U1 snRNP determines mRNA length and regulates isoform expression.

U1 snRNP (U1), in addition to its splicing role, protects pre-mRNAs from drastic premature termination by cleavage and polyadenylation (PCPA) at cryptic polyadenylation signals (PASs) in introns. Here, a high-throughput sequencing strategy of differentially expressed transcripts (HIDE-seq) mapped PCPA sites genome wide in divergent organisms. Surprisingly, whereas U1 depletion terminated most nascent gene transcripts within ~1 kb, moderate functional U1 level decreases, insufficient to inhibit splicing, dose-dependently shifted PCPA downstream and elicited mRNA 3' UTR shortening and proximal 3' exon switching characteristic of activated immune and neuronal cells, stem cells, and cancer. Activated neurons' signature mRNA shortening could be recapitulated by U1 decrease and antagonized by U1 overexpression. Importantly, we show that rapid and transient transcriptional upregulation inherent to neuronal activation physiology creates U1 shortage relative to pre-mRNAs. Additional experiments suggest cotranscriptional PCPA counteracted by U1 association with nascent transcripts, a process we term telescripting, ensuring transcriptome integrity and regulating mRNA length.

Neuronal SAM68 differentially regulates alternative last exon splicing and ensures proper synapse development and function.

Alternative splicing in the 3'UTR of mammalian genes plays a crucial role in diverse biological processes, including cell differentiation and development. SAM68 is a key splicing regulator that controls the diversity of 3'UTR isoforms through alternative last exon (ALE) selection. However, the tissue/cell type-specific mechanisms underlying the splicing control at the 3' end and its functional significance remain unclear. Here, we show that SAM68 regulates ALE splicing in a dose-dependent manner and the neuronal splicing is differentially regulated depending on the characteristics of the target transcript. Specifically, we found that SAM68 regulates interleukin-1 receptor-associated protein splicing through the interaction with U1 small nuclear ribonucleoprotein. In contrast, the ALE splicing of protocadherin-15 (Pcdh15), a gene implicated in several neuropsychiatric disorders, is independent of U1 small nuclear ribonucleoprotein but modulated by the calcium/calmodulin-dependent protein kinase signaling pathway. We found that the aberrant ALE selection of Pcdh15 led to a conversion from a membrane-bound to a soluble isoform and consequently disrupted its localization into excitatory and inhibitory synapses. Notably, the neuronal expression of the soluble form of PCDH15 preferentially affected the number of inhibitory synapses. Moreover, the soluble form of PCDH15 interacted physically with α-neurexins and further disrupted neuroligin-2-induced inhibitory synapses in artificial synapse formation assays. Our findings provide novel insights into the role of neuron-specific alternative 3'UTR isoform selections in synapse development.

Synthesis, growth inhibitory activity against tumor cells, and structure-activity relationship of CGK733 and its analogs.

CGK733 was reported as a compound that inhibited ATM/ATR kinase activities and blocked their checkpoint signaling pathways with great selectivity. However, this paper was subsequently retracted, and the truth about the activity of CGK733 remains unclear. We synthesized various analogs of CGK733 with a modification of the carboxylic acid moiety and/or the aniline derivative moiety to accumulate knowledge of the structure-activity relationship of this compound. Growth inhibitory activity of CGK733 and novel 35 analogs against HeLa S3 cells was evaluated, and the structure-activity relationship revealed that analogs with the 2-naphthyl or 4-fluorophenyl group instead of the benzhydryl group have activity comparable to CGK733 and that the 3-nitro group on the aniline moiety significantly affects the activity.

[A case of acalculous gangrenous cholecystitis due to artery dissection].

A 62-year-old male presented with right intercostal muscle pain. Clinical examination revealed muscular defense in the same area. Abdominal ultrasonography revealed a distended gallbladder and ascites effusion, but no gallstones or polyps were present. Contrast-enhanced computerized tomography was performed, which revealed luminal obstruction due to arterial dissection of the celiac artery and intrinsic hepatic artery. This finding suggested gangrenous cholecystitis; thus, urgent cholecystectomy was performed. Only a few cases of celiac artery dissection and only one case of gangrenous cholecystitis without stones have been reported. We report here an extremely rare case of celiac artery dissection.

