Chimera RNA transcribed from integrated HPV18 genome with adjacent host genomic region promotes oncogenic gene expression through condensate formation.

Most cervical cancers are caused by human papillomavirus (HPV) infection. In HeLa cells, the HPV18 viral genome is integrated at chromosome 8q24.21 and activates transcription of the proto-oncogene c-Myc. However, the mechanism of how the integrated HPV genome and its transcribed RNAs exhibit transcription activation function has not been fully elucidated. In this study, we found that HPV18 transcripts contain an enhancer RNA-like function to activate proximal genes including CCAT1-5L and c-Myc. We showed that the human genome-integrated HPV18 genes are activated by transcription coregulators including BRD4 and Mediator. The transcribed HPV18 RNAs form a liquid-like condensate at chromosome 8q24.21 locus, which in turn accumulates RNA polymerase II. Moreover, we focused on a relatively uncharacterized transcript from the upstream region of CCAT1, named URC. The URC RNA is transcribed as a chimera RNA with HPV18 and is composed of the 3'-untranslated region of the HPV18 transcript. We experimentally showed that the URC contributes to stabilization of HPV18 RNAs by supplying a polyadenylation site for the HPV18 transcript. Our findings suggest that integrated HPV18 at 8q24.21 locus produces HPV18-URC chimera RNA and promotes tumorigenesis through RNA-based condensate formation.

MED26-containing Mediator may orchestrate multiple transcription processes through organization of nuclear bodies.

Mediator is a coregulatory complex that plays essential roles in multiple processes of transcription regulation. One of the human Mediator subunits, MED26, has a role in recruitment of the super elongation complex (SEC) to polyadenylated genes and little elongation complex (LEC) to non-polyadenylated genes, including small nuclear RNAs (snRNAs) and replication-dependent histone (RDH) genes. MED26-containing Mediator plays a role in 3' Pol II pausing at the proximal region of transcript end sites in RDH genes through recruitment of Cajal bodies (CBs) to histone locus bodies (HLBs). This finding suggests that Mediator is involved in the association of CBs with HLBs to facilitate 3' Pol II pausing and subsequent 3'-end processing by supplying 3'-end processing factors from CBs. Thus, we argue the possibility that Mediator is involved in the organization of nuclear bodies to orchestrate multiple processes of gene transcription.

Zrsr2 and functional U12-dependent spliceosome are necessary for follicular development.

ZRSR2 is a splicing factor involved in recognition of 3'-intron splice sites that is frequently mutated in myeloid malignancies and several tumors; however, the role of mutations of Zrsr2 in other tissues has not been analyzed. To explore the biological role of ZRSR2, we generated three Zrsr2 mutant mouse lines. All Zrsr2 mutant lines exhibited blood cell anomalies, and in two lines, oogenesis was blocked at the secondary follicle stage. RNA-seq of Zrsr2 mu secondary follicles showed aberrations in gene expression and showed altered alternative splicing (AS) events involving enrichment of U12-type intron retention (IR), supporting the functional Zrsr2 action in minor spliceosomes. IR events were preferentially associated with centriole replication, protein phosphorylation, and DNA damage checkpoint. Notably, we found alterations in AS events of 50 meiotic genes. These results indicate that ZRSR2 mutations alter splicing mainly in U12-type introns, which may affect peripheral blood cells, and impede oogenesis and female fertility.

Wilms' tumor 1-associating protein complex regulates alternative splicing and polyadenylation at potential G-quadruplex-forming splice site sequences.

Wilms' tumor 1-associating protein (WTAP) is a core component of the N6-methyladenosine (m6A)-methyltransferase complex, along with VIRMA, CBLL1, ZC3H13 (KIAA0853), RBM15/15B, and METTL3/14, which generate m6A, a key RNA modification that affects various processes of RNA metabolism. WTAP also interacts with splicing factors; however, despite strong evidence suggesting a role of Drosophila WTAP homolog fl(2)d in alternative splicing (AS), its role in splicing regulation in mammalian cells remains elusive. Here we demonstrate using RNAi coupled with RNA-seq that WTAP, VIRMA, CBLL1, and ZC3H13 modulate AS, promoting exon skipping and intron retention in AS events that involve short introns/exons with higher GC content and introns with weaker polypyrimidine-tract and branch points. Further analysis of GC-rich sequences involved in AS events regulated by WTAP, together with minigene assay analysis, revealed potential G-quadruplex formation at splice sites where WTAP has an inhibitory effect. We also found that several AS events occur in the last exon of one isoform of MSL1 and WTAP, leading to competition for polyadenylation. Proteomic analysis also suggested that WTAP/CBLL1 interaction promotes recruitment of the 3'-end processing complex. Taken together, our results indicate that the WTAP complex regulates AS and alternative polyadenylation via inhibitory mechanisms in GC-rich sequences.

