Blending and separating dynamics of RNA-binding proteins develop architectural splicing networks spreading throughout the nucleus.

The eukaryotic nucleus has a highly organized structure. Although the spatiotemporal arrangement of spliceosomes on nascent RNA drives splicing, the nuclear architecture that directly supports this process remains unclear. Here, we show that RNA-binding proteins (RBPs) assembled on RNA form meshworks in human and mouse cells. Core and accessory RBPs in RNA splicing make two distinct meshworks adjacently but distinctly distributed throughout the nucleus. This is achieved by mutual exclusion dynamics between the charged and uncharged intrinsically disordered regions (IDRs) of RBPs. These two types of meshworks compete for spatial occupancy on pre-mRNA to regulate splicing. Furthermore, the optogenetic enhancement of the RBP meshwork causes aberrant splicing, particularly of genes involved in neurodegeneration. Genetic mutations associated with neurodegenerative diseases are often found in the IDRs of RBPs, and cells harboring these mutations exhibit impaired meshwork formation. Our results uncovered the spatial organization of RBP networks to drive RNA splicing.

Regulated splicing of large exons is linked to phase-separation of vertebrate transcription factors.

Although large exons cannot be readily recognized by the spliceosome, many are evolutionarily conserved and constitutively spliced for inclusion in the processed transcript. Furthermore, whether large exons may be enriched in a certain subset of proteins, or mediate specific functions, has remained unclear. Here, we identify a set of nearly 3,000 SRSF3-dependent large constitutive exons (S3-LCEs) in human and mouse cells. These exons are enriched for cytidine-rich sequence motifs, which bind and recruit the splicing factors hnRNP K and SRSF3. We find that hnRNP K suppresses S3-LCE splicing, an effect that is mitigated by SRSF3 to thus achieve constitutive splicing of S3-LCEs. S3-LCEs are enriched in genes for components of transcription machineries, including mediator and BAF complexes, and frequently contain intrinsically disordered regions (IDRs). In a subset of analyzed S3-LCE-containing transcription factors, SRSF3 depletion leads to deletion of the IDRs due to S3-LCE exon skipping, thereby disrupting phase-separated assemblies of these factors. Cytidine enrichment in large exons introduces proline/serine codon bias in intrinsically disordered regions and appears to have been evolutionarily acquired in vertebrates. We propose that layered splicing regulation by hnRNP K and SRSF3 ensures proper phase-separation of these S3-LCE-containing transcription factors in vertebrates.

Comparison of intraoperative neurophysiological monitoring methods for lumbosacral lipoma surgery in infants.

BACKGROUND: Stable intraoperative neurophysiology monitoring of infants, especially very young infants, is challenging. In this study, motor evoked potentials (MEPs), the bulbocavernosus reflex (BCR), and somatosensory evoked potentials (SEPs) were simultaneously monitored in infants with lumbosacral lipomas, and these methods were compared retrospectively. METHODS: A total of 21 surgeries performed for lumbosacral lipoma in patients less than 1 year old were studied. The mean age at surgery was 133.8 days (range 21-287 days; ≤ 120 days: 9 cases, > 120 days: 12 cases). Transcranial MEPs were measured in the anal sphincter and gastrocnemius, and tibialis anterior and other muscles were added as needed. The BCR was measured by the electromyogram of the anal sphincter muscle with stimulation of the pubic region, and SEPs were measured from the waveforms of stimulation of the posterior tibial nerves. RESULTS: For the BCR, stable potentials could be recorded for all 9 cases at ≤ 120 days of age. In contrast, for MEPs, stable potentials could be recorded in only 4 of 9 cases (p < 0.05). For all patients > 120 days of age, MEPs and the BCR were measurable. SEPs were undetectable in some patients regardless of age. CONCLUSION: The BCR could be more consistently measured than MEPs in infant patients with lumbosacral lipoma at ≤ 120 days of age.

The effect of shunt removal on the quality of life in patients with congenital hydrocephalus.

