SMN controls neuromuscular junction integrity through U7 snRNP.

The neuromuscular junction (NMJ) is an essential synapse whose loss is a key hallmark of the neurodegenerative disease spinal muscular atrophy (SMA). Here, we show that activity of the SMA-determining SMN protein in the assembly of U7 small nuclear ribonucleoprotein (snRNP)-which functions in the 3'-end processing of replication-dependent histone mRNAs-is required for NMJ integrity. Co-expression of U7-specific Lsm10 and Lsm11 proteins selectively enhances U7 snRNP assembly, corrects histone mRNA processing defects, and rescues key structural and functional abnormalities of neuromuscular pathology in SMA mice-including NMJ denervation, decreased synaptic transmission, and skeletal muscle atrophy. Furthermore, U7 snRNP dysfunction drives selective loss of the synaptic organizing protein Agrin at NMJs innervating vulnerable muscles of SMA mice. These findings reveal a direct contribution of U7 snRNP dysfunction to neuromuscular pathology in SMA and suggest a role for histone gene regulation in maintaining functional synaptic connections between motor neurons and muscles.

Stasimon Contributes to the Loss of Sensory Synapses and Motor Neuron Death in a Mouse Model of Spinal Muscular Atrophy.

Reduced expression of the survival motor neuron (SMN) protein causes the neurodegenerative disease spinal muscular atrophy (SMA). Here, we show that adeno-associated virus serotype 9 (AAV9)-mediated delivery of Stasimon-a gene encoding an endoplasmic reticulum (ER)-resident transmembrane protein regulated by SMN-improves motor function in a mouse model of SMA through multiple mechanisms. In proprioceptive neurons, Stasimon overexpression prevents the loss of afferent synapses on motor neurons and enhances sensory-motor neurotransmission. In motor neurons, Stasimon suppresses neurodegeneration by reducing phosphorylation of the tumor suppressor p53. Moreover, Stasimon deficiency converges on SMA-related mechanisms of p53 upregulation to induce phosphorylation of p53 through activation of p38 mitogen-activated protein kinase (MAPK), and pharmacological inhibition of this kinase prevents motor neuron death in SMA mice. These findings identify Stasimon dysfunction induced by SMN deficiency as an upstream driver of distinct cellular cascades that lead to synaptic loss and motor neuron degeneration, revealing a dual contribution of Stasimon to motor circuit pathology in SMA.

Functional characterization of SMN evolution in mouse models of SMA.

Spinal Muscular Atrophy (SMA) is a monogenic neurodegenerative disorder and the leading genetic cause of infantile mortality. While several functions have been ascribed to the SMN (survival motor neuron) protein, their specific contribution to the disease has yet to be fully elucidated. We hypothesized that some, but not all, SMN homologues would rescue the SMA phenotype in mouse models, thereby identifying disease-relevant domains. Using AAV9 to deliver Smn homologs to SMA mice, we identified a conservation threshold that marks the boundary at which homologs can rescue the SMA phenotype. Smn from Danio rerio and Xenopus laevis significantly prevent disease, whereas Smn from Drosophila melanogaster, Caenorhabditis elegans, and Schizosaccharomyces pombe was significantly less efficacious. This phenotypic rescue correlated with correction of RNA processing defects induced by SMN deficiency and neuromuscular junction pathology. Based upon the sequence conservation in the rescuing homologs, a minimal SMN construct was designed consisting of exons 2, 3, and 6, which showed a partial rescue of the SMA phenotype. While a significant extension in survival was observed, the absence of a complete rescue suggests that while the core conserved region is essential, additional sequences contribute to the overall ability of the SMN protein to rescue disease pathology.

Widespread intronic polyadenylation inactivates tumour suppressor genes in leukaemia.

DNA mutations are known cancer drivers. Here we investigated whether mRNA events that are upregulated in cancer can functionally mimic the outcome of genetic alterations. RNA sequencing or 3'-end sequencing techniques were applied to normal and malignant B cells from 59 patients with chronic lymphocytic leukaemia (CLL)1-3. We discovered widespread upregulation of truncated mRNAs and proteins in primary CLL cells that were not generated by genetic alterations but instead occurred by intronic polyadenylation. Truncated mRNAs caused by intronic polyadenylation were recurrent (n = 330) and predominantly affected genes with tumour-suppressive functions. The truncated proteins generated by intronic polyadenylation often lack the tumour-suppressive functions of the corresponding full-length proteins (such as DICER and FOXN3), and several even acted in an oncogenic manner (such as CARD11, MGA and CHST11). In CLL, the inactivation of tumour-suppressor genes by aberrant mRNA processing is substantially more prevalent than the functional loss of such genes through genetic events. We further identified new candidate tumour-suppressor genes that are inactivated by intronic polyadenylation in leukaemia and by truncating DNA mutations in solid tumours4,5. These genes are understudied in cancer, as their overall mutation rates are lower than those of well-known tumour-suppressor genes. Our findings show the need to go beyond genomic analyses in cancer diagnostics, as mRNA events that are silent at the DNA level are widespread contributors to cancer pathogenesis through the inactivation of tumour-suppressor genes.

