Successful skipping of abnormal pseudoexon by antisense oligonucleotides in vitro for a patient with beta-propeller protein-associated neurodegeneration.

Pathogenic variants in WDR45 on chromosome Xp11 cause neurodegenerative disorder beta-propeller protein-associated neurodegeneration (BPAN). Currently, there is no effective therapy for BPAN. Here we report a 17-year-old female patient with BPAN and show that antisense oligonucleotide (ASO) was effective in vitro. The patient had developmental delay and later showed extrapyramidal signs since the age of 15 years. MRI findings showed iron deposition in the globus pallidus and substantia nigra on T2 MRI. Whole genome sequencing and RNA sequencing revealed generation of pseudoexon due to inclusion of intronic sequences triggered by an intronic variant that is remote from the exon-intron junction: WDR45 (OMIM #300526) chrX(GRCh37):g.48935143G > C, (NM_007075.4:c.235 + 159C > G). We recapitulated the exonization of intron sequences by a mini-gene assay and further sought antisense oligonucleotide that induce pseudoexon skipping using our recently developed, a dual fluorescent splicing reporter system that encodes two fluorescent proteins, mCherry, a transfection marker designed to facilitate evaluation of exon skipping and split eGFP, a splicing reaction marker. The results showed that the 24-base ASO was the strongest inducer of pseudoexon skipping. Our data presented here have provided supportive evidence for in vivo preclinical studies.

A novel splice variant of the human MSTN gene encodes a myostatin-specific myostatin inhibitor.

BACKGROUND: Myostatin, encoded by the MSTN gene comprising 3 exons, is a potent negative regulator of skeletal muscle growth. Although a variety of myostatin inhibitors have been invented for increasing muscle mass in muscle wasting diseases, no effective inhibitor is currently available for clinical use. Myostatin isoforms in several animals have been reported to inhibit myostatin, but an isoform has never been identified for the human MSTN gene, a conserved gene among animals. Here, a splice variant of the human MSTN gene was explored. METHODS: Transcripts and proteins were analysed by reverse transcription-PCR amplification and western blotting, respectively. Proteins were expressed from expression plasmid. Myostatin signalling was assayed by the SMAD-responsive luciferase activity. Cell proliferation was assayed by the Cell Counting Kit-8 (CCK-8) assay and cell counting. Cell cycle was analysed by the FastFUCCI system. RESULTS: Reverse transcription-PCR amplification of the full-length MSTN transcript in CRL-2061 rhabdomyosarcoma cells revealed two bands consisting of a thick expected-size product and a thin additional small-size product. Sequencing of the small-size product showed a 963-bp deletion in the 5' end of exon 3, creating exon 3s, which contained unusual splice acceptor TG dinucleotides. The novel variant was identified in other human cell lines, although it was not identified in skeletal muscle. The 251-amino acid isoform encoded by the novel variant (myostatin-b) was identified in CRL-2061 rhabdomyosarcoma cells. Transfection of a myostatin-b expression plasmid into CRL-2061 and myoblast cells inhibited endogenous myostatin signalling (44%, P < 0.001 and 63%, P < 0.001, respectively). Furthermore, myostatin-b inhibited myostatin signalling induced by recombinant myostatin (68.8%, P < 0.001). In remarkable contrast, myostatin-b did not inhibit the myostatin signalling induced by recombinant growth differentiation factor 11 (9.2%, P = 0.70), transforming growth factor ß (+3.1%, P = 0.83) or activin A (+1.1%, P = 0.96). These results indicate the myostatin-specific inhibitory effect of myostatin-b. Notably, the expression of myostatin-b in myoblasts significantly enhanced cell proliferation higher than the mock-transfected cells by the CCK-8 and direct cell counting assays (60%, P < 0.05 and 39%, P < 0.05, respectively). Myostatin-b increased the percentage of S-phase cells significantly higher than that of the mock-transfected cells (53% vs. 80%, P < 0.05). CONCLUSIONS: We cloned a novel human MSTN variant produced by unorthodox splicing. The variant encoded a novel myostatin isoform, myostatin-b, that inhibited myostatin signalling by myostatin-specific manner and enhanced myoblast proliferation by shifting cell cycle. Myostatin-b, which has myostatin-specific inhibitory activity, could be developed as a natural myostatin inhibitor.

