoskeleton in living cells (Huang et al., 2012). Cells expressing this reporter resemble wild-type cells in morphology, growth rate, and cell division (Huang et al., 2012), indicating that any effects of the reporter on actin dynamics are only subtle (Courtemanche et al., 2016). Expression levels of the LifeAct-GFP reporter were similar in wild-type, tdSMN, tdSMN acp1Δ suppressor, and acp1Δ cells (Figure S4), and all four strains contained the three F-actin structures present in S. pombe: endocytic actin patches, cables, and contractile actin rings (Figure 2A). There were clear similarities in the distribution of F-actin in wild-type, tdSMN acp1Δ, and acp1Δ cells, with all three strains having more bipolar actin and fewer cells with contractile actin rings (Figure 2B). In contrast, the actin patches in tdSMN cells often had a monopolar distribution, and the number of tdSMN cells containing bipolar actin decreased two-fold. There were also more tdSMN cells with actin localized at the contractile actin ring compared with wild-type, suppressor, and acp1Δ cells (Figure 2B). As shown in Figure 2C, wild-type and acp1Δ monopolar cells had a homogeneous size around 8 μm, whereas tdSMN cells were more heterogeneous with elongated cells reaching 15 μm or more, and the sizes of tdSMN acp1Δ suppressor cells were more similar to wild-type cells and acp1Δ cells. Of the cells with actin localized at the cytokinetic ring, tdSMN cells were twice as long compared with wild type, whereas the size of the suppressor cells is between that of wild-type and tdSMN cells (Figures 2C and S5). Altogether, these results indicate that tdSMN cells have defective actin localization and delayed cytokinesis, which can be partially restored by deletion of the acp1⁺ gene.

Figure 2.

Cytokinesis Defect of tdSMN Mutant Cells

(A) Comparison of LifeAct-GFP-labeled F-actin structures in wild-type, tdSMN, tdSMN acp1Δ suppressor, and acp1Δ cells. Fluorescence micrographs show that the S. pombe actin networks are observed in all four strains. The bar represents 3 μm.

(B) Quantification of (A) showing the percentage of cells with corresponding actin distribution for each indicated strain. Monopolar, actin localized at one end; bipolar, actin at both ends; cytokinesis, actin localized at the cytokinetic ring. Data are represented as the mean from three separate experiments (n = 100) with error bars showing the SD.

(C) Quantification of (A) showing the percentage of cells with corresponding cell sizes (in μm) for cells having actin localized as monopolar, bipolar, or at the cytokinetic ring. Results are from three independent measurements (n = 100). Quantification data are also shown as histograms in Figure S5.

tdSMN Cells Display Splicing Defects in the Profilin Gene

Actomyosin ring formation is a highly complex and dynamic process requiring actin remodeling and de novo actin assembly at the division site (Pollard, 2016, Suarez and Kovar, 2016). This actin remodeling is orchestrated by actin-binding proteins including profilin (cdc3⁺), cofilin (adf1⁺), and coronin (crn1⁺). Given that the S. pombe genes coding for these regulators contain introns and due to the snRNP assembly and splicing defects observed in the hypomorphic tdSMN mutant (Campion et al., 2010), we wondered whether genes coding for these actin-binding proteins might be mis-spliced. Splicing of those three genes were thus analyzed by RT-PCR and as shown in Figure 3A, strong splicing defects can be observed for profilin with clear accumulation of pre-messenger RNAs and a decrease in the level of mature messenger (mRNA) both in the tdSMN mutant and tdSMN acp1Δ suppressor cells but not in acp1Δ cells (Figure S4C). In contrast, no significant splicing defects could be found for cofilin and coronin genes (Figures 3B and 3C, respectively). The decrease in the level of mature profilin mRNA leads to about 80% decrease in the level of profilin protein in tdSMN and tdSMN acp1Δ cells as measured by Western blot analysis using antibodies directed against S. pombe profilin (Figures 3D and 3E).

Figure 3.

Fission Yeast SMN-deficient Cells Display Splicing Defects in the Profilin Gene

(A–C) RT-PCR analyses were performed on total RNA isolated from the wild-type, tdSMN mutant, and tdSMN acp1Δ suppressor grown at 25°C. The PCR products were separated on a 1.5% agarose gel and visualized by ethidium bromide staining. Tests were performed for introns located in the S. pombe cdc3⁺ gene coding for profilin (A), the adf1⁺ gene coding for cofilin (B), and the crn1⁺ gene coding for coronin (C). The marker corresponds to the invitrogen 1Kb Plus DNA ladder.