BACKGROUND: Although there have been reports investigating the quality of life of patients who underwent ventriculoperitoneal shunting or endoscopic third ventriculostomy (ETV) for congenital hydrocephalus, there have been no studies of the quality of life of patients after ventriculoperitoneal shunt (VPS) removal. In the present study, a survey of pediatric and congenital hydrocephalus patients was conducted to compare the quality of life of patients with a remaining VPS with that of patients who had the shunt removed. METHODS: Between February 2020 and November 2021, an outpatient survey was administered to patients 8 years of age and older who had undergone VPS due to a diagnosis of congenital hydrocephalus. The Hydrocephalus Outcome Questionnaire (HOQ) was used to assess the quality of life for this study. The HOQ scores (overall health score, physical health score, cognitive health score, and social-emotional health score) were compared among three groups: a VPS-remaining group, VPS-removed with endoscopic third ventriculostomy (ETV) group, and VPS-removed without ETV group. RESULTS: The total number of patients who underwent VPS for hydrocephalus was 71, with 47 in the VPS-remaining group, 14 in the shunt-removed with ETV group, and 10 in the shunt-removed without ETV group. The HOQ overall health score was 0.68 for the VPS-remaining group, 0.74 for the shunt-removed with ETV group, and 0.74 for the shunt-removed without ETV. There were no significant differences between the VPS-remaining group and the VPS-removed with or without ETV groups (p = 0.3255, 0.4178, respectively). CONCLUSION: There was no significant difference in the quality of life between patients with a remaining VPS and those who had their VPS removed with or without ETV.

Current status and challenges of neurosurgical procedures for patients with myelomeningocele in real-world Japan.

BACKGROUND: Little is known about the real-world status of neurosurgical treatment of myelomeningocele patients. OBJECTIVE: To investigate the real-world status of neurosurgical treatment of myelomeningocele patients, medical claims data provided by the Japan Medical Data Center (JMDC) were analyzed. METHODS: The health claims data of 556 patients with myelomeningoceles from January 2005 to March 2020 were examined. The number of neurosurgical procedures, including myelomeningocele repair, tethered cord release, cerebrospinal fluid (CSF) shunt, CSF drainage, and endoscopic third ventriculostomy (ETV), was determined. RESULTS: A total of 313 neurosurgical procedures were performed for 135 patients in 74 institutions during the study period. The shunt survival rate was most affected by shunts that were revised when the patient was less than 1 year old, which had a significantly lower survival rate than all of the initial shunts performed when the patient was less than on1 year old; the 1-year shunt survival rate was 35 vs 64% (P = 0.0102). The survival rate was significantly lower in patients younger than 1 year who had CSF drainage before shunting compared to those younger than 1 year who did not have CSF drainage before shunting; the 1-year shunt survival rate was 27 vs 59% (P = 0.0196), and 81% of patients remained free of tethered cord release 10 years later. CONCLUSIONS: In this study, a revised shunt of less than 1 year of age and CSF drainage before shunting were the factors that lowered the shunt survival rate in the real world for CSF shunts for hydrocephalus associated with myelomeningocele.

Extremely low-frequency electromagnetic field induces acetylation of heat shock proteins and enhances protein folding.

The pervasive weak electromagnetic fields (EMF) inundate the industrialized society, but the biological effects of EMF as weak as 10 µT have been scarcely analyzed. Heat shock proteins (HSPs) are molecular chaperones that mediate a sequential stress response. HSP70 and HSP90 provide cells under undesirable situations with either assisting covalent folding of proteins or degrading improperly folded proteins in an ATP-dependent manner. Here we examined the effect of extremely low-frequency (ELF)-EMF on AML12 and HEK293 cells. Although the protein expression levels of HSP70 and HSP90 were reduced after an exposure to ELF-EMF for 3 h, acetylations of HSP70 and HSP90 were increased, which was followed by an enhanced binding affinities of HSP70 and HSP90 for HSP70/HSP90-organizing protein (HOP/STIP1). After 3 h exposure to ELF-EMF, the amount of mitochondria was reduced but the ATP level and the maximal mitochondrial oxygen consumption were increased, which was followed by the reduced protein aggregates and the increased cell viability. Thus, ELF-EMF exposure for 3 h activated acetylation of HSPs to enhance protein folding, which was returned to the basal level at 12 h. The proteostatic effects of ELF-EMF will be able to be applied to treat pathological states in humans.

5-Aminolevulinic acid fluorescence-guided endoscopic surgery for deep-seated intraparenchymal tumors.