Spliceostatin A stabilizes CDKN1B mRNA through the 3' UTR.

Pre-mRNA splicing is one of the most important mechanisms in gene expression in eukaryotes, and therefore splicing inhibition affects various cellular functions. We previously reported that the potent splicing inhibitor spliceostatin A (SSA) causes cell cycle arrest at G1 and G2/M phases. Upregulation of the p27 cyclin dependent kinase inhibitor, encoded by the CDKN1B gene, is one of the reasons for G1 phase arrest caused by SSA treatment. However, the molecular mechanism of p27 upregulation by SSA remains unknown. In this study, we found that SSA treatment caused stabilization of the p27 protein and increase of CDKN1B mRNA. SSA did not affect transcription of CDKN1B gene, but stabilized CDKN1B mRNA. Finally, we revealed that the 3' untranslated region of CDKN1B mRNA was involved in the stabilization. These results suggest that stabilization of CDKN1B mRNA is one of the reasons of upregulation of the p27 protein by SSA.

Relationship between fatty liver change and nutritional status after total gastrectomy in gastric cancer patients: a retrospective study.

BACKGROUND: The relationship between chronological nutritional changes and development of fatty liver after total gastrectomy (TG) in gastric cancer (GC) patients is still unclear. This study aimed to evaluate relationship between development of fatty liver and chronological changes of nutritional parameters during 12 months after TG. METHODS: We retrospectively analyzed medical records of 59 patients with GC who underwent TG at the Kanazawa Medical University Hospital between January 2009 and December 2017. We defined fatty liver change as a mean liver-to-spleen attenuation ratio (L/S ratio) of less than 1.2 in the computed tomography images at 12 months after TG and divided the patients into fatty liver (FL) and non-FL groups from the L/S ratio. We analyzed serum levels of total protein and albumin, and psoas muscle index (PMI) before TG and at 6 and 12 months after TG in the non-FL and FL groups. RESULTS: Six patients showed an L/S ratio of less than 1.2 at 12 months after TG and were included into FL group. There was no significant difference between the groups in serum parameters, L/S ratio, and PMI before TG. In the FL group, the mean levels of total protein and albumin decreased after TG and were significant lower at 6 months, compared with the non-FL group. And then, these levels in the FL group recovered at 12 months. In contrast, the mean levels of total protein and albumin in the non-FL group did not decrease below the preoperative levels throughout the year after surgery. As with laboratory parameters, all patients in the FL group showed decrease of PMI at 6 months after TG. This proportion was significantly higher than that in the non-FL group (100% vs. 40.8%, P = 0.006). CONCLUSIONS: We evaluated that the patients with fatty liver occurring after TG had significantly lower levels of serum nutritional parameters and skeletal muscle index at 6 months, not but 12 months, after TG.

CCNE1 and E2F1 Partially Suppress G1 Phase Arrest Caused by Spliceostatin A Treatment.

The potent splicing inhibitor spliceostatin A (SSA) inhibits cell cycle progression at the G1 and G2/M phases. We previously reported that upregulation of the p27 cyclin-dependent kinase inhibitor encoded by CDKN1B and its C-terminal truncated form, namely p27*, which is translated from CDKN1B pre-mRNA, is one of the causes of G1 phase arrest caused by SSA treatment. However, the detailed molecular mechanism underlying G1 phase arrest caused by SSA treatment remains to be elucidated. In this study, we found that SSA treatment caused the downregulation of cell cycle regulators, including CCNE1, CCNE2, and E2F1, at both the mRNA and protein levels. We also found that transcription elongation of the genes was deficient in SSA-treated cells. The overexpression of CCNE1 and E2F1 in combination with CDKN1B knockout partially suppressed G1 phase arrest caused by SSA treatment. These results suggest that the downregulation of CCNE1 and E2F1 contribute to the G1 phase arrest induced by SSA treatment, although they do not exclude the involvement of other factors in SSA-induced G1 phase arrest.