(D) Western blot of extracts prepared from the indicated strains using antibodies directed against S. pombe profilin. For loading control, GAPDH levels were determined.

(E) Quantification of (D) with three trials, with error bars showing the SD.

(F) Live-cell GFP images of Crn1-GFP fusion in wild-type, tdSMN mutant, tdSMN acp1Δ suppressor, and acp1Δ cells. DNA was stained with DAPI (blue). See Figure S7 for representative images.

(G) Quantification of intensity (a) (n = 30) and lifetime (b) (n = 20) of endocytic Crn1-GFP patches. Data are presented as dot plots with mean. ns (non-significant), *** (p < 0.001), and ** (p < 0.01) represent statistical significance as determined by ANOVA with Tukey's post-hoc test.

Increased Crn1-GFP Fluorescence and Lifetime of Endocytic Patches in tdSMN acp1Δ Suppressor Cells

As mentioned above, profilin, cofilin, and coronin are important molecules for actin-filament remodeling and disassembly. In order to investigate localization of these proteins, we tried to construct tagged versions of the genes by inserting epitopes using homologous recombination. We were unable to obtain tagged genes for profilin and cofilin, and this is due to the fact that the tagged fusion genes are not functional (Wittenmayer et al., 2000, Okreglak and Drubin, 2007). However, we obtained a strain carrying a coronin-GFP fusion gene expressed from its endogenous promoter. As shown by Western blot using anti-GFP antibodies, expression of the fusion protein is similar in wild-type, tdSMN, tdSMN acp1Δ suppressor, and acp1Δ strains (Figure S6). It has been previously reported that Crn1-GFP localizes in patches that are coincident with actin (Sirotkin et al., 2010). Accordingly, Crn1-GFP fluorescence can be observed as patches at the cell tips as well as at the cell division sites in all three strains (Figures 3F and S7). We quantified coronin fluorescence in live cells as a measure of actin accumulation in endocytic patches, and we found that levels of Crn1-GFP are increased in tdSMN acp1Δ suppressor and in acp1Δ cells (Figure 3G, panel a). Moreover, although identical in wild-type and tdSMN cells, the lifetime of patches is also increased in tdSMN acp1Δ and acp1Δ cells (Figure 3G, panel b). Consistent with studies showing that deletion of acp1⁺ increases the peak amount of actin in endocytic patches (Berro and Pollard, 2014), our results indicate that endocytic patches in tdSMN acp1Δ suppressor and acp1Δ cells might contain higher levels of coronin and actin compared with wild-type and tdSMN cells.

Deletion of acp1⁺ in the tdSMN Mutant Rescues Partially the Endocytic Process

Because defects in actin dynamics result in perturbations of endocytosis (Gachet and Hyams, 2005), we measured endocytosis using an internalization assay with FM4-64, an amphiphilic fluorescent dye entering cells through endocytosis (Vida and Emr, 1995). To examine the transport of FM4-64, cells were first incubated with the dye on ice and then chased at 25°C for various times. As shown in Figures 4A and 4B, the dye is transported efficiently into wild-type and acp1Δ cells. It is also incorporated in the suppressor cells, whereas in tdSMN cells, labeling is highly decreased even after 30 min. Quantification analyses (Figure 4C) confirm that FM4-64 was internalized more slowly in tdSMN cells, suggesting that endocytosis is altered in the tdSMN hypomorphic mutant. They show also that deletion of acp1⁺ rescues partly the endocytic process in the tdSMN acp1Δ suppressor cells.

Figure 4.

Fission Yeast SMN-deficient Cells Display an Endocytosis Defect

(A and B) Analysis of endocytosis was performed for the indicated strains using the lipophilic dye, FM4-64. After incubation with FM4-64 on ice for the indicated time, cells were transferred to an imaging chamber, and images were captured in a single focal plan and the light intensity recorded for 20 cells at each time point. Representative images of cells are shown. Scale bars represent 4 μm.