AIM: The usefulness of 5-aminolevulinic acid (5-ALA) fluorescence-assisted surgery for maximum resection of malignant gliomas has been established. However, its usefulness when combined with endoscopic surgery for deep-seated tumors has not been well established. In this study, whether 5-ALA photodynamic diagnosis (PDD) is feasible and useful for endoscopic surgery was investigated. METHODS: A specially designed endoscope for PDD that delivers white light or blue light (375-440 nm) as excitation light was used. The fluorescence emitted by the tumor was evaluated in the cavity during resection or at the tip of the sheath during biopsy. The intensity of fluorescence was classified into three categories: strong, vague, and negative. RESULTS: A total of 30 intraparenchymal tumors were observed with a neuroendoscope and 5-ALA PDD; 16 patients underwent resection, and 14 underwent biopsy. Overall, 67% (20/30) of tumors showed positive fluorescence of protoporphyrin IX. High-grade gliomas (HGGs) including glioblastoma (GBM) and anaplastic astrocytoma (AA) showed strong fluorescence in 47% (7/15), vague fluorescence in 33% (5/15), and negative fluorescence in 20% (3/15) of cases. Low-grade gliomas (LGGs) showed vague fluorescence in 33% (1/3) and negative fluorescence in 67% (2/3). Diffuse large B-cell lymphoma (DLBCL) showed vague fluorescence in 38% (3/8) and negative fluorescence in 63% (5/8). Metastatic tumors showed strong fluorescence in 25% (1/4) and vague fluorescence in 75% (3/4). In the comparison of fluorescence evaluation, a significant difference was observed only in the comparison between HGGs and DLBCL (p = 0.049). CONCLUSION: These results suggest that 5-ALA PDD-assisted endoscopic surgery is feasible and useful for deep-seated intraparenchymal tumors.

A ventricular catheter that migrated into the fourth ventricle successfully removed using a neuroendoscope.

BACKGROUND: In order to remove a foreign body in the ventricle, such as a ventricular drainage catheter, craniotomy and corticotomy are required to access the ventricle. A case in which a catheter in the 4th ventricle was safely removed with a flexible neuroendoscope is reported. CASE DESCRIPTION: A 47-year-old man underwent coil embolization and ventricular drainage for subarachnoid hemorrhage. 10 days after the operation, he tore off the ventricular drainage catheter and the catheter remained intracranially. The tip of the catheter was in the 4th ventricle and the operation to remove remaining catheter with a neuroendoscope was performed. Using a neuroendoscope, we could remove the catheter safely and did not detect the complications. CONCLUSION: To date, there have been no reports of cases in which a drainage catheter in the ventricle was removed using a flexible endoscope. This case suggests that a flexible endoscope is useful for removing a foreign body from the ventricle less invasively.

Position-specific binding of FUS to nascent RNA regulates mRNA length.

More than half of all human genes produce prematurely terminated polyadenylated short mRNAs. However, the underlying mechanisms remain largely elusive. CLIP-seq (cross-linking immunoprecipitation [CLIP] combined with deep sequencing) of FUS (fused in sarcoma) in neuronal cells showed that FUS is frequently clustered around an alternative polyadenylation (APA) site of nascent RNA. ChIP-seq (chromatin immunoprecipitation [ChIP] combined with deep sequencing) of RNA polymerase II (RNAP II) demonstrated that FUS stalls RNAP II and prematurely terminates transcription. When an APA site is located upstream of an FUS cluster, FUS enhances polyadenylation by recruiting CPSF160 and up-regulates the alternative short transcript. In contrast, when an APA site is located downstream from an FUS cluster, polyadenylation is not activated, and the RNAP II-suppressing effect of FUS leads to down-regulation of the alternative short transcript. CAGE-seq (cap analysis of gene expression [CAGE] combined with deep sequencing) and PolyA-seq (a strand-specific and quantitative method for high-throughput sequencing of 3' ends of polyadenylated transcripts) revealed that position-specific regulation of mRNA lengths by FUS is operational in two-thirds of transcripts in neuronal cells, with enrichment in genes involved in synaptic activities.

tRIP-seq reveals repression of premature polyadenylation by co-transcriptional FUS-U1 snRNP assembly.