Frequent Germline and Somatic Single Nucleotide Variants in the Promoter Region of the Ribosomal RNA Gene in Japanese Lung Adenocarcinoma Patients.

Ribosomal RNA (rRNA), the most abundant non-coding RNA species, is a major component of the ribosome. Impaired ribosome biogenesis causes the dysfunction of protein synthesis and diseases called "ribosomopathies," including genetic disorders with cancer risk. However, the potential role of rRNA gene (rDNA) alterations in cancer is unknown. We investigated germline and somatic single-nucleotide variants (SNVs) in the rDNA promoter region (positions -248 to +100, relative to the transcription start site) in 82 lung adenocarcinomas (LUAC). Twenty-nine tumors (35.4%) carried germline SNVs, and eight tumors (9.8%) harbored somatic SNVs. Interestingly, the presence of germline SNVs between positions +1 and +100 (n = 12; 14.6%) was associated with significantly shorter recurrence-free survival (RFS) and overall survival (OS) by univariate analysis (p < 0.05, respectively), and was an independent prognostic factor for RFS and OS by multivariate analysis. LUAC cell line PC9, carrying rDNA promoter SNV at position +49, showed significantly higher ribosome biogenesis than H1650 cells without SNV. Upon nucleolar stress induced by actinomycin D, PC9 retained significantly higher ribosome biogenesis than H1650. These results highlight the possible functional role of SNVs at specific sites of the rDNA promoter region in ribosome biogenesis, the progression of LUAC, and their potential prognostic value.

Sex-Dimorphic Behavioral Alterations and Altered Neurogenesis in U12 Intron Splicing-Defective Zrsr1 Mutant Mice.

Mutant mice with respect to the splicing factor Zrsr1 present altered spermatogenesis and infertility. To investigate whether Zrsr1 is involved in the homeostatic control that the hypothalamus exerts over reproductive functions, we first analyzed both differential gene and isoform expression and alternative splicing alterations in Zrsr1 mutant (Zrsr1mu) hypothalamus; second, we analyzed the spontaneous and social behavior of Zrsr1mu mice; and third, we analyzed adult cell proliferation and survival in the Zrsr1mu hypothalamus. The Zrsr1mu hypothalamus showed altered expression of genes and isoforms related to the glutathione metabolic process, synaptonemal complex assembly, mRNA transport, and altered splicing events involving the enrichment of U12-type intron retention (IR). Furthermore, increased IR in U12-containing genes related with the prolactin, progesterone, and gonadotropin-releasing hormone (GnRH) reproductive signaling pathway was observed. This was associated with a hyperactive phenotype in both males and females, with an anxious phenotype in females, and with increased social interaction in males, instead of the classical aggressive behavior. In addition, Zrsr1mu females but not males exhibited reduced cell proliferation in both the hypothalamus and the subventricular zone. Overall, these results suggest that Zrsr1 expression and function are relevant to organization of the hypothalamic cell network controlling behavior.

The RNA Methyltransferase Complex of WTAP, METTL3, and METTL14 Regulates Mitotic Clonal Expansion in Adipogenesis.

Adipocyte differentiation is regulated by various mechanisms, of which mitotic clonal expansion (MCE) is a key step. Although this process is known to be regulated by cell cycle modulators, the precise mechanism remains unclear. N6-Methyladenosine (m6A) posttranscriptional RNA modification, whose methylation and demethylation are performed by respective enzyme molecules, has recently been suggested to be involved in the regulation of adipogenesis. Here, we show that an RNA N6-adenosine methyltransferase complex consisting of Wilms' tumor 1-associating protein (WTAP), methyltransferase like 3 (METTL3), and METTL14 positively controls adipogenesis by promoting cell cycle transition in MCE during adipogenesis. WTAP, coupled with METTL3 and METTL14, is increased and distributed in nucleus by the induction of adipogenesis dependently on RNA in vitro Knockdown of each of these three proteins leads to cell cycle arrest and impaired adipogenesis associated with suppression of cyclin A2 upregulation during MCE, whose knockdown also impairs adipogenesis. Consistent with this, Wtap heterozygous knockout mice are protected from diet-induced obesity with smaller size and number of adipocytes, leading to improved insulin sensitivity. These data provide a mechanism for adipogenesis through the WTAP-METTL3-METTL14 complex and a potential strategy for treatment of obesity and associated disorders.