A case of infantile Barth syndrome with severe heart failure: Importance of splicing variants in the TAZ gene.

Barth syndrome (BTHS) is an X-linked disorder characterized by cardiomyopathy, skeletal myopathy, and 3-methylglutaconic aciduria. The causative pathogenic variants for BTHS are in TAZ, which encodes a putative acyltransferase named tafazzin and is involved in the remodeling of cardiolipin in the inner mitochondrial membranes. Pathogenic variants in TAZ result in mitochondrial structural and functional abnormalities. We report a case of infantile BTHS with severe heart failure, left ventricular noncompaction, and lactic acidosis, having a missense c.640C>T (p.His214Tyr) variant in TAZ, which is considered a pathogenic variant based on the previously reported amino acid substitution at the same site (c.641A>G, p.His214Arg). However, in this previously reported case, heart function was compensated and not entirely similar to the present case. Silico prediction analysis suggested that c.640C>T could alter the TAZ messenger RNA (mRNA) splicing process. TAZ mRNAs in isolated peripheral mononuclear cells from the patient and in vitro splicing analysis using minigenes of TAZ found an 8 bp deletion at the 3' end of exon 8, which resulted in the formation of a termination codon in the coding region of exon 9 (H214Nfs*3). These findings suggest that splicing abnormalities should always be considered in BTHS.

Sustained therapeutic benefits by transient reduction of TDP-43 using ENA-modified antisense oligonucleotides in ALS/FTD mice.

The abnormal aggregation of TDP-43 into cytoplasmic inclusions in affected neurons is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although how TDP-43 forms cytoplasmic aggregates and causes neurodegeneration in patients with ALS/FTD remains unclear, reducing cellular TDP-43 levels is likely to prevent aggregation and to rescue neurons from TDP-43 toxicity. To address this issue, here we developed gapmer-type antisense oligonucleotides (ASOs) against human TDP-43 using 2'-O,4'-C-ethylene nucleic acids (ENAs), which are modified nucleic acids with high stability, and tested the therapeutic potential of lowering TDP-43 levels using ENA-modified ASOs. We demonstrated that intracerebroventricular administration of ENA-modified ASOs into a mouse model of ALS/FTD expressing human TDP-43 results in the efficient reduction of TDP-43 levels in the brain and spinal cord. Surprisingly, a single injection of ENA-modified ASOs into TDP-43 mice led to long-lasting improvement of behavioral abnormalities and the suppression of cytoplasmic TDP-43 aggregation, even after TDP-43 levels had returned to the initial levels. Our results demonstrate that transient reduction of TDP-43 using ENA-modified ASOs leads to sustained therapeutic benefits in vivo, indicating the possibility of a disease-modifying therapy by lowering TDP-43 levels for the treatment of the TDP-43 proteinopathies, including ALS/FTD.

Human Dystrophin Dp71ab Enhances the Proliferation of Myoblasts Across Species But Not Human Nonmyoblast Cells.

Dystrophin Dp71 is an isoform produced from the Dp71 promoter in intron 62 of the DMD gene, mutations in which cause Duchenne muscular dystrophy. Dp71 is involved in various cellular processes and comprises more than 10 isoforms produced by alternative splicing. Dp71ab, in which both exons 71 and 78 are deleted, has a hydrophobic C-terminus that is hydrophilic in Dp71. Therefore, Dp71ab is believed to have different roles from Dp71. Previously, we reported that Dp71ab enhanced the proliferation of human myoblasts. Here, we further characterized Dp71ab, focusing on the activation of cell proliferation. Dp71ab increased the proliferation of immortalized human myoblasts in a dose-dependent manner. In contrast, Dp71 suppressed proliferation in a dose-dependent manner. Consistent with these opposite effects, eGFP-tagged Dp71ab and mCherry-tagged Dp71 showed different cellular distributions, with Dp71ab mostly in the nucleus. Notably, human Dp71ab enhanced the proliferation of rat and mouse myoblasts. Despite these findings, human Dp71ab did not enhance the proliferation of human nonmyoblast cells, including rhabdomyosarcoma cells. We concluded that Dp71ab is a myoblast-specific proliferation enhancer. In further studies, Dp71ab will be employed for the expansion of myoblasts in clinical settings.