[Role of Surgical Resection after Chemoradiotherapy in Locally Advanced Pancreatic Cancer].

We investigated 34 cases of preoperative chemoradiotherapy(CRT)for locally advanced pancreatic cancer including resectable pancreatic cancer in our department during the past 11 years. For resectable(R)or borderline resectable(BR)pancreatic cancer, survival curves were generally higher in the CRT plus S-1 group treated after CRT than in the CRT group treated with post-CRT chemotherapy, but there was no statistically significant difference. In non-resected cases, local exacerbation was observed, which was one of the causes of a decline in terminal QOL. From the above, at present, it is desirable to remove R or BR pancreatic cancer after CRT, but the significance of surgery may change in the future due to the improvement of multidisciplinary treatment.

[Significance of Preoperative Chemoradiotherapy for Resectable Pancreatic Cancer].

We investigated the effect of chemoradiotherapy(CRT)on pancreatic cancer and the significance of preoperative chemoradiotherapy( NACRT)on resectable pancreatic cancer. The subjects were 36 patients who underwent CRT for locally advanced pancreatic cancer experienced in our department in the past 12 years(. 1)Regarding the antitumor effect of CRT, tumor diameter, tumor marker, and FDG for PET examination were reduced in 72%, 81%, and 96% of cases, respectively. In addition, the effect of Grade 1b plus 2 was observed in 10 of 16 patients who were resected after CRT(response rate 63%). In these successful cases, irradiation of 40 Gy or more and oral administration of S-1 1,500 mg or more were performed during this period. In addition, the survival rate of the NACRT plus S group(16 cases)was the same as that of the SF group (20 cases)of cStage ⅡA or lower at the same time, 50% survival was longer, and local recurrence was less. Based on the above, preoperative chemoradiotherapy combined with S-1 for resectable pancreatic cancer may be a promising preoperative treatment in the future.

Rbm38 Reduces the Transcription Elongation Defect of the SMEK2 Gene Caused by Splicing Deficiency.

Pre-mRNA splicing is an essential mechanism for ensuring integrity of the transcriptome in eukaryotes. Therefore, splicing deficiency might cause a decrease in functional proteins and the production of nonfunctional, aberrant proteins. To prevent the production of such aberrant proteins, eukaryotic cells have several mRNA quality control mechanisms. In addition to the known mechanisms, we previously found that transcription elongation is attenuated to prevent the accumulation of pre-mRNA under splicing-deficient conditions. However, the detailed molecular mechanism behind the defect in transcription elongation remains unknown. Here, we showed that the RNA binding protein Rbm38 reduced the transcription elongation defect of the SMEK2 gene caused by splicing deficiency. This reduction was shown to require the N- and C-terminal regions of Rbm38, along with an important role being played by the RNA-recognition motif of Rbm38. These findings advance our understanding of the molecular mechanism of the transcription elongation defect caused by splicing deficiency.

[A Case of Effective Disease Control of Advanced Gastric Cancer with Distant Lymph Node Metastases Following Nivolumab Treatment].

A 66-year-old man was diagnosed with advanced gastric cancer(L, Less, Type 2, T4a[SE], N2, M1[LYM], H0, P0, cStage Ⅳ)and received treatment with S-1/cisplatin as first-line chemotherapy. This treatment resulted in partial response(PR) after 3 months, with reduction in the sizes of metastatic lymph nodes surrounding the pancreatic head and paraaortic lesion. However, the sizes of metastatic lymph nodes increased after 7 months of chemotherapy. Ramucirumab/nab-paclitaxel was then administered as second-line chemotherapy, and the diameter of the metastatic lymph nodes subsequently decreased after 4 months of the regimen. However, progressive disease was observed at 7 months, and blood transfusion was required because of bleeding from the primary gastric tumor. Therefore, nivolumab was initiated as third-line chemotherapy 14 months after the first treatment. After nivolumab administration, a 28% reduction in metastatic lymph nodes was achieved within 3 months, together with the regression of the primary gastric tumor and improvement in anemia within 6 months. PR was achieved after 12 months of nivolumab administration, and effective disease control was maintained for 16 months without any adverse reaction to nivolumab.

