Dissecting the role of SMN multimerization in its dissociation from the Cajal body using harmine as a tool compound.

SMN, linked to spinal muscular atrophy, is a key component of the Gemin complex essential for snRNP assembly. Following initial snRNP assembly in the cytoplasm, both snRNPs and SMN migrate to the nucleus and associate with Cajal bodies, where final snRNP maturation occurs. It is assumed that SMN must be free from the Cajal bodies for continuous snRNP biogenesis. Previous observation of the SMN granules docked in CB suggests the existence of a separation mechanism. However, the precise processes that regulate the spatial separation of SMN-complexes from Cajal bodies remain unclear. Here we employed a super-resolution microscope alongside the beta-carboline alkaloid harmine, which disrupted the Cajal body in a reversible manner. Upon removal of harmine, SMN and Coilin first appear as small, interconnected condensates. The SMN condensates mature into spheroidal structures encircled by Coilin, eventually segregating into distinct condensates. Expression of a multimerization-deficient SMN mutant leads to enlarged, atypical Cajal bodies where SMN is unable to segregate into separate condensates. These findings underscore the importance of multimerization in facilitating the segregation of SMN from Coilin within Cajal bodies.

The cell type-specific ER membrane protein UGS148 is not essential in mice.

Genomes of higher eukaryotes encode many uncharacterized proteins, and the functions of these proteins cannot be predicted from the primary sequences due to a lack of conserved functional domains. In this study, we focused on a poorly characterized protein UGS148 that is highly expressed in a specialized cell type called tanycytes that line the ventral wall of the third ventricle in the hypothalamus. Immunostaining of UGS148 revealed the fine morphology of tanycytes with highly branched apical ER membranes. Immunoprecipitation revealed that UGS148 associated with mitochondrial ATPase at least in vitro, and ER and mitochondrial signals occasionally overlapped in tanycytes. Mutant mice lacking UGS148 did not exhibit overt phenotypes, suggesting that UGS148 was not essential in mice reared under normal laboratory conditions. We also found that RNA probes that were predicted to uniquely detect UGS148 mRNA cross-reacted with uncharacterized RNAs, highlighting the importance of experimental validation of the specificity of probes during the hybridization-based study of RNA localization.

Development of a Cell-Based Assay Using a Split-Luciferase Reporter for Compound Screening.

Recently, the finding of recurrent mutations in the spliceosome components in cancer has indicated that the spliceosome is a potential target for cancer therapy. However, the number of small molecules known to affect the cellular spliceosome is currently limited probably because of the lack of a robust cell-based approach to identify small molecules that target the spliceosome. We have previously reported the development of a genetic reporter to detect the cellular levels of small nuclear ribonucleoproteins (snRNPs), which are subunits of the spliceosome, using a split luciferase. However, the original protocol was designed for small scale experiments and was not suitable for compound screening. Here, we found that the use of cell lysis buffer used in blue native polyacrylamide gel electrophoresis (BN-PAGE) dramatically improved the sensitivity and the robustness of the assay. Improved assay conditions were used to discover a small molecule that altered the reporter activity. Our method may be used with other cellular macromolecular complexes and may assist in the discovery of small bioactive molecules.

BAY61-3606 Alters snRNP Composition and Enhances Usage of Suboptimal Splice Acceptor Site.

Intron recognition by the spliceosome mainly depends on conserved intronic sequences such as 5' splice sites, 3' splice sites, and branch sites. Therefore, even substitution of just a single nucleotide in a 5' or 3' splice site abolishes the splicing at the mutated site and leads to cryptic splice site usage. A number of disease-causative mutations have been found in 5' and 3' splice sites, but the genes with these mutations still maintain the correct protein-coding sequence, so recovery of splicing at the mutated splice site may produce a normal protein. Mutations in the spliceosome components have been shown to change the balance between the conformational transition and disassembly of the spliceosome, which affects the decision about whether the reaction of the incorporated substrate will proceed. In addition, the lower disassembly rate caused by such mutations induces splicing of the mutated splice site. We hypothesized that small compounds targeting the spliceosome may include a compound mimicking the effect of those mutations. Thus, we screened a small-compound library and identified a compound, BAY61-3606, that changed the cellular small nuclear ribonucleoprotein composition and also showed activity of enhancing splicing at the mutated 3' splice site of the reporter gene, as well as splicing at the suboptimal 3' splice site of endogenous cassette exons. These results indicate that further analysis of the mechanism of action of BAY61-3606 could enable modulation of the fidelity of splicing.

