The neurodegenerative diseases ALS and SMA are linked at the molecular level via the ASC-1 complex.

Understanding the molecular pathways disrupted in motor neuron diseases is urgently needed. Here, we employed CRISPR knockout (KO) to investigate the functions of four ALS-causative RNA/DNA binding proteins (FUS, EWSR1, TAF15 and MATR3) within the RNAP II/U1 snRNP machinery. We found that each of these structurally related proteins has distinct roles with FUS KO resulting in loss of U1 snRNP and the SMN complex, EWSR1 KO causing dissociation of the tRNA ligase complex, and TAF15 KO resulting in loss of transcription factors P-TEFb and TFIIF. However, all four ALS-causative proteins are required for association of the ASC-1 transcriptional co-activator complex with the RNAP II/U1 snRNP machinery. Remarkably, mutations in the ASC-1 complex are known to cause a severe form of Spinal Muscular Atrophy (SMA), and we show that an SMA-causative mutation in an ASC-1 component or an ALS-causative mutation in FUS disrupts association between the ASC-1 complex and the RNAP II/U1 snRNP machinery. We conclude that ALS and SMA are more intimately tied to one another than previously thought, being linked via the ASC-1 complex.

U1 snRNP is mislocalized in ALS patient fibroblasts bearing NLS mutations in FUS and is required for motor neuron outgrowth in zebrafish.

Mutations in FUS cause amyotrophic lateral sclerosis (ALS), but the molecular pathways leading to neurodegeneration remain obscure. We previously found that U1 snRNP is the most abundant FUS interactor. Here, we report that components of the U1 snRNP core particle (Sm proteins and U1 snRNA), but not the mature U1 snRNP-specific proteins (U1-70K, U1A and U1C), co-mislocalize with FUS to the cytoplasm in ALS patient fibroblasts harboring mutations in the FUS nuclear localization signal (NLS). Similar results were obtained in HeLa cells expressing the ALS-causing FUS R495X NLS mutation, and mislocalization of Sm proteins is RRM-dependent. Moreover, as observed with FUS, knockdown of any of the U1 snRNP-specific proteins results in a dramatic loss of SMN-containing Gems. Significantly, knockdown of U1 snRNP in zebrafish results in motor axon truncations, a phenotype also observed with FUS, SMN and TDP-43 knockdowns. Our observations linking U1 snRNP to ALS patient cells with FUS mutations, SMN-containing Gems, and motor neurons indicate that U1 snRNP is a component of a molecular pathway associated with motor neuron disease. Linking an essential canonical splicing factor (U1 snRNP) to this pathway provides strong new evidence that splicing defects may be involved in pathogenesis and that this pathway is a potential therapeutic target.

Dynamic, inter-subunit interactions between the N-terminal and central mutation regions of cardiac ryanodine receptor.

Naturally occurring mutations in the cardiac ryanodine receptor (RyR2) have been linked to certain types of cardiac arrhythmias and sudden death. Two mutation hotspots that lie in the N-terminal and central regions of RyR2 are predicted to interact with one another and to form an important channel regulator switch. To monitor the conformational dynamics involving these regions, we generated a fluorescence resonance energy transfer (FRET) pair. A yellow fluorescent protein (YFP) was inserted into RyR2 after residue Ser437 in the N-terminal region, and a cyan fluorescent protein (CFP) was inserted after residue Ser2367 in the central region, to form a dual YFP- and CFP-labeled RyR2 (RyR2(S437-YFP/S2367-CFP)). We transfected HEK293 cells with RyR2(S437-YFP/S2367-CFP) cDNAs, and then examined them by using confocal microscopy and by measuring the FRET signal in live cells. The FRET signals are influenced by modulators of RyR2, by domain peptides that mimic the effects of disease causing RyR2 mutations, and by various drugs. Importantly, FRET signals were also readily detected in cells co-transfected with single CFP (RyR2(S437-YFP)) and single YFP (RyR2(S2367-CFP)) labeled RyR2, indicating that the interaction between the N-terminal and central mutation regions is an inter-subunit interaction. Our studies demonstrate that FRET analyses of this CFP- and YFP-labeled RyR2 can be used not only for investigating the conformational dynamics associated with RyR2 channel gating, but potentially, also for identifying drugs that are capable of stabilizing the conformations of RyR2.

Aberrant interaction of calmodulin with the ryanodine receptor develops hypertrophy in the neonatal cardiomyocyte.