Spliceostatin A treatment inhibits mitotic clonal expansion and adipogenesis.

Adipogenesis is a differentiation process from mesenchymal stem cells to adipocytes. It has been reported that adipogenesis is regulated by a highly orchestrated transcriptional cascade. However, the effects of modulation of mRNA splicing on adipogenesis remain unknown. To investigate these effects, 3T3-L1 preadipocyte were treated with the potent splicing inhibitor spliceostatin A, which revealed that splicing inhibition suppressed adipogenesis. In addition, treatment of 3T3-L1 cells with spliceostatin A during the early phase of adipogenesis was sufficient to inhibit adipogenesis. In the early phase of adipogenesis, the cells re-entered the cell cycle, which is referred to as mitotic clonal expansion. As mitotic clonal expansion is required for adipogenesis, it was assumed that splicing inhibition would suppress mitotic clonal expansion, and consequently inhibit adipogenesis. As expected, spliceostatin A treatment caused G1 phase arrest and inhibited cell proliferation, i.e., inhibition of mitotic clonal expansion. These results suggest that splicing activity is required for mitotic clonal expansion and adipogenesis.

Global analysis of pre-mRNA subcellular localization following splicing inhibition by spliceostatin A.

Spliceostatin A (SSA) is a methyl ketal derivative of FR901464, a potent antitumor compound isolated from a culture broth of Pseudomonas sp no. 2663. These compounds selectively bind to the essential spliceosome component SF3b, a subcomplex of the U2 snRNP, to inhibit pre-mRNA splicing. However, the mechanism of SSA's antitumor activity is unknown. It is noteworthy that SSA causes accumulation of a truncated form of the CDK inhibitor protein p27 translated from CDKN1B pre-mRNA, which is involved in SSA-induced cell-cycle arrest. However, it is still unclear whether pre-mRNAs are uniformly exported from the nucleus following SSA treatment. We performed RNA-seq analysis on nuclear and cytoplasmic fractions of SSA-treated cells. Our statistical analyses showed that intron retention is the major consequence of SSA treatment, and a small number of intron-containing pre-mRNAs leak into the cytoplasm. Using a series of reporter plasmids to investigate the roles of intronic sequences in the pre-mRNA leakage, we showed that the strength of the 5' splice site affects pre-mRNA leakage. Additionally, we found that the level of pre-mRNA leakage is related to transcript length. These results suggest that the strength of the 5' splice site and the length of the transcripts are determinants of the pre-mRNA leakage induced by SF3b inhibitors.

The neutrophil/lymphocyte ratio as a predictor of peritoneal metastasis during staging laparoscopy for advanced gastric cancer: a retrospective cohort analysis.

BACKGROUND: The use of staging laparoscopy (SL) has become widespread in patients with advanced gastric cancer (GC). This study aimed to evaluate the predictive value of the neutrophil/lymphocyte ratio (NLR) for the presence of peritoneal metastasis during staging laparoscopy in patients with advanced GC. METHODS: This retrospective analysis was performed in 35 patients with advanced GC who underwent SL at Kanazawa Medical University Hospital between January 2009 and December 2017. Clinicopathological characteristics were examined and multivariate analyses were performed to identify preoperative laboratory parameters that were independently associated with the presence of peritoneal metastasis or cytological malignancy (P/CY positive) during SL. RESULTS: A P/CY-positive result was confirmed during SL in 16 patients (45.7%). Patients with type 4 or diffuse type 3 tumors showed a significantly higher P/CY-positive rate than those with other tumor types (58.3% vs. 18.2%, P = 0.02). In the univariate analysis for preoperative laboratory parameters, NLR (P < 0.0001) and total protein (P = 0.03) and albumin (P = 0.04) levels were significantly correlated with a P/CY-positive result. On multivariate analysis, NLR was significantly correlated with a P/CY-positive result (P = 0.0002). In patients with type 4 or diffuse type 3 tumors, a high NLR (> 3.5) was associated with a significantly higher P/CY-positive rate than a low NLR (≤ 3.5) (83.3% vs. 33.3%, P = 0.01). Moreover, in patients without type 4 or diffuse type 3 tumors, the P/CY-positive rates were 100% and 0% in patients with NLR > 3.5 and NLR ≤ 3.5, respectively. CONCLUSIONS: The preoperative NLR was a significant independent predictor of the presence of peritoneal metastasis during SL. Regardless of tumor type, patients with a high NLR could be reasonable candidates for SL. On the other hand, non-diffuse type tumor accompanied by a low NLR may not need to undergo SL.

