Cellular bases of the RNA metabolism dysfunction in motor neurons of a murine model of spinal muscular atrophy: Role of Cajal bodies and the nucleolus

Spinal muscular atrophy (SMA) is a severe motor neuron (MN) disease caused by the deletion or mutation of the survival motor neuron 1 (SMN1) gene, which results in reduced levels of the SMN protein and the selective degeneration of lower MNs. The best-known function of SMN is the biogenesis of spliceosomal snRNPs, the major components of the premRNA splicing machinery. Therefore, SMN deficiency in SMA leads to widespread splicing abnormalities. We used the SMN∆7 mouse model of SMA to investigate the cellular reorganization of polyadenylated mRNAs associated with the splicing dysfunction in MNs. We demonstrate that SMN deficiency induced the abnormal accumulation of poly(A) RNAs in nuclear granules enriched in the splicing regulator Sam68. However, these granules lacked other RNA-binding proteins, such as TDP43, PABPN1, hnRNPA12B, REF and Y14, which are essential for mRNA processing and nuclear export. These effects were associated with changes in the alternative splicing of the Sam68-dependent Bcl-x and Nrnx1 genes, as well as changes in the relative accumulation of the intron-containing Chat, Chodl, Myh9 and Myh14 mRNAs, which are all important for MN functions. Moreover, the massive accumulation of poly(A)


INTRODUCTION
accumulate in nuclear inclusions under pathological conditions, such as oculopharyngeal muscular dystrophy, myotonic dystrophy type 1 and fragile Xassociated tremor/ataxia syndrome (Bengoechea et al., 2012;Klein et al., 2016;Qurashi et al., 2011;Smith et al., 2007). Moreover, we have previously reported that the dysfunction of nuclear RNA processing in the sensory ganglion upon proteasome inhibition induces the nuclear aggregation of polyadenylated mRNAs and the RNAbinding protein Sam68 (src-associated protein in mitosis of 68 kD) into a new nuclear structure called the "poly(A) RNA granule" (PARG) (Casafont et al., 2010;Palanca et al., 2014). The sequestration of crucial RNA-binding proteins in nuclear inclusions or granules may prevent their normal function and contribute to disease pathogenesis (Renoux and Todd, 2012).
Spinal muscular atrophy (SMA) is an autosomal recessive disease characterized by the progressive degeneration and loss of spinal cord and brainstem motor neurons (MNs) (Soler-Botija et al., 2002;Talbot and Tizzano, 2017). SMA is caused by a homozygous deletion or mutation in the survival of motor neuron 1 (SMN1) gene that results in decreased levels of the full-length SMN protein (Burghes and Beattie, 2009;Lefebvre et al., 1995). SMA patients carry a nearly identical SMN1 gene paralogue named SMN2, which differs from SMN1 by a C to T transition in exon 7 (Burghes and Beattie, 2009;Lefebvre et al., 1997). Although both the SMN1 and SMN2 genes encode the SMN protein, approximately 90% of the SMN2 mRNA transcripts generate an alternatively spliced isoform that lacks exon 7 and encodes a truncated form of the SMN protein (SMN∆7) that is rapidly degraded (Monani et al., 2000). Therefore, SMN2 expression cannot fully compensate for the deficiency of the full-length SMN protein.
The best-known function of SMN is the biogenesis of spliceosomal snRNPs (for a review, see (Cauchi, 2010;Matera and Wang, 2014)). Linked to this function, SMN Comentari [Ed1]: Please consistently define abbreviations either before or after the abbreviation throughout the document.