Impaired Spermatogenesis, Muscle, and Erythrocyte Function in U12 Intron Splicing-Defective Zrsr1 Mutant Mice.

The U2AF35-like ZRSR1 has been implicated in the recognition of 3' splice site during spliceosome assembly, but ZRSR1 knockout mice do not show abnormal phenotypes. To analyze ZRSR1 function and its precise role in RNA splicing, we generated ZRSR1 mutant mice containing truncating mutations within its RNA-recognition motif. Homozygous mutant mice exhibited severe defects in erythrocytes, muscle stretch, and spermatogenesis, along with germ cell sloughing and apoptosis, ultimately leading to azoospermia and male sterility. Testis RNA sequencing (RNA-seq) analyses revealed increased intron retention of both U2- and U12-type introns, including U12-type intron events in genes with key functions in spermatogenesis and spermatid development. Affected U2 introns were commonly found flanking U12 introns, suggesting functional cross-talk between the two spliceosomes. The splicing and tissue defects observed in mutant mice attributed to ZRSR1 loss of function suggest a physiological role for this factor in U12 intron splicing.

Suppression of Slit2/Robo1 mediated HUVEC migration by Robo4.

Slit proteins and their receptors, the Roundabout (Robo) family, are known to have a pivotal role in the vascular system. Slit2/Robo1 regulates the migration of human umbilical vein endothelial cells (HUVECs) and tumor-associated endothelial cells. Robo4, the endothelial-specific Robo, is also considered to be involved in vascular cell migration. However, the Slit/Robo signaling pathway is still unclear. Using a Boyden chamber assay, we found that Slit2 induces the migration of HUVECs under a Robo4 knockdown condition. This effect disappeared in Robo1 knockdown cells. The co-existence of the N-terminal extracellular portion of Robo1 blocked the Slit2-evoked migration of HUVECs, while that of Robo4 caused no effect. These results show that the Slit2 signal is transduced through Robo1, while the negative regulation of Robo4 is an intracellular event. Targeted proteomics using an anti-Robo1 monoclonal antibody identified CdGAP, an adhesion-localized Rac1-and Cdc42-specific GTPase activating protein, as a candidate for Slit2/Robo1 signaling. Robo1 and CdGAP were co-immunoprecipitated from CHO cells co-transfected with Robo1 and CdGAP genes. These results suggest that Slit2/Robo1 binding exerts an effect on cell migration, which is negatively regulated by Robo4, and Robo1 may function by interacting with CdGAP in HUVECs.

Identification of Wilms' tumor 1-associating protein complex and its role in alternative splicing and the cell cycle.

Wilms' tumor 1-associating protein (WTAP) is a putative splicing regulator that is thought to be required for cell cycle progression through the stabilization of cyclin A2 mRNA and mammalian early embryo development. To further understand how WTAP acts in the context of the cellular machinery, we identified its interacting proteins in human umbilical vein endothelial cells and HeLa cells using shotgun proteomics. Here we show that WTAP forms a novel protein complex including Hakai, Virilizer homolog, KIAA0853, RBM15, the arginine/serine-rich domain-containing proteins BCLAF1 and THRAP3, and certain general splicing regulators, most of which have reported roles in post-transcriptional regulation. The depletion of these respective components of the complex resulted in reduced cell proliferation along with G2/M accumulation. Double knockdown of the serine/arginine-rich (SR)-like proteins BCLAF1 and THRAP3 by siRNA resulted in a decrease in the nuclear speckle localization of WTAP, whereas the nuclear speckles were intact. Furthermore, we found that the WTAP complex regulates alternative splicing of the WTAP pre-mRNA by promoting the production of a truncated isoform, leading to a change in WTAP protein expression. Collectively, these findings show that the WTAP complex is a novel component of the RNA processing machinery, implying an important role in both posttranscriptional control and cell cycle regulation.

Wilms' tumor 1-associating protein regulates G2/M transition through stabilization of cyclin A2 mRNA.

Wilms' tumor 1-associating protein (WTAP) has been reported to be a ubiquitously expressed nuclear protein. Although a relation to splicing factors has been postulated, its actual physiological function still remains to be elucidated. To investigate the role of WTAP, we generated WTAP-knockout mice and performed small interfering RNA (siRNA)-mediated knockdown analyses in primary cultured cells. In DNA microarrays using human umbilical vein endothelial cells, WTAP-targeted siRNA treatment resulted in markedly reduced expression of cell-cycle-related genes. siRNA-mediated WTAP knockdown down-regulated the stability of cyclin A2 mRNA through a nine-nucleotide essential sequence in cyclin A2 mRNA 3' UTR. WTAP knockdown induced G2 accumulation, which is partially rescued by adenoviral overexpression of cyclin A2. Moreover, WTAP-null mice exhibited proliferative failure with death resulting at approximately embryonic day 6.5, an etiology almost identical to cyclin A2-null mice. Collectively, these findings establish WTAP as an essential factor for the stabilization of cyclin A2 mRNA, thereby regulating G2/M cell-cycle transition.