An Antisense Oligonucleotide against a Splicing Enhancer Sequence within Exon 1 of the MSTN Gene Inhibits Pre-mRNA Maturation to Act as a Novel Myostatin Inhibitor.

Antisense oligonucleotides (ASOs) are agents that modulate gene function. ASO-mediated out-of-frame exon skipping has been employed to suppress gene function. Myostatin, encoded by the MSTN gene, is a potent negative regulator of skeletal muscle growth. ASOs that induce skipping of out-of-frame exon 2 of the MSTN gene have been studied for their use in increasing muscle mass. However, no ASOs are currently available for clinical use. We hypothesized that ASOs against the splicing enhancer sequence within exon 1 of the MSTN gene would inhibit maturation of pre-mRNA, thereby suppressing gene function. To explore this hypothesis, ASOs against sequences of exon 1 of the MSTN gene were screened for their ability to reduce mature MSTN mRNA levels. One screened ASO, named KMM001, decreased MSTN mRNA levels in a dose-dependent manner and reciprocally increased MSTN pre-mRNA levels. Accordingly, KMM001 decreased myostatin protein levels. KMM001 inhibited SMAD-mediated myostatin signaling in rhabdomyosarcoma cells. Remarkably, it did not decrease GDF11 mRNA levels, indicating myostatin-specific inhibition. As expected, KMM001 enhanced the proliferation of human myoblasts. We conclude that KMM001 is a novel myostatin inhibitor that inhibits pre-mRNA maturation. KMM001 has great promise for clinical applications and should be examined for its ability to treat various muscle-wasting conditions.

Dual Fluorescence Splicing Reporter Minigene Identifies an Antisense Oligonucleotide to Skip Exon v8 of the CD44 Gene.

Splicing reporter minigenes are used in cell-based in vitro splicing studies. Exon skippable antisense oligonucleotide (ASO) has been identified using minigene splicing assays, but these assays include a time- and cost-consuming step of reverse transcription PCR amplification. To make in vitro splicing assay easier, a ready-made minigene (FMv2) amenable to quantitative splicing analysis by fluorescence microscopy was constructed. FMv2 was designed to encode two fluorescence proteins namely, mCherry, a transfection marker and split eGFP, a marker of splicing reaction. The split eGFP was intervened by an artificial intron containing a multicloning site sequence. Expectedly, FMv2 transfected HeLa cells produced not only red mCherry but also green eGFP signals. Transfection of FMv2CD44v8, a modified clone of FMv2 carrying an insertion of CD44 exon v8 in the multicloning site, that was applied to screen exon v8 skippable ASO, produced only red signals. Among seven different ASOs tested against exon v8, ASO#14 produced the highest index of green signal positive cells. Hence, ASO#14 was the most efficient exon v8 skippable ASO. Notably, the well containing ASO#14 was clearly identified among the 96 wells containing randomly added ASOs, enabling high throughput screening. A ready-made FMv2 is expected to contribute to identify exon skippable ASOs.

Dystrophin Dp71ab is monoclonally expressed in human satellite cells and enhances proliferation of myoblast cells.