We have shown previously that the inter-domain interaction between the two domains of RyR (ryanodine receptor), CaMBD [CaM (calmodulin)-binding domain] and CaMLD (CaM-like domain), activates the Ca(2+) channel, and this process is called activation-link formation [Gangopadhyay and Ikemoto (2008) Biochem. J. 411, 415-423]. Thus CaM that is bound to CaMBD is expected to interfere the activation-link formation, thereby stabilizing the closed state of the channel under normal conditions. In the present paper, we report that, upon stimulation of neonatal cardiomyocytes with the pro-hypertrophy agonist ET-1 (endothelin-1), CaM dissociates from the RyR, which induces a series of intracellular events: increased frequency of Ca(2+) transients, translocation of the signalling molecules CaM, CaMKII (CaM kinase II) and the transcription factor NFAT (nuclear factor of activated T-cells) to the nucleus. These events then lead to the development of hypertrophy. Importantly, an anti-CaMBD antibody that interferes with activation-link formation prevented all of these intracellular events triggered by ET-1 and prevented the development of hypertrophy. These results indicate that the aberrant formation of the activation link between CaMBD and CaMLD of RyR is a key step in the development of hypertrophy in cultured cardiomyocytes.

Intracellular translocation of calmodulin and Ca2+/calmodulin-dependent protein kinase II during the development of hypertrophy in neonatal cardiomyocytes.

We have recently shown that stimulation of cultured neonatal cardiomyocytes with endothelin-1 (ET-1) first produces conformational disorder within the ryanodine receptor (RyR2) and diastolic Ca(2+) leak from the sarcoplasmic reticulum (SR), then develops hypertrophy (HT) in the cardiomyocytes (Hamada et al., 2009 [3]). The present paper addresses the following question. By what mechanism does crosstalk between defective operation of RyR2 and activation of the HT gene program occur? Here we show that the immuno-stain of calmodulin (CaM) is localized chiefly in the cytoplasmic area in the control cells; whereas, in the ET-1-treated/hypertrophied cells, major immuno-staining is localized in the nuclear region. In addition, fluorescently labeled CaM that has been introduced into the cardiomyocytes using the BioPORTER system moves from the cytoplasm to the nucleus with the development of HT. The immuno-confocal imaging of Ca(2+)/CaM-dependent protein kinase II (CaMKII) also shows cytoplasm-to-nucleus shift of the immuno-staining pattern in the hypertrophied cells. In an early phase of hypertrophic growth, the frequency of spontaneous Ca(2+) transients increases, which accompanies with cytoplasm-to-nucleus translocation of CaM. In a later phase of hypertrophic growth, further increase in the frequency of spontaneous Ca(2+) transients results in the appearance of trains of Ca(2+) spikes, which accompanies with nuclear translocation of CaMKII. The cardio-protective reagent dantrolene (the reagent that corrects the de-stabilized inter-domain interaction within the RyR2 to a normal mode) ameliorates aberrant intracellular Ca(2+) events and prevents nuclear translocation of both CaM and CaMKII, then prevents the development of HT. These results suggest that translocation of CaM and CaMKII from the cytoplasm to the nucleus serves as messengers to transmit the pathogenic signal elicited in the surface membrane and in the RyR2 to the nuclear transcriptional sites to activate HT program.

Defective regulation of the ryanodine receptor induces hypertrophy in cardiomyocytes.

Recent studies on cardiac hypertrophy animal model suggest that inter-domain interactions within the ryanodine receptor (RyR2) become defective concomitant with the development of hypertrophy (e.g. de-stabilization of the interaction between N-terminal and central domains of RyR2; T. Oda, M. Yano, T. Yamamoto, T. Tokuhisa, S. Okuda, M. Doi, T. Ohkusa, Y. Ikeda, S. Kobayashi, N. Ikemoto, M. Matsuzaki, Defective regulation of inter-domain interactions within the ryanodine receptor plays a key role in the pathogenesis of heart failure, Circulation 111 (2005) 3400-3410). To determine if de-stabilization of the inter-domain interaction in fact causes hypertrophy, we introduced DPc10 (a peptide corresponding to the G(2460)-P(2495) region of RyR2, which is known to de-stabilize the N-terminal/central domain interaction) into rat neonatal cardiomyocytes by mediation of peptide carrier BioPORTER. After incubation for 24h the peptide induced hypertrophy, as evidenced by significant increase in cell size and [(3)H]leucine uptake. K201 or dantrolene, the reagents known to correct the de-stabilized inter-domain interaction to a normal mode, prevented the DPc10-induced hypertrophy. These results suggest that disruption of the normal N-terminal/central inter-domain interaction within the RyR2 is a causative mechanism of cardiomyocyte hypertrophy.