Mild SMN missense alleles are only functional in the presence of SMN2 in mammals.

Spinal muscular atrophy (SMA) is caused by reduced levels of full-length SMN (FL-SMN). In SMA patients with one or two copies of the Survival Motor Neuron 2 (SMN2) gene there are a number of SMN missense mutations that result in milder-than-predicted SMA phenotypes. These mild SMN missense mutation alleles are often assumed to have partial function. However, it is important to consider the contribution of FL-SMN as these missense alleles never occur in the absence of SMN2. We propose that these patients contain a partially functional oligomeric SMN complex consisting of FL-SMN from SMN2 and mutant SMN protein produced from the missense allele. Here we show that mild SMN missense mutations SMND44V, SMNT74I or SMNQ282A alone do not rescue mice lacking wild-type FL-SMN. Thus, missense mutations are not functional in the absence of FL-SMN. In contrast, when the same mild SMN missense mutations are expressed in a mouse containing two SMN2 copies, functional SMN complexes are formed with the small amount of wild-type FL-SMN produced by SMN2 and the SMA phenotype is completely rescued. This contrasts with SMN missense alleles when studied in C. elegans, Drosophila and zebrafish. Here we demonstrate that the heteromeric SMN complex formed with FL-SMN is functional and sufficient to rescue small nuclear ribonucleoprotein assembly, motor neuron function and rescue the SMA mice. We conclude that mild SMN missense alleles are not partially functional but rather they are completely non-functional in the absence of wild-type SMN in mammals.

Pharmacokinetics, pharmacodynamics, and efficacy of a small-molecule SMN2 splicing modifier in mouse models of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is caused by the loss or mutation of both copies of the survival motor neuron 1 (SMN1) gene. The related SMN2 gene is retained, but due to alternative splicing of exon 7, produces insufficient levels of the SMN protein. Here, we systematically characterize the pharmacokinetic and pharmacodynamics properties of the SMN splicing modifier SMN-C1. SMN-C1 is a low-molecular weight compound that promotes the inclusion of exon 7 and increases production of SMN protein in human cells and in two transgenic mouse models of SMA. Furthermore, increases in SMN protein levels in peripheral blood mononuclear cells and skin correlate with those in the central nervous system (CNS), indicating that a change of these levels in blood or skin can be used as a non-invasive surrogate to monitor increases of SMN protein levels in the CNS. Consistent with restored SMN function, SMN-C1 treatment increases the levels of spliceosomal and U7 small-nuclear RNAs and corrects RNA processing defects induced by SMN deficiency in the spinal cord of SMNΔ7 SMA mice. A 100% or greater increase in SMN protein in the CNS of SMNΔ7 SMA mice robustly improves the phenotype. Importantly, a ∼50% increase in SMN leads to long-term survival, but the SMA phenotype is only partially corrected, indicating that certain SMA disease manifestations may respond to treatment at lower doses. Overall, we provide important insights for the translation of pre-clinical data to the clinic and further therapeutic development of this series of molecules for SMA treatment.

G3BP-Caprin1-USP10 complexes mediate stress granule condensation and associate with 40S subunits.

Mammalian stress granules (SGs) contain stalled translation preinitiation complexes that are assembled into discrete granules by specific RNA-binding proteins such as G3BP. We now show that cells lacking both G3BP1 and G3BP2 cannot form SGs in response to eukaryotic initiation factor 2α phosphorylation or eIF4A inhibition, but are still SG-competent when challenged with severe heat or osmotic stress. Rescue experiments using G3BP1 mutants show that phosphomimetic G3BP1-S149E fails to rescue SG formation, whereas G3BP1-F33W, a mutant unable to bind G3BP partner proteins Caprin1 or USP10, rescues SG formation. Caprin1/USP10 binding to G3BP is mutually exclusive: Caprin binding promotes, but USP10 binding inhibits, SG formation. G3BP interacts with 40S ribosomal subunits through its RGG motif, which is also required for G3BP-mediated SG formation. We propose that G3BP mediates the condensation of SGs by shifting between two different states that are controlled by the phosphorylation of S149 and by binding to Caprin1 or USP10.

Disease mechanisms and therapeutic approaches in spinal muscular atrophy.