Dystrophin Dp71 is the smallest isoform of the DMD gene, mutations in which cause Duchenne muscular dystrophy (DMD). Dp71 has also been shown to have roles in various cellular processes. Stem cell-based therapy may be effective in treating DMD, but the inability to generate a sufficient number of stem cells remains a significant obstacle. Although Dp71 is comprised of many variants, Dp71 in satellite cells has not yet been studied. Here, the full-length Dp71 consisting of 18 exons from exons G1 to 79 was amplified by reverse transcription-PCR from total RNA of human satellite cells. The amplified product showed deletion of both exons 71 and 78 in all sequenced clones, indicating monoclonal expression of Dp71ab. Western blotting of the satellite cell lysate showed a band corresponding to over-expressed Dp71ab. Transfection of a plasmid expressing Dp71ab into human myoblasts significantly enhanced cell proliferation when compared to the cells transfected with the mock plasmid. However, transfection of the Dp71 expression plasmid encoding all 18 exons did not enhance myoblast proliferation. These findings indicated that Dp71ab, but not Dp71, is a molecular enhancer of myoblast proliferation and that transfection with Dp71ab may generate a high yield of stem cells for DMD treatment.

Intronic Alternative Polyadenylation in the Middle of the DMD Gene Produces Half-Size N-Terminal Dystrophin with a Potential Implication of ECG Abnormalities of DMD Patients.

The DMD gene is one of the largest human genes, being composed of 79 exons, and encodes dystrophin Dp427m which is deficient in Duchenne muscular dystrophy (DMD). In some DMD patient, however, small size dystrophin reacting with antibody to N-terminal but not to C-terminal has been identified. The mechanism to produce N-terminal small size dystrophin remains unknown. Intronic polyadenylation is a mechanism that produces a transcript with a new 3' terminal exon and a C-terminal truncated protein. In this study, intronic alternative polyadenylation was disclosed to occur in the middle of the DMD gene and produce the half-size N-terminal dystrophin Dp427m, Dpm234. The 3'-rapid amplification of cDNA ends revealed 421 bp sequence in the downstream of DMD exon 41 in U-251 glioblastoma cells. The cloned sequence composing of the 5' end sequence of intron 41 was decided as the terminal exon, since it encoded poly (A) signal followed by poly (A) stretch. Subsequently, a fragment from DMD exon M1 to intron 41 was obtained by PCR amplification. This product was named Dpm234 after its molecular weight. However, Dpm234 was not PCR amplified in human skeletal and cardiac muscles. Remarkably, Dpm234 was PCR amplified in iPS-derived cardiomyocytes. Accordingly, Western blotting of cardiomyocyte proteins showed a band of 234 kDa reacting with dystrophin antibody to N-terminal, but not C-terminal. Clinically, DMD patients with mutations in the Dpm234 coding region were found to have a significantly higher likelihood of two ECG abnormal findings. Intronic alternative splicing was first revealed in Dp427m to produce small size dystrophin.

Schwann cell-specific Dp116 is expressed in glioblastoma cells, revealing two novel DMD gene splicing patterns.

BACKGROUND: The DMD gene is one of the largest human genes, being composed of 79 exons. Dystrophin Dp116 expressed from the promoter in intron 55 is a Schwann cell-specific isoform. The pathophysiological roles of Dp116 are largely unknown, because of its limited expression. This study assessed the expression of Dp116 in glioblastoma cells and evaluated the splicing patterns of the DMD gene in these cells. METHODS: Full-length Dp116 cDNA was PCR amplified from U-251 glioblastoma cells. Dp116 protein was analyzed by Western blotting. RESULTS: Full-length Dp116 cDNA, extending from exon S1 to exon 79, was PCR amplified to avoid confusion with other DMD isoforms. The full-length Dp116 transcript was amplified as nearly 3 kb in size. Western blotting of U-251 cell lysates revealed a signal at a position corresponding to vector-expressed Dp116 protein, indicating that Dp116 is expressed in glioblastoma cells. Sequencing of the amplified product revealed five splice variants, all skipping exon 78. The most abundant transcript lacked only exon 78 (Dp116b), whereas the second most abundant transcript lacked both exons 71 and 78 (Dp116ab). A third transcript lacking exons 71-74 and 78 was also identified (Dp116bc). Two novel splicing patterns were also observed, one with a deletion of exons 68 and 69 (Dp116bΔ68-69) and the other with a 100 bp deletion in the 5' terminal end of exon 75 (75s), which was produced by the activation of a cryptic splice acceptor site (Dp116b75s). However, the splicing patterns in glioblastoma cells of DMD exons in Dp116 and Dp71 showed no significant differences. CONCLUSIONS: Dp116 is expressed in glioblastoma cells as five splicing variants, with Dp116b being the most abundant. Two novel splicing patterns of DMD exons were observed.