Divergent synthesis of kinase inhibitor derivatives, leading to discovery of selective Gck inhibitors.

We accomplished divergent synthesis of potent kinase inhibitor BAY 61-3606 (1) and 27 derivatives via conjugation of imidazo[1,2-c]pyrimidine and indole ring compounds with aromatic (including pyridine) derivatives by means of palladium-catalyzed cross-coupling reaction. Spleen tyrosine kinase (Syk) and germinal center kinase (Gck, MAP4K2) inhibition assays showed that some of the synthesized compounds were selective Gck inhibitors.

Epidermal Growth Factor-dependent Activation of the Extracellular Signal-regulated Kinase Pathway by DJ-1 Protein through Its Direct Binding to c-Raf Protein.

DJ-1 is an oncogene and also a causative gene for familial Parkinson disease. DJ-1 has various functions, and the oxidative status of cysteine at position 106 (Cys-106) is crucial for determination of the activation level of DJ-1. Although DJ-1 requires activated Ras for its oncogenic activity and although it activates the extracellular signal-regulated kinase (ERK) pathway, a cell growth pathway downstream of Ras, the precise mechanism underlying activation of the ERK pathway by DJ-1 is still not known. In this study, we found that DJ-1 directly bound to the kinase domain of c-Raf but not to Ras and that Cys-106 mutant DJ-1 bound to c-Raf more weakly than did wild-type DJ-1. Co-localization of DJ-1 with c-Raf in the cytoplasm was enhanced in epidermal growth factor (EGF)-treated cells. Knockdown of DJ-1 expression attenuated the phosphorylation level of c-Raf in EGF-treated cells, resulting in reduced activation of MEK and ERK1/2. Although EGF-treated DJ-1 knock-out cells also showed attenuated c-Raf activation, reintroduction of wild-type DJ-1, but not C106S DJ-1, into DJ-1 knock-out cells restored c-Raf activation in a DJ-1 binding activity in a c-Raf-dependent manner. DJ-1 was not responsible for activation of c-Raf in phorbol myristate acetate-treated cells. Furthermore, DJ-1 stimulated self-phosphorylation activity of c-Raf in vitro, but DJ-1 was not a target for Raf kinase. Oxidation of Cys-106 in DJ-1 was not affected by EGF treatment. These findings showed that DJ-1 is a positive regulator of the EGF/Ras/ERK pathway through targeting c-Raf.

Prefoldin plays a role as a clearance factor in preventing proteasome inhibitor-induced protein aggregation.

Prefoldin is a molecular chaperone composed of six subunits, PFD1-6, and prevents misfolding of newly synthesized nascent polypeptides. Although it is predicted that prefoldin, like other chaperones, modulates protein aggregation, the precise function of prefoldin against protein aggregation under physiological conditions has never been elucidated. In this study, we first established an anti-prefoldin monoclonal antibody that recognizes the prefoldin complex but not its subunits. Using this antibody, it was found that prefoldin was localized in the cytoplasm with dots in co-localization with polyubiquitinated proteins and that the number and strength of dots were increased in cells that had been treated with lactacystin, a proteasome inhibitor, and thapsigargin, an inducer of endoplasmic reticulum stress. Knockdown of prefoldin increased the level of SDS-insoluble ubiquitinated protein and reduced cell viability in lactacystin and thapsigargin-treated cells. Opposite results were obtained in prefoldin-overexpressed cells. It has been reported that mice harboring a missense mutation L110R of MM-1α/PFD5 exhibit neurodegeneration in the cerebellum. Although the prefoldin complex containing L110R MM-1α was properly formed in vitro and in cells derived from L110R MM-1α mice, the levels of ubiquitinated proteins and cytotoxicity were higher in L110R MM-1α cells than in wild-type cells under normal conditions and were increased by lactacystin and thapsigargin treatment, and growth of L110R MM-1α cells was attenuated. Furthermore, the polyubiquitinated protein aggregation level was increased in the brains of L110R MM-1α mice. These results suggest that prefoldin plays a role in quality control against protein aggregation and that dysfunction of prefoldin is one of the causes of neurodegenerative diseases.

A split luciferase-based reporter for detection of a cellular macromolecular complex.