Splicing inhibition decreases phosphorylation level of Ser2 in Pol II CTD.

Phosphorylation of the C-terminal domain of the largest subunit of RNA polymerase II (Pol II), especially Ser2 and Ser5 residues, plays important roles in transcription and mRNA processing, including 5' end capping, splicing and 3' end processing. These phosphorylation events stimulate mRNA processing, however, it is not clear whether splicing activity affects the phosphorylation status of Pol II. In this study, we found that splicing inhibition by potent splicing inhibitors spliceostatin A (SSA) and pladienolide B or by antisense oligos against snRNAs decreased phospho-Ser2 level, but had little or no effects on phospho-Ser5 level. In contrast, transcription and translation inhibitors did not decrease phospho-Ser2 level, therefore inhibition of not all the gene expression processes cause the decrease of phospho-Ser2. SSA treatment caused early dissociation of Pol II and decrease in phospho-Ser2 level of chromatin-bound Pol II, suggesting that splicing inhibition causes downregulation of phospho-Ser2 through at least these two mechanisms.

[Prognosis of Conversion Gastrectomy Cases for Stage IV Gastric Cancer].

BACKGROUND: We evaluated the efficacy of surgical resection following response to primary chemotherapy for prospective registered Stage IV gastric cancer patients. PATIENTS AND METHODS: We analyzed the details and prognosis of 6 patients having advanced gastric cancer clinically diagnosed as resectable following primary chemotherapy between 2011 and 2015. RESULTS: The reason for being diagnosed as unresectable before chemotherapy was metastasis to distant sites, including paraaortic lymph node metastasis in 3 cases, peritoneal metastasis in 2 cases, and liver metastasis in 1 case.Two patients were able to undergo R0 resection, and the remaining 4 patients were unable to undergo complete resection.The median survival time (MST)of the patients who underwent R0 resection was 567.5 days, and the MST of the patients who could not undergo R0 resection was 474 days. CONCLUSION: Careful consideration of conversion gastrectomy may be important in inducing longterm survival in clinical Stage IV gastric cancer patients.

U1 snRNP protects pre-mRNAs from premature cleavage and polyadenylation.

In eukaryotes, U1 small nuclear ribonucleoprotein (snRNP) forms spliceosomes in equal stoichiometry with U2, U4, U5 and U6 snRNPs; however, its abundance in human far exceeds that of the other snRNPs. Here we used antisense morpholino oligonucleotide to U1 snRNA to achieve functional U1 snRNP knockdown in HeLa cells, and identified accumulated unspliced pre-mRNAs by genomic tiling microarrays. In addition to inhibiting splicing, U1 snRNP knockdown caused premature cleavage and polyadenylation in numerous pre-mRNAs at cryptic polyadenylation signals, frequently in introns near (<5 kilobases) the start of the transcript. This did not occur when splicing was inhibited with U2 snRNA antisense morpholino oligonucleotide or the U2-snRNP-inactivating drug spliceostatin A unless U1 antisense morpholino oligonucleotide was also included. We further show that U1 snRNA-pre-mRNA base pairing was required to suppress premature cleavage and polyadenylation from nearby cryptic polyadenylation signals located in introns. These findings reveal a critical splicing-independent function for U1 snRNP in protecting the transcriptome, which we propose explains its overabundance.