(C) Quantification of (A) and (B). Data are represented as the mean ± SEM from three separate experiments (n = 20).

Discussion

Yeast models have been proven to be relevant research tools to study biological pathways and to identify components and mechanisms of many essential cellular processes. They have also been used for identifying the functions of genes related to human diseases, including neurodegenerative diseases (for review, see Fruhmann et al., 2017). In this work, using S. pombe, we demonstrate a clear correlation between an impaired function of SMN in snRNP assembly and defects in actin dynamics. We show that SMN deficiency gives rise to splicing defects in the profilin gene leading to decreased cellular levels of profilin. No interaction could be detected between S. pombe SMN and profilin by two-hybrid assays (Figure S8), indicating that the observed splicing defects are responsible for the alteration of actin dynamics in the tdSMN mutant. Indeed, profilins constitute a family of small monomeric G-actin-binding proteins that are structurally highly conserved and essential for the regulation of filamentous F-actin networks (Jockusch et al., 2007). In both mammalian and yeast cells, overproduction of profilin enhances linear actin arrays and inhibits short-branched actin networks, whereas lower level of profilin favors such branched actin patches (Burke et al., 2014, Suarez et al., 2015, Rotty et al., 2015). It is also known that modifying profilin levels alters specific F-actin networks and leads to defects in cytokinesis in various cells including fission yeast, worms, and mice (Balasubramanian et al., 1994, Severson et al., 2002, Witke et al., 2001). Our results are thoroughly consistent with profilin being required for proper distribution of actin to different homeostatic F-actin networks (Suarez and Kovar, 2016, Pollard, 2016, Carlier and Shekhar, 2017).

As splicing of profilin is not rescued by deletion of acp1⁺, the acp1Δ modifier/protective effect is likely downstream. Accordingly, the heterodimeric capping protein binds to free F-actin barbed-ends with high affinity (Winkelman et al., 2014, Shekhar et al., 2015) and competes also with other barbed-ends regulators such as formins (Kovar et al., 2005) or Aip1 (Berro and Pollard, 2014). It is also known that binding of profilin to barbed-ends enhances the dissociation of actin from barbed ends and thus destabilizes the actin filaments (Jégou et al., 2011, Pernier et al., 2016). Our results are consistent with these observations and support the model for actin dynamics depicted in Figure 5. In this model, due to decreased levels of profilin in the tdSMN mutant, profilin is unable to compete with proteins binding to barbed ends including the capping protein, which can therefore bind to excessive numbers of barbed ends, enhancing stabilization of actin filaments. As a consequence, the pool of actin available for the formation of new actin networks is low, and this generates the endocytosis and cytokinesis delays observed in the tdSMN mutant. Upon deletion of the acp1⁺ gene in tdSMN cells, the capping protein is not functional enabling actin depolymerization from barbed ends. This refills the pool of polymerizable actin restoring actin turnover/distribution to other networks and alleviates partially the tdSMN growth defect. The tdSMN acp1Δ suppressor exhibits also increased binding of coronin as shown by enhanced fluorescence of actin patches in cells expressing an endogenous Crn1-GFP reporter (Figure 3G). Because it has been reported that coronin cooperates with actin-binding regulators to sever older filaments (Gandhi and Goode, 2008), the suppressive mechanism of acp1⁺ deletion in the tdSMN background could indeed be due to increased availability of actin molecules. Our results are in agreement with the view that profilin orchestrates actin homeostasis and with the emerging notion that competition for limited amounts of actin monomers represents an important regulatory mechanism for the organization of actin cytoskeletal networks and for cellular growth (Suarez and Kovar, 2016, Pollard, 2016, Carlier and Shekhar, 2017).

Figure 5.

Model of Actin Dynamics in S. pombe Wild-Type, tdSMN Mutant, and tdSMN acp1Δ Suppressor Cells

Upper panel: correct actin turnover is achieved in wild-type cells because profilin can compete with the functional capping protein, for binding to barbed ends of actin filaments. Middle panel: in the tdSMN mutant, defective splicing downregulates profilin, which perturbs actin turnover by allowing excessive binding of capping protein. Lower panel: deletion of the acp1 subunit of the capping protein in the tdSMN mutant removes functional capping protein and thus restores actin dynamics by allowing depolymerisation of actin from barbed ends. This generates increased redistribution of actin monomer to the different actin networks. Pointed (−) and barbed (+) ends are indicated. See also Discussion for details.