RNA processing occurs co-transcriptionally through the dynamic recruitment of RNA processing factors to RNA polymerase II (RNAPII). However, transcriptome-wide identification of protein-RNA interactions specifically assembled on transcribing RNAPII is challenging. Here, we develop the targeted RNA immunoprecipitation sequencing (tRIP-seq) method that detects protein-RNA interaction sites in thousands of cells. The high sensitivity of tRIP-seq enables identification of protein-RNA interactions at functional subcellular levels. Application of tRIP-seq to the FUS-RNA complex in the RNAPII machinery reveals that FUS binds upstream of alternative polyadenylation (APA) sites of nascent RNA bound to RNAPII, which retards RNAPII and suppresses the recognition of the polyadenylation signal by CPSF. Further tRIP-seq analyses demonstrate that the repression of APA is achieved by a complex composed of FUS and U1 snRNP on RNAPII, but not by either one alone. Moreover, our analysis reveals that FUS mutations in familial amyotrophic lateral sclerosis (ALS) that impair the FUS-U1 snRNP interaction aberrantly activate the APA sites. tRIP-seq provides new insights into the regulatory mechanism of co-transcriptional RNA processing by RNA processing factors.

Molecular Hydrogen Enhances Proliferation of Cancer Cells That Exhibit Potent Mitochondrial Unfolded Protein Response.

Molecular hydrogen ameliorates pathological states in a variety of human diseases, animal models, and cell models, but the effects of hydrogen on cancer have been rarely reported. In addition, the molecular mechanisms underlying the effects of hydrogen remain mostly unelucidated. We found that hydrogen enhances proliferation of four out of seven human cancer cell lines (the responders). The proliferation-promoting effects were not correlated with basal levels of cellular reactive oxygen species. Expression profiling of the seven cells showed that the responders have higher gene expression of mitochondrial electron transport chain (ETC) molecules than the non-responders. In addition, the responders have higher mitochondrial mass, higher mitochondrial superoxide, higher mitochondrial membrane potential, and higher mitochondrial spare respiratory capacity than the non-responders. In the responders, hydrogen provoked mitochondrial unfolded protein response (mtUPR). Suppression of cell proliferation by rotenone, an inhibitor of mitochondrial ETC complex I, was rescued by hydrogen in the responders. Hydrogen triggers mtUPR and induces cell proliferation in cancer cells that have high basal and spare mitochondrial ETC activities.

Familial tendency in patients with lipoma of the filum terminale.

PURPOSE: Lipoma of the filum terminale (FL) is an abnormality in which fat is deposited in the filum terminale. This lipoma is often detected by skin abnormalities in the lumbosacral area such as a sacrococcygeal dimple. Some cases may develop tethered cord and become symptomatic. However, the genetic basis of FL is still unclear. METHODS: This study aimed to determine whether there was a family history of FL or other forms of spina bifida among 54 families of 56 patients with FL and to examine whether there is a familial predisposition in FL. In addition, sex, age at diagnosis, presence of symptoms, presence of sacrococcygeal dimple, and the level of conus medullaris between familial and spontaneous cases were evaluated. RESULTS: Of the 54 families of FL patients, there were 48 siblings. Among the 48 siblings, 2 had "occult" FL. The frequency of FL among siblings was estimated to be 4.2% (2/48), which was significantly higher than the sum of previously reported cases of spontaneous FL (0.91%; p = 0.017). However, there was no significant difference in sex, age at diagnosis, presence of symptoms, presence of sacrococcygeal dimple, diameter of filum terminale, or level of conus medullaris between familial and spontaneous cases. CONCLUSION: To our knowledge, this is the first report on familial FL and examination of the frequency of FL among siblings. The high probability of FL among siblings of FL patients suggests that genetic factors may play a role in FL development.

Resection of a recurrent medulloblastoma in the anterior middle part of the aqueduct with a flexible endoscope: a case report.

Resection or biopsy of intraventricular brain tumors using an endoscope has become common, but the limitations of these procedures are not clear. Manipulation to access a tumor that arises from the blind angle of the rigid endoscope, such as the anterior part of the aqueduct, is limited. We report here that we successfully resected a recurrent medulloblastoma in the anterior part of the aqueduct using only a flexible endoscope. This method appears to be suitable for poorly vascularized and suctionable tumors that arise in the blind angle of a rigid endoscope.