Histopathological studies of aortic dissection in streptozotocin-induced diabetic APA hamsters.

Syrian hamsters of the APA strain (APA hamsters) are known to show continuous diabetes accompanied by its complications, such as glomerulosclerosis and atherosclerosis, following a single injection of streptozotocin (SZ). Recently, we observed Stanford type B aortic dissection in three diabetic APA hamsters and histopathological analysis was performed. The histopathologic observations in the false lumen, such as proliferation of granulation tissues, neointima and pseudoneointima, corresponded to the non-thrombosed type of human aortic dissection, and blood clots of the thrombosed type were similar to the remodeling structures of aortic dissection found in human cases. Thus, this model may be useful for investigating the etiology and pathogenesis of aortic dissection accompanying diabetes mellitus in humans.

Vascular endothelial growth factor- and thrombin-induced termination factor, Down syndrome critical region-1, attenuates endothelial cell proliferation and angiogenesis.

Activation and dysfunction of the endothelium underlie many vascular disorders including atherosclerosis, tumor growth, and inflammation. Endothelial cell activation is mediated by many different extra-cellular signals, which result in overlapping yet distinct patterns of gene expression. Here we show, in DNA microarray analyses, that vascular endothelial growth factor (VEGF) and thrombin result in dramatic and rapid upregulation of Down syndrome critical region (DSCR)-1 gene encoding exons 4-7, a negative feedback regulator of calcium-calcineurin-NF-AT signaling. VEGF- and thrombin-mediated induction of DSCR-1 involves the cooperative binding of NF-ATc and GATA-2/3 to neighboring consensus motifs in the upstream promoter. Constitutive expression of DSCR-1 in endothelial cells markedly impaired NF-ATc nuclear localization, proliferation, and tube formation. Under in vivo conditions, overexpression of DSCR-1 reduced vascular density in matrigel plugs and melanoma tumor growth in mice. Taken together, these findings support a model in which VEGF- and thrombin-mediated induction of endothelial cell proliferation triggers a negative feedback loop consisting of DSCR-1 gene induction and secondary inhibition of NF-AT signaling. As a natural brake in the angiogenic process, this negative pathway may lend itself to therapeutic manipulation in pathological states.

Interaction between hex and GATA transcription factors in vascular endothelial cells inhibits flk-1/KDR-mediated vascular endothelial growth factor signaling.

Recent evidence supports a role for GATA transcription factors as important signal intermediates in differentiated endothelial cells. The goal of this study was to identify proteins that interact with endothelial-derived GATA transcription factors. Using yeast two-hybrid screening, we identified hematopoietically expressed homeobox (Hex) as a GATA-binding partner in endothelial cells. The physical association between Hex and GATA was confirmed with immunoprecipitation in cultured cells. Hex overexpression resulted in decreased flk-1/KDR expression, both at the level of the promoter and the endogenous gene, and attenuated vascular endothelial growth factor-mediated tube formation in primary endothelial cell cultures. In electrophoretic mobility shift assays, Hex inhibited the binding of GATA-2 to the flk-1/KDR 5'-untranslated region GATA motif. Finally, in RNase protection assays, transforming growth factor beta1, which has been previously shown to decrease flk-1 expression by interfering with GATA binding activity, was shown to increase Hex expression in endothelial cells. Taken together, the present study provides evidence for a novel association between Hex and GATA and suggests that transforming growth factor beta-mediated repression of flk-1/KDR and vascular endothelial growth factor signaling involves the inducible formation of inhibitory Hex-GATA complexes.

Protective effects of probucol treatment on pancreatic beta-cell function of SZ-induced diabetic APA hamsters.