The spliceosome is a highly dynamic macromolecular ribonucleoprotein (RNP) machine that catalyzes pre-mRNA splicing by assembling U1, U2, U4, U5, and U6 small nuclear RNPs (snRNPs). To process large numbers of introns with a limited number of snRNPs, synthesis and recycling of snRNPs must be maintained within an appropriate range to avoid their shortage. However, the mechanism that maintains cellular snRNP levels is unknown. Molecules that modulate cellular snRNP levels may help to define this mechanism but are not available. Therefore, the goal of the current study was to develop a reporter for snRNP levels using split luciferase based on proteomic analysis of snRNPs. We constructed an expression library of a luciferase fragment fused to core components of U5 snRNP and used it to isolate pre-mRNA processing factor 6 (PRPF6) and small nuclear ribonucleoprotein 40 kDa (U5-40K) that specifically reconstitute luciferase activity in the U5 snRNP complex. Here we show that this reporter detects the effects of small molecules on the levels of the U5 snRNP reporter protein complex. Our approach provides an alternative assay to discover small molecules targeting a macromolecular complex when the structure of the complex is not precisely identified.

Spliceosome discards intermediates via the DEAH box ATPase Prp43p.

To promote fidelity in nuclear pre-mRNA splicing, the spliceosome rejects and discards suboptimal substrates that have engaged the spliceosome. Whereas DExD/H box ATPases have been implicated in rejecting suboptimal substrates, the mechanism for discarding suboptimal substrates has remained obscure. Corroborating evidence that suboptimal, mutated lariat intermediates can be exported to the cytoplasm for turnover, we have found that the ribosome can translate mutated lariat intermediates. By glycerol gradient analysis, we have found that the spliceosome can dissociate mutated lariat intermediates in vivo in a manner that requires the DEAH box ATPase Prp43p. Through an in vitro assay, we demonstrate that Prp43p promotes the discard of suboptimal and optimal 5' exon and lariat intermediates indiscriminately. Finally, we demonstrate a requirement for Prp43p in repressing splicing at a cryptic splice site. We propose a model for the fidelity of exon ligation in which the DEAH box ATPase Prp22p slows the flow of suboptimal intermediates through exon ligation and Prp43p generally promotes discard of intermediates, thereby establishing a pathway for turnover of stalled intermediates. Because Prp43p also promotes spliceosome disassembly after exon ligation, this work establishes a parallel between the discard of suboptimal intermediates and the dissociation of a genuine excised intron product.

Prefoldin subunits are protected from ubiquitin-proteasome system-mediated degradation by forming complex with other constituent subunits.

The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation.

Exon ligation is proofread by the DExD/H-box ATPase Prp22p.

To produce messenger RNA, the spliceosome excises introns from precursor (pre)-mRNA and splices the flanking exons. To establish fidelity, the spliceosome discriminates against aberrant introns, but current understanding of such fidelity mechanisms is limited. Here we show that an ATP-dependent activity represses formation of mRNA from aberrant intermediates having mutations in any of the intronic consensus sequences. This proofreading activity is disabled by mutations that impair the ATPase or RNA unwindase activity of Prp22p, a conserved spliceosomal DExD/H-box ATPase. Further, cold-sensitive prp22 mutants permit aberrant mRNA formation from a mutated 3' splice-site intermediate in vivo. We conclude that Prp22p generally represses splicing of aberrant intermediates, in addition to its known ATP-dependent role in promoting release of genuine mRNA. This dual function for Prp22p validates a general model in which fidelity can be enhanced by a DExD/H-box ATPase.

DJ-1-Mediated protective effect of protocatechuic aldehyde against oxidative stress in SH-SY5Y cells.

DJ-1 was identified as a causal gene for a familial form of early onset Parkinson's disease (PD), park 7. DJ-1 plays roles in transcriptional regulation and the anti-oxidative stress reaction. In this study, we found that protocatechuic aldehyde (PAL), a traditional Chinese medicine compound, bound to DJ-1 in vitro and that PAL protected SH-SY5Y cells but not DJ-1-knockdown SH-SY5Y cells from oxidative stress-induced cell death, indicating that the protective effect of PAL is mediated by DJ-1. Furthermore, PAL inhibited production of reactive oxygen species and the inhibition was abated in DJ-1-knockdown cells. PAL increased and decreased phosphorylation of AKT and PTEN, respectively, in SH-SY5Y cells, suggesting that the AKT pathway is one of the specific signaling pathways in PAL-induced neuroprotection. Moreover, PAL prevented superfluous oxidation of cysteine 106 of DJ-1, an essential amino acid for DJ-1's function. The present study demonstrates that PAL has potential neuroprotective effects through DJ-1.