Role of dynamic nuclear deformation on genomic architecture reorganization.

Higher-order genomic architecture varies according to cell type and changes dramatically during differentiation. One of the remarkable examples of spatial genomic reorganization is the rod photoreceptor cell differentiation in nocturnal mammals. The inverted nuclear architecture found in adult mouse rod cells is formed through the reorganization of the conventional architecture during terminal differentiation. However, the mechanisms underlying these changes remain largely unknown. Here, we found that the dynamic deformation of nuclei via actomyosin-mediated contractility contributes to chromocenter clustering and promotes genomic architecture reorganization during differentiation by conducting an in cellulo experiment coupled with phase-field modeling. Similar patterns of dynamic deformation of the nucleus and a concomitant migration of the nuclear content were also observed in rod cells derived from the developing mouse retina. These results indicate that the common phenomenon of dynamic nuclear deformation, which accompanies dynamic cell behavior, can be a universal mechanism for spatiotemporal genomic reorganization.

Competitive regulation of alternative splicing and alternative polyadenylation by hnRNP H and CstF64 determines acetylcholinesterase isoforms.

Acetylcholinesterase (AChE), encoded by the ACHE gene, hydrolyzes the neurotransmitter acetylcholine to terminate synaptic transmission. Alternative splicing close to the 3΄ end generates three distinct isoforms of AChET, AChEH and AChER. We found that hnRNP H binds to two specific G-runs in exon 5a of human ACHE and activates the distal alternative 3΄ splice site (ss) between exons 5a and 5b to generate AChET. Specific effect of hnRNP H was corroborated by siRNA-mediated knockdown and artificial tethering of hnRNP H. Furthermore, hnRNP H competes for binding of CstF64 to the overlapping binding sites in exon 5a, and suppresses the selection of a cryptic polyadenylation site (PAS), which additionally ensures transcription of the distal 3΄ ss required for the generation of AChET. Expression levels of hnRNP H were positively correlated with the proportions of the AChET isoform in three different cell lines. HnRNP H thus critically generates AChET by enhancing the distal 3΄ ss and by suppressing the cryptic PAS. Global analysis of CLIP-seq and RNA-seq also revealed that hnRNP H competitively regulates alternative 3΄ ss and alternative PAS in other genes. We propose that hnRNP H is an essential factor that competitively regulates alternative splicing and alternative polyadenylation.

Splicing regulation and dysregulation of cholinergic genes expressed at the neuromuscular junction.

We humans have evolved by acquiring diversity of alternative RNA metabolisms including alternative means of splicing and transcribing non-coding genes, and not by acquiring new coding genes. Tissue-specific and developmental stage-specific alternative RNA splicing is achieved by tightly regulated spatiotemporal regulation of expressions and activations of RNA-binding proteins that recognize their cognate splicing cis-elements on nascent RNA transcripts. Genes expressed at the neuromuscular junction are also alternatively spliced. In addition, germline mutations provoke aberrant splicing by compromising binding of RNA-binding proteins, and cause congenital myasthenic syndromes (CMS). We present physiological splicing mechanisms of genes for agrin (AGRN), acetylcholinesterase (ACHE), MuSK (MUSK), acetylcholine receptor (AChR) α1 subunit (CHRNA1), and collagen Q (COLQ) in human, and their aberration in diseases. Splicing isoforms of AChET , AChEH , and AChER are generated by hnRNP H/F. Skipping of MUSK exon 10 makes a Wnt-insensitive MuSK isoform, which is unique to human. Skipping of exon 10 is achieved by coordinated binding of hnRNP C, YB-1, and hnRNP L to exon 10. Exon P3A of CHRNA1 is alternatively included to generate a non-functional AChR α1 subunit in human. Molecular dissection of splicing mutations in patients with CMS reveals that exon P3A is alternatively skipped by hnRNP H, polypyrimidine tract-binding protein 1, and hnRNP L. Similarly, analysis of an exonic mutation in COLQ exon 16 in a CMS patient discloses that constitutive splicing of exon 16 requires binding of serine arginine-rich splicing factor 1. Intronic and exonic splicing mutations in CMS enable us to dissect molecular mechanisms underlying alternative and constitutive splicing of genes expressed at the neuromuscular junction. This is an article for the special issue XVth International Symposium on Cholinergic Mechanisms.