Motor neuron diseases are neurological disorders characterized primarily by the degeneration of spinal motor neurons, skeletal muscle atrophy, and debilitating and often fatal motor dysfunction. Spinal muscular atrophy (SMA) is an autosomal-recessive motor neuron disease of high incidence and severity and the most common genetic cause of infant mortality. SMA is caused by homozygous mutations in the survival motor neuron 1 (SMN1) gene and retention of at least one copy of the hypomorphic gene paralog SMN2. Early studies established a loss-of-function disease mechanism involving ubiquitous SMN deficiency and suggested SMN upregulation as a possible therapeutic approach. In recent years, greater knowledge of the central role of SMN in RNA processing combined with deep characterization of animal models of SMA has significantly advanced our understanding of the cellular and molecular basis of the disease. SMA is emerging as an RNA disease not limited to motor neurons, but one that involves dysfunction of motor circuits that comprise multiple neuronal subpopulations and possibly other cell types. Advances in SMA research have also led to the development of several potential therapeutics shown to be effective in animal models of SMA that are now in clinical trials. These agents offer unprecedented promise for the treatment of this still incurable neurodegenerative disease.

SMN control of RNP assembly: from post-transcriptional gene regulation to motor neuron disease.

At the post-transcriptional level, expression of protein-coding genes is controlled by a series of RNA regulatory events including nuclear processing of primary transcripts, transport of mature mRNAs to specific cellular compartments, translation and ultimately, turnover. These processes are orchestrated through the dynamic association of mRNAs with RNA binding proteins and ribonucleoprotein (RNP) complexes. Accurate formation of RNPs in vivo is fundamentally important to cellular development and function, and its impairment often leads to human disease. The survival motor neuron (SMN) protein is key to this biological paradigm: SMN is essential for the biogenesis of various RNPs that function in mRNA processing, and genetic mutations leading to SMN deficiency cause the neurodegenerative disease spinal muscular atrophy. Here we review the expanding role of SMN in the regulation of gene expression through its multiple functions in RNP assembly. We discuss advances in our understanding of SMN activity as a chaperone of RNPs and how disruption of SMN-dependent RNA pathways can cause motor neuron disease.

SMN is essential for the biogenesis of U7 small nuclear ribonucleoprotein and 3'-end formation of histone mRNAs.

Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by a deficiency in the survival motor neuron (SMN) protein. SMN mediates the assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs) and possibly other RNPs. Here, we investigated SMN requirement for the biogenesis and function of U7--an snRNP specialized in the 3'-end formation of replication-dependent histone mRNAs that normally are not polyadenylated. We show that SMN deficiency impairs U7 snRNP assembly and decreases U7 levels in mammalian cells. The SMN-dependent U7 reduction affects endonucleolytic cleavage of histone mRNAs leading to abnormal accumulation of 3'-extended and polyadenylated transcripts followed by downstream changes in histone gene expression. Importantly, SMN deficiency induces defects of histone mRNA 3'-end formation in both SMA mice and human patients. These findings demonstrate that SMN is essential for U7 biogenesis and histone mRNA processing in vivo and identify an additional RNA pathway disrupted in SMA.

A cell system for phenotypic screening of modifiers of SMN2 gene expression and function.

Spinal muscular atrophy (SMA) is an inherited neurodegenerative disease caused by homozygous inactivation of the SMN1 gene and reduced levels of the survival motor neuron (SMN) protein. Since higher copy numbers of the nearly identical SMN2 gene reduce disease severity, to date most efforts to develop a therapy for SMA have focused on enhancing SMN expression. Identification of alternative therapeutic approaches has partly been hindered by limited knowledge of potential targets and the lack of cell-based screening assays that serve as readouts of SMN function. Here, we established a cell system in which proliferation of cultured mouse fibroblasts is dependent on functional SMN produced from the SMN2 gene. To do so, we introduced the entire human SMN2 gene into NIH3T3 cell lines in which regulated knockdown of endogenous mouse Smn severely decreases cell proliferation. We found that low SMN2 copy number has modest effects on the cell proliferation phenotype induced by Smn depletion, while high SMN2 copy number is strongly protective. Additionally, cell proliferation correlates with the level of SMN activity in small nuclear ribonucleoprotein assembly. Following miniaturization into a high-throughput format, our cell-based phenotypic assay accurately measures the beneficial effects of both pharmacological and genetic treatments leading to SMN upregulation. This cell model provides a novel platform for phenotypic screening of modifiers of SMN2 gene expression and function that act through multiple mechanisms, and a powerful new tool for studies of SMN biology and SMA therapeutic development.

Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration.

Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

eIF5A promotes translation elongation, polysome disassembly and stress granule assembly.