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analysis of the fluorescence hybridization signal intensities at P14. Multiple measurements of the poly(A) RNA signal intensities were performed in the peripheral cytoplasm, where the Nissl substance is largely distributed in MNs. A significant reduction of the relative poly(A) RNA concentration was detected in the Nissl substance of PARG-containing MNs, compared with PARG-free MNs, from both SMN∆7 and WT mice (Fig. 2H).
Electron microscopy analysis of PARG-containing MNs revealed structural features of severe neuronal dysfunction, including a paucity of the protein synthesis machinery, nuclear shape aberrations with numerous nuclear envelope invaginations Importantly, PARGs localized in euchromatin domains, wherein co-transcriptional premRNA processing occurs (Girard et al., 2012;Moore and Proudfoot, 2009), and they frequently appeared in close proximity to the nucleolus (Fig. 3B) and interchromatin granule clusters (Fig. 3A, right inset), the ultrastructural counterpart of nuclear speckles (Lamond and Spector, 2003).

The PARG is a distinct nuclear compartment
To establish the identity of the PARG as a distinct nuclear structure in SMA MNs, we performed double labeling for poly(A) RNA in combination with molecular markers of nuclear compartments, such as coilin (Cajal bodies), SMN (gems), the TMG-cap (nuclear speckles) and the 20S proteasome (clastosomes). In a recent study, we demonstrated that the reduced levels of SMN in MNs from SMN∆7 mice were associated with a severe depletion of Cajal bodies (Tapia O et al., 2017), nuclear structures involved in the biogenesis of both spliceosomal snRNPs and nucleolar snoRNPs (for a review, see (Lafarga et al., 2017;Machyna et al., 2013). Co-staining for poly(A) RNA and coilin confirmed the depletion of typical Cajal bodies in SMN∆7 MNs. Nevertheless, small coilin-positive and poly(A) RNA-negative residual Cajal bodies were occasionally found adjacent to PARGs, but as two clearly distinct nuclear structures (Fig. 4A). Similarly, gems, SMN-positive and coilin-negative nuclear bodies (Lafarga et al., 2017;Liu and Dreyfuss, 1996), were never observed in SMN∆7 MNs, and SMN was not concentrated in PARGs (Fig. 4B). Moreover, PARGs did not concentrate spliceosomal snRNPs, which typically appeared to be enriched in nuclear speckles immunolabeled for the TMG-cap of spliceosomal snRNAs (Fig. 4C). Finally, the catalytic 20S proteasome, a molecular marker of clastosomes, which are nuclear bodies enriched in ubiquitylated proteins and active 20S proteasomes (Lafarga et al., 2002), was not concentrated in PARGs (Fig. 4D).

PARGs concentrate the RNA-binding protein Sam68.
Since the RNA-binding protein Sam68 is a regulator of SMN2 alternative splicing (Pedrotti et al., 2010), we investigated its nuclear reorganization in SMN-deficient MNs from SMN∆7 mice. In WT MNs, co-staining for Sam68 and poly(A) RNA revealed a predominant nuclear localization of Sam68, which excluded the nucleolus and poly(A) RNA-positive nuclear speckles ( Fig. 5A-C). The nuclear distribution was nonhomogeneous, with extensive areas of diffuse staining, and a few irregular domains in which higher levels of Sam68 accumulation were observed (Fig. 5B). Although the basic nuclear pattern of Sam68 immunostaining was preserved in SMN∆7 MNs, this Comentari [Ed4]: Please use a consistent capitalization and punctuation format for section headings throughout the manuscript. Some journals request a specific style, so please review the journal's guidelines.
splicing regulator was strongly concentrated in PARGs ( Fig. 5D-F). Representing the fluorescence intensity profiles of poly(A) RNA and Sam68 across a line confirmed the colocalization of both signals in PARGs from SMN∆7 MNs, as well as the absence of Sam68 in nuclear speckles (Fig. 5G, H). Similarly, immunogold electron microscopy for Sam68 showed that both the compact and the ring-shaped PARGs were decorated with numerous gold particles (Fig. 5I, J).
We next investigated whether the accumulation of Sam68 in PARGs was associated with changes in Sam68 mRNA and protein levels in tissue extracts from the spinal cord at P5 and P14. Although we observed a trend indicating the downregulation of Sam68 mRNA levels in SMN∆7 mice, this decrease was not significant in the qRT-PCR validation (Fig. 5K). Similarly, western blotting analysis revealed no significant changes in Sam68 protein levels in samples from SMN∆7 mice compared with those from WT littermates (Fig. 5L). However, as expected, a severe reduction of SMN protein levels was observed in SMN∆7 mice (Fig. 5L).
Previous studies have demonstrated that Sam68 regulates the alternative splicing of two important genes for neuronal function, Bcl-x and Nrxn1, which encode an apoptotic regulatory factor and the presynaptic membrane protein neurexin, respectively (Iijima et al., 2011;Paronetto et al., 2007a). These studies also suggest that the relocation of Sam68 in nuclear foci affects the alternative splicing of its premRNA targets (Paronetto et al., 2007a). This finding prompted us to investigate whether the partial relocation of this splicing regulator in PARGs is associated with changes in the balance of Bcl-x and Nrxn1 splicing isoforms. The Bcl-x transcript is alternatively spliced to generate the antiapoptotic Bcl-x(L) variant or the proapoptotic Bcl-x(s) variant (Boise et al., 1993). Real-time PCR quantification of these two variants in spinal cord extracts showed a significant decrease of the Bcl-x(s)/Bcl-x(L) ratio in SMN∆7 mice compared with WT mice (Fig. 5M). Similarly, we found a significant reduction of the Nrxn1 4(-)/Nrxn1 4(+) ratio in SMN∆7 mice (Fig. 5M), which reflects a higher relative abundance of the isoform 4(+), which includes exon 20 in Nrxn1 alternatively spliced segment 4 (AS4).

