The contribution and therapeutic implications of IGHMBP2 mutations on IGHMBP2 biochemical activity and ABT1 association.

Mutations within immunoglobulin mu DNA binding protein (IGHMBP2), an RNA-DNA helicase, result in SMA with respiratory distress type I (SMARD1) and Charcot Marie Tooth type 2S (CMT2S). The underlying biochemical mechanism of IGHMBP2 is unknown as well as the functional significance of IGHMBP2 mutations in disease severity. Here we report the biochemical mechanisms of IGHMBP2 disease-causing mutations D565N and H924Y, and their potential impact on therapeutic strategies. The IGHMBP2-D565N mutation has been identified in SMARD1 patients, while the IGHMBP2-H924Y mutation has been identified in CMT2S patients. For the first time, we demonstrate a correlation between the altered IGHMBP2 biochemical activity associated with the D565N and H924Y mutations and disease severity and pathology in patients and our Ighmbp2 mouse models. We show that IGHMBP2 mutations that alter the association with activator of basal transcription (ABT1) impact the ATPase and helicase activities of IGHMBP2 and the association with the 47S pre-rRNA 5' external transcribed spacer. We demonstrate that the D565N mutation impairs IGHMBP2 ATPase and helicase activities consistent with disease pathology. The H924Y mutation alters IGHMBP2 activity to a lesser extent while maintaining association with ABT1. In the context of the compound heterozygous patient, we demonstrate that the total biochemical activity associated with IGHMBP2-D565N and IGHMBP2-H924Y proteins is improved over IGHMBP2-D565N alone. Importantly, we demonstrate that the efficacy of therapeutic applications may vary based on the underlying IGHMBP2 mutations and the relative biochemical activity of the mutant IGHMBP2 protein.

ABT1 modifies SMARD1 pathology via interactions with IGHMBP2 and stimulation of ATPase and helicase activity.

SMA with respiratory distress type 1 (SMARD1) and Charcot-Marie-Tooth type 2S (CMT2S) are results of mutations in immunoglobulin mu DNA binding protein 2 (IGHMBP2). IGHMBP2 is a UPF1-like helicase with proposed roles in several cellular processes, including translation. This study examines activator of basal transcription 1 (ABT1), a modifier of SMARD1-nmd disease pathology. Microscale thermophoresis and dynamic light scattering demonstrate that IGHMBP2 and ABT1 proteins directly interact with high affinity. The association of ABT1 with IGHMBP2 significantly increases the ATPase and helicase activity as well as the processivity of IGHMBP2. The IGHMBP2/ABT1 complex interacts with the 47S pre-rRNA 5' external transcribed spacer and U3 small nucleolar RNA (snoRNA), suggesting that the IGHMBP2/ABT1 complex is important for pre-rRNA processing. Intracerebroventricular injection of scAAV9-Abt1 decreases FVB-Ighmbp2nmd/nmd disease pathology, significantly increases lifespan, and substantially decreases neuromuscular junction denervation. To our knowledge, ABT1 is the first disease-modifying gene identified for SMARD1. We provide a mechanism proposing that ABT1 decreases disease pathology in FVB-Ighmbp2nmd/nmd mutants by optimizing IGHMBP2 biochemical activity (ATPase and helicase activity). Our studies provide insight into SMARD1 pathogenesis, suggesting that ABT1 modifies IGHMBP2 activity as a means to regulate pre-rRNA processing.

The Ighmbp2D564N mouse model is the first SMARD1 model to demonstrate respiratory defects.