Stress granules (SGs) are cytoplasmic foci at which untranslated mRNAs accumulate in cells exposed to environmental stress. We have identified ornithine decarboxylase (ODC), an enzyme required for polyamine synthesis, and eIF5A, a polyamine (hypusine)-modified translation factor, as proteins required for arsenite-induced SG assembly. Knockdown of deoxyhypusine synthase (DHS) or treatment with a deoxyhypusine synthase inhibitor (GC7) prevents hypusine modification of eIF5A as well as arsenite-induced polysome disassembly and stress granule assembly. Time-course analysis reveals that this is due to a slowing of stress-induced ribosome run-off in cells lacking hypusine-eIF5A. Whereas eIF5A only marginally affects protein synthesis under normal conditions, it is required for the rapid onset of stress-induced translational repression. Our results reveal that hypusine-eIF5A-facilitated translation elongation promotes arsenite-induced polysome disassembly and stress granule assembly in cells subjected to adverse environmental conditions.

Angiogenin-induced tRNA-derived stress-induced RNAs promote stress-induced stress granule assembly.

Angiogenin (ANG) is a secreted ribonuclease that cleaves tRNA to initiate a stress-response program in mammalian cells. Here we show that ANG inhibits protein synthesis and promotes arsenite- and pateamine A-induced assembly of stress granules (SGs). These effects are abrogated in cells transfected with the ANG inhibitor RNH1. Transfection of natural or synthetic 5'- but not 3'-tRNA fragments (tRNA-derived stress-induced RNAs; tiRNAs) induces the phospho-eukaryotic translation initiation factor 2alpha-independent assembly of SGs. Natural 5'-tiRNAs but not 3'-tiRNAs are capped with a 5'-monophosphate that is required for optimal SG assembly. These findings reveal that SG assembly is a component of the ANG- and tiRNA-induced stress response program.

Real-time and quantitative imaging of mammalian stress granules and processing bodies.

Nuclear mRNA domains such as nucleoli, speckles, Cajal bodies, and gems demonstrate that RNA function and morphology are inextricably linked; granular mRNA structures are self-generated in tandem with metabolic activity. Similarly, cytoplasmic compartmentalization of mRNA into mRNP structures such as stress granules (SGs) and processing bodies (PBs) reiterate the link between function and structure; the assembly of SGs and PBs requires mRNA released from disassembling polysomes on translational arrest. SGs contain mRNA still associated with some of the translational machinery, specifically 40S subunits and a subset of translation initiation factors including eIF3, eIF4F, eIF4B, and PABP. PBs also contain mRNA and eIF4E but lack other preinitiation factors and contain instead a number of proteins associated with mRNA decay such as DCP1a, DCP2, hedls/GE-1, p54/RCK. Many other proteins (e.g., argonaute, FAST, RAP-55, TTP) and microRNAs are present in both SGs and PBs, sometimes shepherding specific mRNA transcripts between the translation and decay machineries. Recently, we described markers and methods to visualize SGs and PBs in fixed cells (Kedersha and Anderson, 2007), but understanding the dynamic nature of SGs and PBs requires live cell imaging. This presents unique challenges, because it requires the overexpression of fluorescently tagged SG/PB marker proteins, which can shift the mRNA equilibrium toward SGs or PBs, thus obscuring the result. We describe stably expressed, fluorescently tagged SG and PB markers that exhibit similar behavior to their endogenous counterparts, thus allowing real-time imaging of SGs and PBs.

A functional RNAi screen links O-GlcNAc modification of ribosomal proteins to stress granule and processing body assembly.

Stress granules (SGs) and processing bodies (PBs) are microscopically visible ribonucleoprotein granules that cooperatively regulate the translation and decay of messenger RNA. Using an RNA-mediated interference-based screen, we identify 101 human genes required for SG assembly, 39 genes required for PB assembly, and 31 genes required for coordinate SG and PB assembly. Although 51 genes encode proteins involved in mRNA translation, splicing and transcription, most are not obviously associated with RNA metabolism. We find that several components of the hexosamine biosynthetic pathway, which reversibly modifies proteins with O-linked N-acetylglucosamine (O-GlcNAc) in response to stress, are required for SG and PB assembly. O-GlcNAc-modified proteins are prominent components of SGs but not PBs, and include RACK1 (receptor for activated C kinase 1), prohibitin-2, glyceraldehyde-3-phosphate dehydrogenase and numerous ribosomal proteins. Our results suggest that O-GlcNAc modification of the translational machinery is required for aggregation of untranslated messenger ribonucleoproteins into SGs. The lack of enzymes of the hexosamine biosynthetic pathway in budding yeast may contribute to differences between mammalian SGs and related yeast EGP (eIF4E, 4G and Pab1 containing) bodies.