PARGs did not concentrate other RNA-binding proteins involved in mRNA processing and export
Having demonstrated the concentration of Sam68 in PARGs, we then proceeded to Previous studies have demonstrated that SMA severity correlates with the decreased assembly of spliceosomal snRNP complexes, which leads to widespread defects in the splicing of genes expressed in MNs (Doktor et al., 2017;Lotti et al., 2012;Zhang et al., 2008). On this basis, we investigated whether the nuclear accumulation of polyadenylated mRNAs in PARGs was associated with the splicing dysfunction of four genes, Chat, Chodl, Myh9 and Myh14, which are important for MN maturation and synapse development and function (Bäumer et al., 2009;Newell-Litwa et al., 2015).
These genes encode choline acetyltransferase (Chat) and chondrolectin (Chodl), which are processed by the major U2-dependent spliceosome, and the non-muscle myosin II isoforms IIA (Myh9) and IIC (Myh14), which are processed by the minor U12dependent spliceosome. Splicing efficiency was analyzed by estimating the percentage of the unspliced (exon-intron sequence) forms of the Chodl, Chat, Myh9 and Myh14 premRNAs by qRT-PCR in spinal cord RNA extracts. Importantly, we found that in relation to WT samples, samples from P5 SMN7 animals had a significant increase in the proportion of unspliced forms of the Chat and Myh14 mRNAs, while the accumulation of the unspliced forms was extended to the four examined premRNAs, Chodl, Chat, Myh9 and Myh14, during the late symptomatic stage (P14) (Fig. 8A, B).
No significant changes in unspliced Actb (the beta-actin housekeeping gene) premRNAs were detected when samples from WT and SMN∆7 mice were compared at P5 or P14 ( Fig. 8A, B).