Our work shows also that tdSMN cells contain extensively polymerized actin networks, which are more resistant to depolymerization as shown by increased growth on plates containing LatA (Figure 1E). Although F-actin dynamics and localization are also impaired in SMA mouse models (Ackermann et al., 2013, Gabanella et al., 2016), it is interesting to note that levels of F-actin are significantly reduced upon SMN knockdown at the presynaptic site at the NMJ structure as well as in NSC34 and HEK293T cells (Ackermann et al., 2013, Hosseinibarkooie et al., 2016). As depicted in Figure 6, this apparent contradiction in the amounts of F-actin found in the S. pombe tdSMN mutant and SMA cells could be explained by the fact that splicing defects of an unidentified actin regulator, different of profilin, might be responsible for the observed alterations in actin dynamics in mammalian models of SMA. According to this view, defective splicing has never been observed for profilin genes in studies aimed to characterize splicing defects in mammalian SMA models (Zhang et al., 2008, Baumer et al., 2009, Lotti et al., 2012).

Figure 6.

Schematic Depiction of Events Leading to Alterations and Rescues of Actin Dynamics in SMN-deficient S. pombe and Mammalian SMA Models

In the tdSMN mutant, due to splicing defects, profilin levels are decreased, giving rise to higher levels of F-actin and altering actin dynamics. Correct actin dynamics can be restored by deletion of the acp1⁺ gene. For SMA in mammals, we propose that splicing defects lead to decreased levels of an unidentified actin regulator, and this results in lower levels of F-actin and alterations in actin dynamics, which can be suppressed by overexpression of PLS3 or CORO1C (Hosseinibarkooie et al., 2016, Kaifer et al., 2017).

A stabilized actin network could also explain the observed perturbation of the endocytic pathway in tdSMN cells (Figure 4). As already mentioned in the Introduction, defects in endocytosis were also observed when SMN levels were compromised in various SMA models (Dimitriadi et al., 2016, Hosseinibarkooie et al., 2016, Riessland et al., 2017, Janzen et al., 2018). In mice particularly, it has been shown that endocytic defects can be restored by overexpression of Plastin 3 or Coronin 1C, which are F-actin-binding and -bundling proteins. Remarkably, the capping protein was also co-immunoprecipitated from PLS3-overexpressing HEK293T cells, indicating that it is part of a unique functional complex (Hosseinibarkooie et al., 2016). It is thus possible that restoration of actin dynamics might also represent the mechanism leading to the rescue of endocytic trafficking observed in SMA mice.

Several studies have also identified mutations in the PFN1 gene in human familial amyotrophic lateral sclerosis (ALS) patients (Smith et al., 2015). Accordingly, novel mouse models carrying profilin-1 mutations show abnormal F/G-actin ratio in spinal cords and have physical and mental ALS-like phenotypes (Fil et al., 2017). Given that cytoskeletal defects in the brain and spinal cord tissues emerge as one of the most important causes of motor neuron vulnerability, it is tempting to speculate that SMA could also be caused by splicing alterations in genes playing a role in actin dynamics (Figure 6). In this regard, a recent transcriptomic study has reported a significant downregulation of the axon-guidance pathway in spinal cords of postnatal day 5 SMA mice (Doktor et al., 2017). This is particularly intriguing because formation of axons is dependent on crosstalks between actin filaments and the microtubule cytoskeleton (Blanquie and Bradke, 2018). Future work will be necessary to define precisely the splicing pattern of many attractive candidates including genes coding for the various actin-binding partners, many isoforms being implicated in the assembly of different actin cellular structures.

Limitations of the Study

Our study shows that splicing defects in the profilin gene occur in the fission yeast tdSMN hypomorphic mutant and lead to alterations in actin dynamics. However, given that splicing capabilities are lowered in tdSMN cells, it is likely that additional splicing defects take place in other pre-mRNAs, indicating that multiple cellular processes might be affected in the tdSMN mutant. Moreover, the direct relevance of this work to SMA disease progression remains to be determined using in vivo studies in mammalian systems.

Methods

All methods can be found in the accompanying Transparent Methods supplemental file.

Published: January 24, 2020