To clarify whether oxidative stress is involved in the pathogenesis of islet lesions of diabetic animals, the effects of probucol (PB), an antioxidant and anti-hyperlipidemia agent, on the islets in streptozotocin (SZ)-induced diabetic APA hamsters in the acute and chronic phases of diabetes were examined. The control (CB group) and diabetic (SZ group) hamsters were treated with PB (1% in the diet) for 4 weeks from several days after SZ injection as the acute diabetic group, or 8 weeks from 6 weeks after SZ injection as the chronic diabetic group. Glucose tolerance test revealed that PB treatment decreased the high serum glucose level after glucose injection in the diabetic APA hamsters in the acute diabetic phase. Immunohistochemistry revealed that PB treatment significantly increased the percentage of the insulin positive area in the diabetic hamsters pancreata in both the acute and chronic phases. In addition, 4-hydroxy-2-nonenal (4HNE; an oxidative stress marker) positive cells were slightly reduced by PB treatment in the acute diabetic phase. Double-immunostaining for insulin and PCNA (proliferating cell nuclear antigen) revealed that elevation of the percentage of insulin and PCNA double-positive cells against insulin-positive cells was seen in the islets of PB-treated diabetic hamsters, but the difference was not significant compared with untreated diabetic hamsters (p = 0.07). In semi-quantitative RT-PCR, the expression of two genes, Reg (Regenerating gene) and INGAP (islet neogenesis associated protein), in the diabetic APA hamsters was significantly increased compared to the control groups in both diabetic phases. PB treatment significantly reduced Reg expression in the chronic diabetic phase. These data suggest that PB treatment in SZ-injected diabetic hamsters partially restored beta-cell function through acting as an antioxidant and induced higher expression of Reg and INGAP genes in the pancreas of hamsters.

The effect of probucol on atherosclerosis in streptozotocin-induced diabetic-hyperlipidemic APA hamsters in different stages of atherosclerosis.

We investigated the effect of probucol (PB) on atherosclerosis in streptozotocin (SZ)-induced diabetic-hyperlipidemic APA hamsters in three different stages, the early, middle and late stages of atherosclerosis. Male APA hamsters were injected intraperitoneally with SZ or vehicle alone (citrate buffer; CB) as a control at the age of 8 weeks. At 6 weeks after injection (WAI) of SZ or CB (the early stage), 14 WAI (the middle stage) and 26 WAI (the late stage), animals were assigned to PB treated- or non-treated groups (CBPB, SZPB, CB, SZ). After 8 weeks of PB administration with diet, the aorta was taken from each animal for assessment of atheromatous lesions and blood samples were subjected to serum biochemical analysis and the measurement of blood lipid peroxide (LPO). In the middle stage, PB treatment significantly decreased serum total cholesterol level, slightly decreased LPO, and also tended to reduce the lesion area, although no statistical difference was seen. There was no marked effect of PB treatment in the early and late stages. These findings suggest that single use of PB has little effect on atherosclerosis of a hyperglycemia-hyperlipidemia animal model.

Effects of group musical therapy on inpatients with chronic psychoses: a controlled study.

The objective of the present study was to examine the efficacy of group musical therapy for inpatients with DSM-IV schizophrenia or schizoaffective psychosis. Thirty-four therapy group subjects in a ward for long-stay female patients received 15 group musical therapy sessions over 4 months, while 32 waiting group subjects from another ward with the same function were to wait for the sessions until the studied course was completed. The assessment included measures of psychotic symptoms, objective quality of life and subjective musical experiences. Comparison of the groups indicated that significant advantages in the therapy group subjects were detected in some measures concerning personal relations and a subjective sense of participation in a chorus activity. However, the follow-up evaluation suggested that the improvement might not be durable. These findings suggested that the musical therapy had some, but possibly only short-lived, effects on personal relations and musical experiences of chronic psychotic patients.

Functional and histochemical analysis on pancreatic islets of APA hamsters with SZ-induced hyperglycemia and hyperlipidemia.

To clarify how Syrian hamsters of the APA strain (APA hamsters) keep a diabetic condition for a long period, the functional and histochemical changes in the pancreatic islets of diabetic APA hamsters were examined. By glucose tolerance test, no glucose-induced insulin secretion was seen in the diabetic APA hamsters. By immunohistochemistry, it was revealed that at 24 hr after SZ-injection, the number of islets had decreased and that remnant islets had become markedly smaller. The islets had hardly any insulin-immunoreactive cells and consisted of cells stained by anti-glucagon and somatostatin antibodies. One, three and six months after SZ-injection, a small number of cells with vacuolative changes, which were positive for PAS staining, were observed in most islets and the vacuolated cells were stained mainly by anti-insulin antibody. In addition, a number of PCNA-positive cells were observed, especially in the periphery of the vacuolated cells, while TUNEL-positive cells were not detected. This data suggests that beta-cells proliferating as a result of the replication of the resident beta-cells in islets had fallen into degeneration and necrosis by a stress, such as the glycogen deposition in hyperglycemia and hyperlipidemia. Consequently, secretion of insulin was maintained at low levels, which allowed the hamsters to live without insulin therapy in the diabetic condition for over 6 months.