Identification of the shortest splice variant of Dp71, together with five known variants, in glioblastoma cells.

BACKGROUND: Dystrophin Dp71 mRNA is produced from the most distal alternative promoter of the DMD gene, mutations in which cause Duchenne muscular dystrophy (DMD). Dp71 is characterized by a wide variety of splice variants. In addition to being associated with cognitive disturbance in patients with DMD, Dp71 may also play a role in tumorigenesis. This study analyzed Dp71 transcripts in glioblastoma, the most common and most lethal type of cerebral malignancy. METHODS: Dp71 mRNA in the U-251 glioblastoma cell line was analyzed by reverse-transcription polymerase chain reaction (RT-PCR). The amplified products were subcloned and sequenced. RESULTS: RT-PCR amplification of the 5' end of the Dp71 transcript yielded a product of expected size, indicating transcription from the Dp71 promoter in glioblastoma. Amplification of full-length Dp71, from exon G1 to DMD exon 79, yielded a product of expected size, as well as a faint, smaller sized band. Sequencing of 17 clones revealed six different alternatively spliced variants, with only one clone being of full-length Dp71 containing all 18 exons. Ten clones lacked exon 78 (Dp71b), indicating that Dp71b was a major type of Dp71 in glioblastoma. In addition, three clones lacked both exons 71 and 78 (Dp71ab), one clone lacked exons 71, 73 and 78 (Dp71ab △73), one clone lacked exons 71-74 and 78 (Dp71bc), and one clone lacked exons 68-76 and 78 (Dp71b△68-76). This novel transcript was the shortest Dp71 variant, with a predicted stop codon in exon 77 and was predicted to produce a 24.8 kDa protein, consisting of 216 amino acids including 15 amino acids from exon 77. This novel product was classified as Dp71g because of its unique C-terminal amino acid sequence. CONCLUSIONS: Six splice variants of Dp71 were identified in glioblastoma cells, with Dp71b being the most abundant. Deletion of exon 78 was an apparent default splicing pathway in glioblastoma, being observed in 16 of 17 clones. Glioblastoma cells contained the shortest Dp71 transcript (Dp71b△68-76) identified to date, with a unique C-terminal amino acid sequence. These findings suggest the need to assess the function of Dp71 variants in glioblastoma.

Comprehensive behavioral analysis of mice deficient in Rapgef2 and Rapgef6, a subfamily of guanine nucleotide exchange factors for Rap small GTPases possessing the Ras/Rap-associating domain.

Rapgef2 and Rapgef6 define a subfamily of guanine nucleotide exchange factors for Rap small GTPases, characterized by the possession of the Ras/Rap-associating domain. Previous genomic analyses suggested their possible involvement in the etiology of schizophrenia. We recently demonstrated the development of an ectopic cortical mass (ECM), which resembles the human subcortical band heterotopia, in the dorsal telencephalon-specific Rapgef2 conditional knockout (Rapgef2-cKO) brains. Additional knockout of Rapgef6 in Rapgef2-cKO mice resulted in gross enlargement of the ECM whereas knockout of Rapgef6 alone (Rapgef6-KO) had no discernible effect on the brain morphology. Here, we performed a battery of behavioral tests to examine the effects of Rapgef2 or Rapgef6 deficiency on higher brain functions. Rapgef2-cKO mice exhibited hyperlocomotion phenotypes. They showed decreased anxiety-like behavior in the elevated plus maze and the open-field tests as well as increased depression-like behavior in the Porsolt forced swim and tail suspension tests. They also exhibited increased sociability especially in novel environments. They showed defects in cognitive function as evidenced by reduced learning ability in the Barnes circular maze test and by impaired working memory in the T maze tests. In contrast, although Rapgef6 and Rapgef2 share similarities in biochemical roles, Rapgef6-KO mice exhibited mild behavioral abnormalities detected with a number of behavioral tests, such as hyperlocomotion phenotype in the open-field test and the social interaction test with a novel environment and working-memory defects in the T-maze test. In conclusion, although there were differences in their brain morphology and the magnitude of the behavioral abnormalities, Rapgef2-cKO mice and Rapgef6-KO mice exhibited hyperlocomotion phenotype and working-memory defect, both of which could be recognized as schizophrenia-like behavior.