[Radical Resection of cT3a Gallbladder Cancer after Neoadjuvant Chemotherapy - A Case Report].

We report a case of a radical resection of cT3a gallbladder cancer after neoadjuvant chemotherapy(NAC). A 68-year-old man was referred to our hospital with a chief complaint of right hypochondralgia.Imaging findings were consistent with acute cholecystitis with a stone at the neck of the gallbladder, and advanced gallbladder cancer with infiltration into segments 4 and 5 from the fundus of the gallbladder, Gfb, cT3a(liver), cN1(8a), cM0, cStage III B, was diagnosed on staging laparoscopy. The patient received 3 courses of GEM plus CDDP NAC.The response to the treatment included reduction of the main tumor by 35%, diminished accumulation of FDG at the 8a lymph node, and decrease in serum CA19-9, from 163 U/mL to 75 U/mL. Cholecystectomy with the gallbladder bed and regional lymphadenectomy were performed.The histologic examination revealed extensive necrosis and degeneration of cancer cells in the infiltrating lesions, and the therapeutic effect was judged as Grade I b.The patient has now survived for 11 months without recurrence.

[Treatment Outcomes of Advanced Gastric Cancer after Neoadjuvant Chemotherapy with S-1 and Cisplatin].

BACKGROUND: The prognosis after neoadjuvant chemotherapy(NAC)is expected to improve in patients with resectable advanced gastric cancer who are at high risk of recurrence or those with unfavorable prognostic factors. PATIENTS AND METHODS: This retrospective study examined treatment outcomes and survival of 25 patients with advanced gastric cancer who received NAC with S-1 and cisplatin(CDDP)between October 2008 and December 2015. RESULTS: Among patients with clinical Stage II (4 patients)and III (21 patients)tumors, 13 had partial response(PR)and 12 had stable disease(SD). Neither complete response(CR)nor progressive disease(PD)was noted. CR of lymph node metastases was observed in 6 patients, PR in 9 patients, and SD in 7 patients. R0 resection was performed in 16 patients, R1 in 3 patients, and R2 in 6 patients. Histologic grades of primary tumors were Grade 0(1 patient), Grade 1a(16 patients), Grade 1b(5 patients), Grade 2(3 patients), and Grade 3(none). The 3-year survival rate after R0 resection was 46%, 3-year progression-free survival rate was 68%, and 3-year recurrence-free survival rate was 69%. Significant differences were observed for pathologic stages ypN0/1, 2, and 3(p=0.04), tumor down-stage(p=0.02), and overall tumor fStage I , II / III , and IV (p<0.01). CONCLUSION: It is conceivable that R0 resection and downstaging after NAC will improve prognosis.

Identification of the specific interactors of the human lariat RNA debranching enzyme 1 protein.

In eukaryotes, pre-mRNA splicing is an essential step for gene expression. We have been analyzing post-splicing intron turnover steps in higher eukaryotes. Here, we report protein interaction between human Debranching enzyme 1 (hDbr1) and several factors found in the Intron Large (IL) complex, which is an intermediate complex of the intron degradation pathway. The hDbr1 protein specifically interacts with xeroderma pigmentosum, complementeation group A (XPA)-binding protein 2 (Xab2). We also attempted to identify specific interactors of hDbr1. Co-immunoprecipitation experiments followed by mass spectrometry analysis identified a novel protein as one of the specific interactors of hDbr1. This protein is well conserved among many species and shows the highest similarity to yeast Drn1, so it is designated as human Dbr1 associated ribonuclease 1 (hDrn1). hDrn1 directly interacts with hDbr1 through protein-protein interaction. Furthermore, hDrn1 shuttles between the nucleus and the cytoplasm, as hDbr1 protein does. These findings suggest that hDrn1 has roles in both the nucleus and the cytoplasm, which are highly likely to involve hDbr1.