DISCUSSION
The present study demonstrates that the SMN deficiency in SMN∆7 MNs affects the  (Lafarga et al., 2017;Liu and Dreyfuss, 1996), have been reported in fetal MNs (Young et al., 2001), but they are absent in postnatal and mature MNs from both WT and SMN∆7 mice (Tapia O et al., 2017). Moreover, we did not detect SMN in PARGs.
PARG-containing MNs were already observed at P5, but they increased in number during the late symptomatic stage (P14), presumably reflecting the wellestablished asynchrony at the beginning of MN degeneration in SMN∆7 mice (Tarabal et al., 2014). We propose that the formation of PARGs in SMN∆7 MNs reflects a stressrelated dysfunction of RNA metabolism, essentially in premRNA splicing. Consistent with this view, premRNA splicing has emerged as a well-known and important target of several stressing agents, resulting in alternative splicing dysregulation and splicing inhibition (Biamonti and Caceres, 2009a). In the case of SMA, several studies demonstrated that the hyperactivation of the endoplasmic reticulum stress pathway and widespread defects in splicing underlie the neurodegeneration observed in SMA MNs (Doktor et al., 2017;Jangi et al., 2017;Lotti et al., 2012;Ng et al., 2015;Zhang et al., 2008). Moreover, we have previously reported the formation of PARGs following proteasome inhibition-induced proteotoxic stress in rat sensory ganglion neurons, an experimental condition that produces a dysfunction of RNA metabolism and a disruption of the protein synthesis machinery (Casafont et al., 2010;Palanca et al., 2014).
An important finding in this study is the accumulation of Sam68 in PARGs.
Sam68 is a member of the STAR (signal transducer and activator of RNA) family of RNA-binding proteins that bind both RNA and DNA and are involved in signal transduction, transcription and alternative splicing regulation (Hartmann et al., 1999;Matter et al., 2002;Rajan et al., 2008;Richard, 2010). As a splicing regulator, Sam68 may promote exon inclusion or exclusion in certain neural premRNAs, including SMN2, Bcl-x and Nrxn 1 (Iijima et al., 2011;Paronetto et al., 2007b;Richard, 2010). Regarding SMN genes, Sam68 is a physiological regulator of SMN2, but not of SMN1, splicing.
Thus, Sam68 directly binds to the SMN2 premRNA and acts as a splicing repressor of exon 7 inclusion in SMN2 transcripts (Pagliarini et al., 2015;Pedrotti et al., 2010). In the present study, we detected no significant changes in the mRNA and protein levels of proteins that link premRNA splicing to nuclear export, (Dreyfuss et al., 2002;Zhou et al., 2000) are not detectable in these granules. Moreover, we observed a reduction of the levels of PABPN1, a protein that, in addition to polyadenylation, is also involved in mRNA export from the nucleus (Apponi et al., 2010). Therefore, a deficiency of this export factor in SMN∆7 MNs could also contribute to the accumulation of poly(A)
Our results extend the splicing defects in SMA MNs. Thus, we demonstrate here the accumulation of incompletely spliced (intron-containing) Chat, Chodl, Myh9 and In conclusion, SMN deficiency in SMN∆7 MNs causes a severe mRNA metabolism dysfunction, resulting in the abnormal nuclear accumulation of polyadenylated RNAs in PARGs, as well as the cytoplasmic depletion of these RNAs.
We propose that the accumulation of incompletely processed polyadenylated mRNAs interferes with their export from the nucleus and affects their translation efficiency. The present study provides additional support for the hypothesis that the dysfunction of nuclear mRNA metabolism plays a critical role in MN degeneration and consequently in SMA pathogenesis.

In situ hybridization and quantification
Comentari [Ed6]: Please be consistent in the manufacturer information provided (e.g., state/province and country only or city, state/province, and country) for specialized equipment, software, and reagents. Some journals request a specific style, so please review the journal's guidelines. for 30 min and the primary antibody for 2 h at 37°C. After washing, the sections were incubated with the specific secondary antibodies coupled to either 10 nm or 15 nm gold particles (BioCell, UK; diluted 1:50 in PBS containing 1% BSA). Following immunogold labeling, the grids were stained with lead citrate and uranyl acetate and examined with a JEOL 201 electron microscope. As controls, ultrathin sections were treated as described above without the primary antibody.

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SDS-PAGE and immunoblotting
Spinal cords from WT (n=3) and SMN7 mice (n=5)         although the nature of the RNA generated in these foci has not been reported (Busà et al., 2010). Collectively, these results support the idea that the formation of distinct categories of Sam68-positive nuclear bodies/foci is part of the cellular stress response to severe RNA metabolism dysfunction.