Oxidative status of DJ-1-dependent activation of dopamine synthesis through interaction of tyrosine hydroxylase and 4-dihydroxy-L-phenylalanine (L-DOPA) decarboxylase with DJ-1.

Parkinson disease (PD) is caused by loss of dopamine, which is synthesized from tyrosine by two enzymes, tyrosine hydroxylase (TH) and 4-dihydroxy-L-phenylalanine decarboxylase (DDC). DJ-1 is a causative gene for the familial form of PD, but little is known about the roles of DJ-1 in dopamine synthesis. In this study, we found that DJ-1 directly bound to TH and DDC and positively regulated their activities in human dopaminergic cells. Mutants of DJ-1 found in PD patients, including heterozygous mutants, lost their activity and worked as dominant-negative forms toward wild-type DJ-1. When cells were treated with H(2)O(2), 6-hydroxydopamine, or 1-methyl-4-phenylpyridinium, changes in activities of TH and DDC accompanied by oxidation of cysteine 106 of DJ-1 occurred. It was found that DJ-1 possessing Cys-106 with SH and SOH forms was active and that DJ-1 possessing Cys-106 with SO(2)H and SO(3)H forms was inactive in terms of stimulation of TH and DDC activities. These findings indicate an essential role of DJ-1 in dopamine synthesis and contribution of DJ-1 to the sporadic form of PD.

What is the switch for coupling transcription and splicing? RNA Polymerase II C-terminal domain phosphorylation, phase separation and beyond.

Phosphorylation of the RNA polymerase II C-terminal domain (Pol II CTD) has important roles in the kinetic coupling of splicing with transcription, which is essential for many genes to maintain correct splicing patterns. However, because of the extensively repeated low complexity sequences of Pol II CTD, it was unclear how phosphorylation-dependent molecular interactions were able to provide sufficient specificity to spatiotemporally partition various cotranscriptional events. Here we try to view the molecular mechanisms governing cotranscriptional splicing from the role of phase separation based on recent studies showing the ability of Pol II CTD to form droplets. This article is categorized under: RNA Processing > Splicing Regulation/Alternative Splicing RNA Processing > Splicing Mechanisms RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

DJ-1 binds to mitochondrial complex I and maintains its activity.

Parkinson's disease (PD) is caused by neuronal cell death, and oxidative stress and mitochondrial dysfunction are thought to be responsible for onset of PD. DJ-1, a causative gene product of a familial form of Parkinson's disease, PARK7, plays roles in transcriptional regulation and anti-oxidative stress. The possible mitochondrial function of DJ-1 has been proposed, but its exact function remains unclear. In this study, we found that DJ-1 directly bound to NDUFA4 and ND1, nuclear and mitochondrial DNA-encoding subunits of mitochondrial complex I, respectively, and was colocalized with complex I and that complex I activity was reduced in DJ-1-knockdown NIH3T3 and HEK293 cells. These findings suggest that DJ-1 is an integral mitochondrial protein and that DJ-1 plays a role in maintenance of mitochondrial complex I activity.

Tenascin-X induces cell detachment through p38 mitogen-activated protein kinase activation.

Extracellular matrix glycoprotein tenascin-X (TNX) is the largest member of the tenascin family. In this study, we investigated the adhesive properties of TNX and the signaling pathway to be induced to mouse fibroblast L cells on TNX substrate. Approximately 45% of evaluable cells used in the cell adhesion assay were attached to purified TNX but did not spread and were rounded on TNX. The remaining 55% of cells were detached from the TNX substrate and were floating in the conditioned medium. In rounded cells on TNX, phosphorylation of focal adhesion kinase (FAK) was diminished compared with that in cells on control phosphate buffered saline (PBS). To better understand the pathways that lead to the detachment of cells on the TNX substrate, we examined phosphorylation of p38 mitogen-activated protein (MAP) kinase. Phosphorylation of p38 MAP kinase was observed in the rounded cells on TNX in a dose-dependent manner, and the maximum effect was observed at 30 min on TNX. Inhibition of p38 MAP kinase alpha expression by RNA interference partially suppressed the TNX-induced cell detachment. These results suggest that the p38 MAP kinase is a major mediator of TNX-induced cell detachment.

Specific cleavage of DJ-1 under an oxidative condition.