Interaction of the Lys(3614)-Asn(3643) calmodulin-binding domain with the Cys(4114)-Asn(4142) region of the type 1 ryanodine receptor is involved in the mechanism of Ca2+/agonist-induced channel activation.

In the present study we show that the interaction of the CaM (calmodulin)-binding domain (Lys(3614)-Asn(3643)) with the Cys(4114)-Asn(4142) region (a region included in the CaM-like domain) serves as an intrinsic regulator of the RyR1 (type-1 ryanodine receptor). We tested the effects of antibodies raised against the two putative key regions of RyR1 [anti-(Lys(3614)-Asn(3643)) and anti-(Cys(4114)-Asn(4142)) antibodies]. Both antibodies produced significant inhibition of [3H]ryanodine-binding activity of RyR1. This suggests that the inter-domain interaction between the two domains, Lys(3614)-Asn(3643) and Cys(4114)-Asn(4142), activates the channel, and that the binding of antibody to either side of the interacting domain pair interfered with the formation of a 'channel-activation link' between the two regions. In order to spectroscopically monitor the mode of interaction of these domains, the site of inter-domain interaction was fluorescently labelled with MCA [(7-methoxycoumarin-4-yl)acetyl] in a site-directed manner. The accessibility of the bound MCA to a large molecular mass fluorescence quencher, BSA-QSY (namely, the size of a gap between the interacting domains) decreased with an increase of [Ca2+] in a range of 0.03-2.0 microM, as determined by Stern-Volmer fluorescence quenching analysis. The Ca2+-dependent decrease in the quencher accessibility was more pronounced in the presence of 150 microM 4-CmC (4-chlorometacresol), and was reversed by 1 mM Mg2+ (a well-known inhibitor of Ca2+/agonist-induced channel activation). These results suggest that the Lys(3614)-Asn(3643) and Cys(4114)-Asn(4142) regions of RyR1 interact with each other in a Ca2+- and agonist-dependent manner, and this serves as a mechanism of Ca2+- and agonist-dependent activation of the RyR1 Ca2+ channel.

In Vitro System for Coupling RNAP II Transcription to Primary microRNA Processing and a Three-Way System for RNAP II Transcription/Splicing/microRNA Processing.

In the genome, primary microRNAs (pri-miRNAs) are encoded either as independent transcriptional units with their own promoters (intergenic miRNAs) or within the introns of other genes (intronic miRNAs). Here, we report two methods, one that we established for coupled RNAP II transcription and pri-miRNA processing and the other that is a three-way system for RNAP II transcription, pri-miRNA processing, and pre-mRNA splicing. In these systems, CMV-DNA constructs encoding the processing substrates are incubated in HeLa cell nuclear extracts in the presence of 32P-UTP to generate the nascent RNAP II transcripts, which are processed efficiently by the endogenous RNA processing machineries in nuclear extracts.

Interactome analyses revealed that the U1 snRNP machinery overlaps extensively with the RNAP II machinery and contains multiple ALS/SMA-causative proteins.

Mutations in multiple RNA/DNA binding proteins cause Amyotrophic Lateral Sclerosis (ALS). Included among these are the three members of the FET family (FUS, EWSR1 and TAF15) and the structurally similar MATR3. Here, we characterized the interactomes of these four proteins, revealing that they largely have unique interactors, but share in common an association with U1 snRNP. The latter observation led us to analyze the interactome of the U1 snRNP machinery. Surprisingly, this analysis revealed the interactome contains ~220 components, and of these, >200 are shared with the RNA polymerase II (RNAP II) machinery. Among the shared components are multiple ALS and Spinal muscular Atrophy (SMA)-causative proteins and numerous discrete complexes, including the SMN complex, transcription factor complexes, and RNA processing complexes. Together, our data indicate that the RNAP II/U1 snRNP machinery functions in a wide variety of molecular pathways, and these pathways are candidates for playing roles in ALS/SMA pathogenesis.