Rapgef2, a guanine nucleotide exchange factor for Rap1 small GTPases, plays a crucial role in adherence junction (AJ) formation in radial glial cells through ERK-mediated upregulation of the AJ-constituent protein expression.

Rapgef2 and Rapgef6 define a subfamily of guanine nucleotide exchange factors for Rap1, characterized by possession of the Ras/Rap-associating domains and implicated in the etiology of schizophrenia. We previously found that dorsal telencephalon-specific Rapgef2 conditional knockout mice exhibits severe defects in formation of apical surface adherence junctions (AJs) and localization of radial glial cells (RGCs). In this study, we analyze the underlying molecular mechanism by using primary cultures of RGCs established from the developing cerebral cortex. The results show that Rapgef2-deficient RGCs exhibit a decreased ability of neurosphere formation, morphological changes represented by regression of radial glial (RG) fibers and reduced expression of AJ-constituent proteins such as N-cadherin, zonula occludens-1, E-cadherin and ß-catenin. Moreover, siRNA-mediated knockdown of Rapgef2 or Rap1A inhibits the AJ protein expression and RG fiber formation while overexpression of Rapgef2, Rapgef6, Rap1AG12V or Rap1BG12V in Rapgef2-deficient RGCs restores them. Furthermore, Rapgef2-deficient RGCs exhibit a reduction in phosphorylation of extracellular signal-regulated kinase (ERK) leading to downregulation of the expression of c-jun, which is implicated in the AJ protein expression. These results indicate a crucial role of the Rapgef2-Rap1A-ERK-c-jun pathway in regulation of the AJ formation in RGCs.

Phosphatidylinositol 3,5-bisphosphate is involved in methylglyoxal-induced activation of the Mpk1 mitogen-activated protein kinase cascade in Saccharomyces cerevisiae.

Methylglyoxal (MG) is a natural metabolite derived from glycolysis, and this 2-oxoaldehyde has been implicated in some diseases including diabetes. However, the physiological significance of MG for cellular functions is yet to be fully elucidated. We previously reported that MG activates the Mpk1 (MAPK) cascade in the yeast Saccharomyces cerevisiae To gain further insights into the cellular functions and responses to MG, we herein screened yeast-deletion mutant collections for susceptibility to MG. We found that mutants defective in the synthesis of phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) are more susceptible to MG. PtdIns(3,5)P2 levels increased following MG treatment, and vacuolar morphology concomitantly changed to a single swollen shape. MG activated the Pkc1-Mpk1 MAPK cascade in which a small GTPase Rho1 plays a crucial role, and the MG-induced phosphorylation of Mpk1 was impaired in mutants defective in the PtdIns(3,5)P2 biosynthetic pathway. Of note, heat shock-induced stress also provoked Mpk1 phosphorylation in a Rho1-dependent manner; however, PtdIns(3,5)P2 was dispensable for the heat shock-stimulated activation of this signaling pathway. Our results suggest that PtdIns(3,5)P2 is specifically involved in the MG-induced activation of the Mpk1 MAPK cascade and in the cellular adaptation to MG-induced stress.