DJ-1 was initially identified by us as a novel oncogene and has recently been found to be a causative gene for familial Parkinson's disease (PD) PARK7. DJ-1 plays roles in transcriptional regulation and in oxidative stress function, and its oxidative state at cysteine residues determines activities of DJ-1. In this study, we found that recombinant DJ-1 expressed in and purified from E. coli was specifically cleaved between glycine and proline at amino acid numbers 157 and 158, respectively, by treatment of DJ-1 with H2O2. A substitution mutant of DJ-1 from cysteine to serine at amino acid number 106, a major oxidation site of DJ-1, was found not to be cleaved under an oxidative condition, suggesting oxidation-dependent cleavage of DJ-1. Cleavage of DJ-1 was also observed in human SH-SY5Y cells that had been treated with H2O2. These results suggest that oxidative stress-induced cleavage of DJ-1 regulates functions of DJ-1.

A Splicing Reporter Tuned to Non-AG Acceptor Sites Reveals that Luteolin Enhances the Recognition of Non-canonical Acceptor Sites.

Removal of an intron requires precise recognition of the splice donor and acceptor sites located at the 5' and 3' termini of introns. Although the roles of these sequences differ, mutations in both sites easily block normal splicing and produce an aberrant mRNA. For example, many splice-site mutations occur in patients with inherited diseases. Several approaches have been evaluated to restore expression of a functional protein; however, because of the strict requirement for an AG dinucleotide at the 3' terminus of a U2-type intron, no method is available to correct splicing at a mutated sequence. To identify compounds that allow splicing at the non-AG acceptor site, in the present study we constructed a reporter gene with a modified polypyrimidine tract. However, the modified polypyrimidine tract mediated splicing at adjacent non-canonical acceptor sites, including the original mutated site. Further, we show that certain flavones such as luteolin and apigenin enhanced aberrant splicing at the non-canonical acceptor site of the reporter gene. These results suggest that the reporter gene and luteolin may be useful for further screening to identify molecules that correct aberrant splicing caused by a disease-associated splice acceptor site mutation.

A novel nucleolar protein, PAPA-1, induces growth arrest as a result of cell cycle arrest at the G1 phase.

We have identified a novel nucleolar protein, PAP-1-associated protein-1 (PAPA-1), after screening the interacting proteins with Pim-1-associated protein-1 (PAP-1), a protein that is a phosphorylation target of Pim-1 kinase. PAPA-1 comprises 345 amino acids with a basic amino-acid cluster. PAPA-1 was found to be localized in the nucleolus in transfected HeLa cells, and the lysine/histidine cluster was essential for nucleolar localization of PAPA-1. PAPA-1 protein and mRNA expression decreased upon serum restimulation of starvation-synchronized cells, which displayed maximum level of PAPA-1 expression at G0 and early G1 phase of the cell cycle. Ectopic expression of PAPA-1 induced growth suppression of cells, and the effect was dependent on its nucleolar localization in established HeLa cell lines that inducibly express PAPA-1 or its deletion mutant under the control of a tetracycline-inducible promoter. Furthermore, when PAPA-1-inducible HeLa cells were synchronized by thymidine, colcemid or mimosine, and then PAPA-1 was expressed, the proportion of cells at the G1 phase was obviously increased. These results suggest that PAPA-1 induces growth and cell cycle arrests at the G1 phase of the cell cycle.

Prefoldin prevents aggregation of α-synuclein.

Protein aggregation is observed in various neurodegeneration diseases, including Parkinson's disease (PD). Alpha-synuclein, a causative gene product of familial PD, is a major component of large aggregates (inclusion bodies) in PD. Prefoldin, a molecular chaperone comprised of six subunits, PFD1~6, prevents misfolding of newly synthesized nascent polypeptides and also prevents aggregation of protein such as a pathogenic form of Huntingtin, a causative gene product of Huntington disease. In this study, we first found that aggregation of TagRFP-tagged wild-type α-synuclein and its pathogenic mutants, but not that of GFP-tagged α-synuclein, occurred in transfected Neuro-2a cells. The fluorescence of GFP is weakened under the condition of pH 4.5-5.0, and TagRFP is a stable red fluorescence protein under an acidic condition. Aggregated TagRFP-wild-type α-synuclein and its pathogenic mutants in Neuro-2a cells were ubiquitinated and were colocalized with the prefoldin complex in the lysosome under this condition. Furthermore, knockdown of PFD2 and PFD5 disrupted prefoldin formation in α-synuclein-expressing cells, resulting in accumulation of aggregates of wild-type and pathogenic α-synuclein and in induction of cell death. The levels of aggregation and cell death in pathogenic α-synuclein-transfected cells tended to be higher than those in wild-type α-synuclein-transfected cells. These results suggest that prefoldin works as a protective factor in aggregated α-synuclein-induced cell death.