5-ALA fluorescence-guided endoscopic surgery for mixed germ cell tumors.

5-Aminolevulinic acid (5-ALA) fluorescence-guided surgery is widely used for detection and planning of resection of malignant gliomas and other brain tumors. However, no reports have described 5-ALA fluorescence-guided surgery or direct visualization of germ cell tumors. Here, we report two cases of germ cell tumors in which a positive 5-ALA fluorescent signal was visualized with a neuroendoscope. Both cases had a tumor in the pineal region that was associated with hydrocephalus. The patients underwent surgery after administration of 5-ALA. After ventricular puncture of the anterior horn, we could observe the ventricular wall and tumor using the Karl Storz Photodynamic diagnosis system endoscope. Then, biopsy of the pineal tumor and endoscopic third ventriculostomy were performed in both cases. In case 1, a 22-year-old man, part of the ventricular wall and tumor tissue showed red fluorescence. In case 2, a 16-year-old man, part of the fornix and infundibular recess showed red fluorescence, and the tumor showed relatively weak red fluorescence. The histopathological diagnosis of both cases was pure germinoma. This is the first report of direct visualization of mixed germinomas with 5-ALA fluorescence-guided endoscopic surgery. This method not only allows visualization of the tumor mass, but may also be useful for detailed observation in the ventricular wall.

IntSplice: prediction of the splicing consequences of intronic single-nucleotide variations in the human genome.

Precise spatiotemporal regulation of splicing is mediated by splicing cis-elements on pre-mRNA. Single-nucleotide variations (SNVs) affecting intronic cis-elements possibly compromise splicing, but no efficient tool has been available to identify them. Following an effect-size analysis of each intronic nucleotide on annotated alternative splicing, we extracted 105 parameters that could affect the strength of the splicing signals. However, we could not generate reliable support vector regression models to predict the percent-splice-in (PSI) scores for normal human tissues. Next, we generated support vector machine (SVM) models using 110 parameters to directly differentiate pathogenic SNVs in the Human Gene Mutation Database and normal SNVs in the dbSNP database, and we obtained models with a sensitivity of 0.800±0.041 (mean and s.d.) and a specificity of 0.849±0.021. Our IntSplice models were more discriminating than SVM models that we generated with Shapiro-Senapathy score and MaxEntScan::score3ss. We applied IntSplice to a naturally occurring and nine artificial intronic mutations in RAPSN causing congenital myasthenic syndrome. IntSplice correctly predicted the splicing consequences for nine of the ten mutants. We created a web service program, IntSplice (http://www.med.nagoya-u.ac.jp/neurogenetics/IntSplice) to predict splicing-affecting SNVs at intronic positions from -50 to -3.

H-InvDB in 2013: an omics study platform for human functional gene and transcript discovery.

H-InvDB (http://www.h-invitational.jp/) is a comprehensive human gene database started in 2004. In the latest version, H-InvDB 8.0, a total of 244 709 human complementary DNA was mapped onto the hg19 reference genome and 43 829 gene loci, including nonprotein-coding ones, were identified. Of these loci, 35 631 were identified as potential protein-coding genes, and 22 898 of these were identical to known genes. In our analysis, 19 309 annotated genes were specific to H-InvDB and not found in RefSeq and Ensembl. In fact, 233 genes of the 19 309 turned out to have protein functions in this version of H-InvDB; they were annotated as unknown protein functions in the previous version. Furthermore, 11 genes were identified as known Mendelian disorder genes. It is advantageous that many biologically functional genes are hidden in the H-InvDB unique genes. As large-scale proteomic projects have been conducted to elucidate the functions of all human proteins, we have enhanced the proteomic information with an advanced protein view and new subdatabase of protein complexes (Protein Complex Database with quality index). We propose that H-InvDB is an important resource for finding novel candidate targets for medical care and drug development.

Fat necrosis appearing as intraorbital tumour: case report.

A 33-year-old male presented with an extremely rare case of intraorbital fat necrosis. A magnetic resonance imaging scan showed a 10-mm mass lesion within the right lateral rectal muscle. Surgical removal was performed. Histological analysis showed diffuse adipose cells surrounded by macrophage cells. Fat necrosis was diagnosed.