Evidence that C9ORF72 Dipeptide Repeat Proteins Associate with U2 snRNP to Cause Mis-splicing in ALS/FTD Patients.

Hexanucleotide repeat expansion in the C9ORF72 gene results in production of dipeptide repeat (DPR) proteins that may disrupt pre-mRNA splicing in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. At present, the mechanisms underlying this mis-splicing are not understood. Here, we show that addition of proline-arginine (PR) and glycine-arginine (GR) toxic DPR peptides to nuclear extracts blocks spliceosome assembly and splicing, but not other types of RNA processing. Proteomic and biochemical analyses identified the U2 small nuclear ribonucleoprotein particle (snRNP) as a major interactor of PR and GR peptides. In addition, U2 snRNP, but not other splicing factors, mislocalizes from the nucleus to the cytoplasm both in C9ORF72 patient induced pluripotent stem cell (iPSC)-derived motor neurons and in HeLa cells treated with the toxic peptides. Bioinformatic studies support a specific role for U2-snRNP-dependent mis-splicing in C9ORF72 patient brains. Together, our data indicate that DPR-mediated dysfunction of U2 snRNP could account for as much as ∼44% of the mis-spliced cassette exons in C9ORF72 patient brains.

In vitro splicing of erythropoietin by the Mycobacterium tuberculosis RecA intein without substituting amino acids at the splice junctions.

Protein splicing is a self-catalyzed process involving the excision of an intervening polypeptide sequence, the intein, and joining of the flanking polypeptide sequences, the extein, by a peptide bond. We have studied the in vitro splicing of erythropoietin (EPO) using a truncated form of the Mycobacterium tuberculosis RecA mini-intein in which the homing endonuclease domain was replaced with a hexahistidine sequence (His-tag). The intein was inserted adjacent to cysteine residues to assure that the spliced product had the natural amino acid sequence. When expressed in Escherichia coli, intein-containing EPO was found entirely as inclusion bodies but could be refolded in soluble form in the presence of 0.5 M arginine. Protein splicing of the refolded protein could be induced with a reducing agent such as DTT or tris(2-carboxyethyl)phosphine and led to the formation of EPO and mini-intein along with some cleavage products. Protein splicing mediated by the RecA intein requires the presence of a cysteine residue adjacent to the intein insertion site. We compared the efficiencies of protein splicing adjacent to three of the four cysteine residues of EPO (Cys29, Cys33 and Cys161) and found that insertion of intein adjacent to Cys29 allowed far more efficient protein splicing than insertion adjacent to Cys33 or Cys161. For ease of purification, our experiments involved a His-tagged EPO fusion protein and a His-tagged intein and the spliced products (25 kDa EPO and 24 kDa mini-intein) were identified by Western blotting using anti-EPO and anti-His-tag antibodies and by mass spectroscopy. The optimal splicing yield at Cys29 (40%) occurred at pH 7.0 after refolding at 4 degrees C and splicing for 18 h at 25 degrees C in the presence of 1 mM DTT.

Role of the Met3534-Ala4271 region of the ryanodine receptor in the regulation of Ca2+ release induced by calmodulin binding domain peptide.

CaMBP, a peptide corresponding to the 3614-3643 calmodulin (CaM) binding region of the ryanodine receptor (RyR1), is known to activate RyR1 Ca2+ channel. To analyze the mechanism of channel regulation by the CaMBP-RyR1 interaction, we investigated a), CaMBP binding to RyR1, b), induced local conformational changes in the CaMBP binding region of RyR1 using the fluorescent conformational probe badan attached to CaMBP (CaMBP-badan), and c), effects of "a" and "b" on SR Ca2+ release. We also monitored the interaction of CaMBP-badan with CaM and a peptide corresponding to the Met3534-Ala4271 region of RyR1 (R3534-4271) as a control. At lower peptide concentrations (< or =15 microM), CaMBP binding to RyR1 increased the intensity of badan fluorescence emission at a shorter wavelength (the state resembling CaMBP-badan/Ca-CaM) and induced Ca2+ release. Further increase in CaMBP concentration (up to approximately 50 microM) produced more binding of CaMBP accompanied by further increase in the badan fluorescence emission but at a longer wavelength (the state resembling CaMBP-badan/apo-CaM) and inhibited Ca2+ release. Binding of CaMBP-badan to R3534-4271 increased the intensity of badan fluorescence, showing the similar concentration-dependent red-shift of the emission maximum. It is proposed that CaMBP interacts with two classes of binding sites located in the Met3534-Ala4271 region of RyR1, which activate and inhibit the Ca2+ channel, respectively.