Spinal muscular atrophy with respiratory distress type I (SMARD1) is a neurodegenerative disease defined by respiratory distress, muscle atrophy and sensory and autonomic nervous system defects. SMARD1 is a result of mutations within the IGHMBP2 gene. We have generated six Ighmbp2 mouse models based on patient-derived mutations that result in SMARD1 and/or Charcot-Marie Tooth Type 2 (CMT2S). Here we describe the characterization of one of these models, Ighmbp2D564N (human D565N). The Ighmbp2D564N/D564N mouse model mimics important aspects of the SMARD1 disease phenotype, including motor neuron degeneration and muscle atrophy. Ighmbp2D564N/D564N is the first SMARD1 mouse model to demonstrate respiratory defects based on quantified plethysmography analyses. SMARD1 disease phenotypes, including the respiratory defects, are significantly diminished by intracerebroventricular (ICV) injection of ssAAV9-IGHMBP2 and the extent of phenotypic restoration is dose-dependent. Collectively, this model provides important biological insight into SMARD1 disease development.

SMN-inducing compounds for the treatment of spinal muscular atrophy.

Spinal muscular atrophy (SMA) is a leading genetic cause of infant mortality. A neurodegenerative disease, it is caused by loss of SMN1, although low, but essential, levels of SMN protein are produced by the nearly identical gene SMN2. While no effective treatment or therapy currently exists, a new wave of therapeutics has rapidly progressed from cell-based and preclinical animal models to the point where clinical trials have initiated for SMA-specific compounds. There are several reasons why SMA has moved relatively rapidly towards novel therapeutics, including: SMA is monogenic; the molecular understanding of SMN gene regulation has been building for nearly 20 years; and all SMA patients retain one or more copies of SMN2 that produces low levels of full-length, fully functional SMN protein. This review primarily focuses upon the biology behind the disease and examines SMN1- and SMN2-targeted therapeutics.

Disruption of the Survival Motor Neuron (SMN) gene in pigs using ssDNA.

Spinal Muscular Atrophy (SMA) is an autosomal recessive neurodegenerative disease that is a result of a deletion or mutation of the SMN1 (Survival Motor Neuron) gene. A duplicated and nearly identical copy, SMN2, serves as a disease modifier as increasing SMN2 copy number decreases the severity of the disease. Currently many therapeutic approaches for SMA are being developed. Therapeutic strategies aim to modulate splicing of SMN2-derived transcripts, increase SMN2 gene expression, increase neuro-protection of motor neurons, stabilize the SMN protein, replace the SMN1 gene and reconstitute the motor neuron population. It is our goal to develop a pig animal model of SMA for the development and testing of therapeutics and evaluation of toxicology. In the development of a SMA pig model, it was important to demonstrate that the human SMN2 gene would splice appropriately as the model would be based on the presence of the human SMN2 transgene. In this manuscript, we show splicing of the human SMN1 and SMN2 mini-genes in porcine cells is consistent with splicing in human cells, and we report the first genetic knockout of a gene responsible for a neurodegenerative disease in a large animal model using gene targeting with single-stranded DNA and somatic cell nuclear transfer.

C. elegans mitotic cyclins have distinct as well as overlapping functions in chromosome segregation.

Mitotic cyclins in association with the Cdk1 protein kinase regulate progression through mitosis in all eukaryotes. Here, we address to what extent mitotic cyclins in the nematode Caenorhabditis elegans provide overlapping functions or distinct biological activities. C. elegans expresses a single A-type cyclin (CYA-1), three typical B-type cyclins (CYB-1, CYB-2.1 and CYB-2.2), and one B3-subfamily member (CYB-3). While we observed clear redundancies between the cyb genes, cyb-1 and cyb-3 also contribute specific essential functions in meiosis and mitosis. CYB-1 and CYB-3 show similar temporal and spatial expression, both cyclins localize prominently to the nucleus, and both associate with CDK-1 and display histone H1 kinase activity in vitro. We demonstrate that inhibition of cyb-1 by RNAi interferes with chromosome congression and causes aneuploidy. In contrast, cyb-3(RNAi) embryos fail to initiate sister chromatid separation. Inhibition of both cyclins simultaneously results in a much earlier and more dramatic arrest. However, only the combination of cyb-1, cyb-3 and cyb-2.1/cyb-2.2 RNAi fully resembles cdk-1 inhibition. This combination of redundant and specific phenotypes supports that in vivo phosphorylation of certain Cdk targets can be achieved by multiple Cdk1/cyclin complexes, while phosphorylation of other targets requires a unique Cdk1/cyclin combination.