Crucial Role of Rapgef2 and Rapgef6, a Family of Guanine Nucleotide Exchange Factors for Rap1 Small GTPase, in Formation of Apical Surface Adherens Junctions and Neural Progenitor Development in the Mouse Cerebral Cortex.

Cerebral neocortex development in mammals requires highly orchestrated events involving proliferation, differentiation, and migration of neural progenitors and neurons. Rapgef2 and Rapgef6 constitute a unique family of guanine nucleotide exchange factors for Rap1 small GTPase, which is known to play crucial roles in migration of postmitotic neurons. We previously reported that conditional knockout of Rapgef2 in dorsal telencephalon (Rapgef2-cKO) resulted in the formation of an ectopic cortical mass (ECM) resembling that of subcortical band heterotopia. Here we show that double knockout of Rapgef6 in Rapgef2-cKO mice (Rapgef2/6-dKO) results in marked enlargement of the ECM. While Rapgef2-cKO affects late-born neurons only, Rapgef2/6-dKO affects both early-born and late-born neurons. The Rapgef2-cKO cortex at embryonic day (E) 15.5, and the Rapgef2/6-dKO cortex at E13.5 and E15.5 show disruption of the adherens junctions (AJs) on the apical surface, detachment of radial glial cells (RGCs) from the apical surface and disorganization of the radial glial fiber system, which are accompanied by aberrant distribution of RGCs and intermediate progenitors, normally located in the ventricular zone and the subventricular zone, respectively, over the entire cerebral cortex. Moreover, intrauterine transduction of Cre recombinase into the Rapgef2(flox/flox) brains also results in the apical surface AJ disruption and the RGC detachment from the apical surface, both of which are effectively suppressed by cotransduction of the constitutively active Rap1 mutant Rap1(G12V). These results demonstrate a cell-autonomous role of the Rapgef2/6-Rap1 pathway in maintaining the apical surface AJ structures, which is necessary for the proper development of neural progenitor cells.

Critical function of RA-GEF-2/Rapgef6, a guanine nucleotide exchange factor for Rap1, in mouse spermatogenesis.

Small GTPase Rap1 has been implicated in the proper differentiation of testicular germ cells. In the present study, we investigated the functional significance of RA-GEF-2/Rapgef6, a guanine nucleotide exchange factor for Rap1, in testicular differentiation using mice lacking RA-GEF-2. RA-GEF-2 was expressed predominantly on the luminal side of the seminiferous tubules in wild-type mice. No significant differences were observed in the body weights or hormonal parameters of RA-GEF-2(-)(/)(-) and wild-type mice. However, the testes of RA-GEF-2(-)(/)(-) male mice were significantly smaller than those of wild-type mice and were markedly atrophied as well as hypospermatogenic. The concentration and motility of epididymal sperm were also markedly reduced and frequently had an abnormal shape. The pregnancy rate and number of fetuses were markedly lower in wild-type females after they mated with RA-GEF-2(-)(/)(-) males than with wild-type males, which demonstrated the male infertility phenotype of RA-GEF-2(-)(/)(-) mice. Furthermore, a significant reduction and alteration were observed in the expression level and cell junctional localization of N-cadherin, respectively, in RA-GEF-2(-)(/)(-) testes, which may, at least in part, account for the defects in testicular differentiation and spermatogenesis in these mice.

The role of acetylation in the subcellular localization of an oncogenic isoform of translation factor eIF5A.

Mammalian cells express two isoforms of eIF5A, eIF5A1 and eIF5A2, but little is known about the function of eIF5A2. Here we report that eIF5A2 is reversibly acetylated at lysine-47. HDAC6 and SIRT2 were identified as the enzymes responsible for deacetylating eIF5A2. Analysis using acetylation-deficient mutants indicated that acetylation regulates the subcellular localization of eIF5A2.

Acetylation regulates subcellular localization of eukaryotic translation initiation factor 5A (eIF5A).