Dantrolene stabilizes domain interactions within the ryanodine receptor.

Interdomain interactions between N-terminal and central domains serving as a "domain switch" are believed to be essential to the functional regulation of the skeletal muscle ryanodine receptor-1 Ca(2+) channel. Mutational destabilization of the domain switch in malignant hyperthermia (MH), a genetic sensitivity to volatile anesthetics, causes functional instability of the channel. Dantrolene, a drug used to treat MH, binds to a region within this proposed domain switch. To explore its mechanism of action, the effect of dantrolene on MH-like channel activation by the synthetic domain peptide DP4 or anti-DP4 antibody was examined. A fluorescence probe, methylcoumarin acetate, was covalently attached to the domain switch using DP4 as a delivery vehicle. The magnitude of domain unzipping was determined from the accessibility of methylcoumarin acetate to a macromolecular fluorescence quencher. The Stern-Volmer quenching constant (K(Q)) increased with the addition of DP4 or anti-DP4 antibody. This increase was reversed by dantrolene at both 37 and 22 degrees C and was unaffected by calmodulin. [(3)H]Ryanodine binding to the sarcoplasmic reticulum and activation of sarcoplasmic reticulum Ca(2+) release, both measures of channel activation, were enhanced by DP4. These activities were inhibited by dantrolene at 37 degrees C, yet required the presence of calmodulin at 22 degrees C. These results suggest that the mechanism of action of dantrolene involves stabilization of domain-domain interactions within the domain switch, preventing domain unzipping-induced channel dysfunction. We suggest that temperature and calmodulin primarily affect the coupling between the domain switch and the downstream mechanism of regulation of Ca(2+) channel opening rather than the domain switch itself.

Fluorescence probe study of Ca2+-dependent interactions of calmodulin with calmodulin-binding peptides of the ryanodine receptor.

We have used a highly environment-sensitive fluorescent probe 6-bromoacetyl-2-dimethylaminonaphthalene (badan) to study the interaction between calmodulin (CaM) and a CaM-binding peptide of the ryanodine receptor (CaMBP) and its sub-fragments F1 and F4. Badan was attached to the Thr34Cys mutant of CaM (CaM-badan). Ca(2+) increase in a physiological range of Ca(2+) (0.1-2 microM) produced about 40 times increase in the badan fluorescence. Upon binding to CaMBP, the badan fluorescence of apo-CaM showed a small increase at a slow rate; whereas that of Ca-CaM showed a large decrease at a very fast rate. Upon binding of CaM to the badan-labeled CaMBP, the badan fluorescence showed a small and slow increase at low Ca(2+), and a large and fast increase at high Ca(2+). Thus, the badan probe attached to CaM Cys(34) can be used to monitor conformational changes occurring not only in CaM, but also those in the CaM-CaMBP interface. Based on our results we propose that both the interaction interface and the global conformation of the CaM-CaMBP complex are altered by calcium.

An in vitro screening system for protein splicing inhibitors based on green fluorescent protein as an indicator.

This paper describes an in vitro fluorometric assay system for protein splicing based on the RecA intein of Mycobacterium tuberculosis and a modified green fluorescent protein (GFP). The assay takes advantage of the fact that polypeptides inserted adjacent to residue 129 of GFP cause the protein to form inclusion bodies when expressed in Escherichia coli and to be incapable of fluorophore formation. However, when the inserted polypeptide is an intein, the renatured fusion protein can undergo protein splicing and chromophore formation. Comparison of chromophore formation by renatured GFP-intein fusion and renatured GFP showed that under optimal conditions (pH 6.5 and 20 degrees C) protein splicing is significantly slower than GFP chromophore formation. Taking advantage of the reversible inhibition of protein splicing by zinc ion, a fluorometric protein splicing assay was developed in which the denatured fusion protein of GFP and the RecA intein was purified on a metal ion affinity column and renatured in the presence of 2 mM ZnCl2. When diluted into appropriate buffers, protein splicing could be initiated by the addition of a molar excess of EDTA and followed fluorometrically. This assay should be valuable as a high-throughput screening system for protein splicing inhibitors as potential antimycobacterial agents and as tools for studying the mechanism of protein splicing.