Identification and characterisation of a nuclear localisation signal in the SMN associated protein, Gemin4.

Gemin4 is a ubiquitously expressed multifunctional protein that is involved in U snRNP assembly, apoptosis, nuclear/cytoplasmic transportation, transcription, and RNAi pathways. Gemin4 is one of the core components of the Gemin-complex, which also contains survival motor neuron (SMN), the seven Gemin proteins (Gemin2-8), and Unrip. Mutations in the SMN1 gene cause the autosomal recessive disorder spinal muscular atrophy (SMA). Although the functions assigned to Gemin4 predominantly occur in the nucleus, the mechanisms that mediate the nuclear import of Gemin4 remain unclear. Here, using a novel panel of Gemin4 constructs we identify a canonical nuclear import sequence (NLS) in the N-terminus of Gemin4. The Gemin4 NLS is necessary and independently sufficient to mediate nuclear import of Gemin4. This is the first functional NLS identified within the SMN-Gemin complex.

Identification and characterization of the porcine (Sus scrofa) survival motor neuron (SMN1) gene: an animal model for therapeutic studies.

Spinal muscular atrophy (SMA) is an autosomal recessive disorder that is characterized by the degeneration of the motor neurons of the spinal cord leading to muscle atrophy. SMA is a result of a loss-of-function of the gene survival motor neuron-1 (SMN1). We have chosen to generate a transgenic swine model of SMA for the development and testing of therapeutics and evaluation of toxicology. To this end, we report the first cloning and identification of the swine SMN1 gene and show that there is significant sequence homology between swine and human SMN throughout the coding region. Reverse transcriptase-polymerase chain reaction results demonstrated slight changes in SMN RNA expression during development and in different tissues. In contrast, protein expression profiles were dramatically different based upon different tissues and developmental stages, consistent with human SMN expression. Porcine SMN localization is consistent with human SMN, localizing diffusely within the cytoplasm and in punctate nuclear structures characteristic of nuclear gems. Importantly, transient transfection of porcine SMN1 in 3813 SMA type 1 fibroblasts demonstrate that porcine SMN1 can rescue the deficiency of SMN protein and gem formation in these cells. These studies provide the first characterization of the porcine SMN1 gene and SMN protein and suggest that a transgenic swine SMA model is feasible.

Protein phosphatase 1 binds to the RNA recognition motif of several splicing factors and regulates alternative pre-mRNA processing.

Alternative splicing emerges as one of the most important mechanisms to generate transcript diversity. It is regulated by the formation of protein complexes on pre-mRNA. We demonstrate that protein phosphatase 1 (PP1) binds to the splicing factor transformer2-beta1 (tra2-beta1) via a phylogenetically conserved RVDF sequence located on the RNA recognition motif (RRM) of tra2-beta1. PP1 binds directly to tra2-beta1 and dephosphorylates it, which regulates the interaction between tra2-beta1 and other proteins. Eight other proteins, including SF2/ASF and SRp30c, contain an evolutionary conserved PP1 docking motif in the beta-4 strand of their RRMs indicating that binding to PP1 is a new function of some RRMs. Reducing PP1 activity promotes usage of numerous alternative exons, demonstrating a role of PP1 activity in splice site selection. PP1 inhibition promotes inclusion of the survival of motoneuron 2 exon 7 in a mouse model expressing the human gene. This suggests that reducing PP1 activity could be a new therapeutic principle to treat spinal muscular atrophy and other diseases caused by missplicing events. Our data indicate that the binding of PP1 to evolutionary conserved motifs in several RRMs is the link between known signal transduction pathways regulating PP1 activity and pre-mRNA processing.