Eukaryotic translation initiation factor 5A (eIF5A) is a protein subject to hypusination, which is essential for its function. eIF5A is also acetylated, but the role of that modification is unknown. Here, we report that acetylation regulates the subcellular localization of eIF5A. We identified PCAF as the major cellular acetyltransferase of eIF5A, and HDAC6 and SIRT2 as its major deacetylases. Inhibition of the deacetylases or impaired hypusination increased acetylation of eIF5A, leading to nuclear accumulation. As eIF5A is constitutively hypusinated under physiological conditions, we suggest that reversible acetylation plays a major role in controlling the subcellular localization of eIF5A.

Glyoxalase system in yeasts: structure, function, and physiology.

The glyoxalase system consists of glyoxalase I and glyoxalase II. Glyoxalase I catalyzes the conversion of methylglyoxal (CH(3)COCHO), a metabolite derived from glycolysis, with glutathione to S-D-lactoylglutathione, while glyoxalase II hydrolyses this glutathione thiolester to D-lactic acid and glutathione. Since methylglyoxal is toxic due to its high reactivity, the glyoxalase system is crucial to warrant the efficient metabolic flux of this reactive aldehyde. The budding yeast Saccharomyces cerevisiae has the sole gene (GLO1) encoding the structural gene for glyoxalase I. Meanwhile, this yeast has two isoforms of glyoxalase II encoded by GLO2 and GLO4. The expression of GLO1 is regulated by Hog1 mitogen-activated protein kinase and Msn2/Msn4 transcription factors under highly osmotic stress conditions. The physiological significance of GLO1 expression in response to osmotic stress is to combat the increase in the levels of methylglyoxal in cells during the production of glycerol as a compatible osmolyte. Deficiency in GLO1 in S. cerevisiae causes pleiotropic phenotypes in terms of stress response, because the steady state level of methylglyoxal increases in glo1Δ cells thereby constitutively activating Yap1 transcription factor. Yap1 is crucial for oxidative stress response, although methylglyoxal per se does not enhance the intracellular oxidation level in yeast, but it directly modifies cysteine residues of Yap1 that are critical for the nucleocytoplasmic localization of this b-ZIP transcription factor. Consequently, glyoxalase I can be defined as a negative regulator of Yap1 through modulating the intracellular methylglyoxal level.

Calcineurin/Crz1 destabilizes Msn2 and Msn4 in the nucleus in response to Ca(2+) in Saccharomyces cerevisiae.

Although methylglyoxal is derived from glycolysis, it has adverse effects on cellular function. Hence, the intrinsic role of methylglyoxal in vivo remains to be determined. Glyoxalase 1 is a pivotal enzyme in the metabolism of methylglyoxal in all types of organisms. To learn about the physiological roles of methylglyoxal, we have screened conditions that alter the expression of the gene encoding glyoxalase 1, GLO1, in Saccharomyces cerevisiae. We show that the expression of GLO1 is induced following treatment with Ca2+ and is dependent on the MAPK (mitogen-activated protein kinase) Hog1 protein and the Msn2/Msn4 transcription factors. Intriguingly, the Ca2+-induced expression of GLO1 was enhanced in the presence of FK506, a potent inhibitor of calcineurin. Consequently, the Ca2+-induced expression of GLO1 in a mutant that is defective in calcineurin or Crz1, the sole transcription factor downstream of calcineurin, was much greater than that in the wild-type strain even without FK506. This phenomenon was dependent upon a cis-element, the STRE (stress-response element), in the promoter that is able to mediate the response to Ca2+ signalling together with Hog1 and Msn2/Msn4. The level of Ca2+-induced expression of GLO1 reached a maximum in cells overexpressing MSN2 even when FK506 was not present, whereas in cells overexpressing CRZ1 the level was greatly reduced and increased markedly when FK506 was present. We also found that the levels of Msn2 and Msn4 proteins in Ca2+-treated cells decreased gradually and that FK506 blocked the degradation of Msn2/Msn4. We propose that Crz1 destabilizes Msn2/Msn4 in the nuclei of cells in response to Ca2+ signalling.