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.

Dual function of SF3B2 on chromatin and RNA to regulate transcription in head and neck squamous cell carcinoma.

RNA is spliced concomitantly with transcription and the process is organized by RNA splicing factors, transcriptional regulators, and chromatin regulators. RNA is spliced in close proximity to transcription machinery. Hence, some RNA splicing factors may play a role in transcription. Here, we show that the splicing factor SF3B2 binds to gene regulatory elements and mRNA to modulate transcription and RNA stability in head and neck squamous cell carcinoma cells. High SF3B2 expression leads to poor prognosis in patients with head and neck squamous cell carcinoma and to progression of tumor growth in mice. SF3B2 promotes tumor growth, owing to its involvement in activation of gene expression associated with mitochondrial electron transport and transcription regulatory region DNA binding. SF3B2 is enriched around the promoter element on chromatin and the transcription termination site on RNA. SF3B2 is involved in the regulation of RNA stability. According to the SF3B2-binding profile, SF3B2 regulates RNA polymerase II activity, in addition to regulating RNA splicing. Mechanistically, SF3B2 promotes the binding of structural maintenance of chromosomes 1A and CCCTC-binding factor (CTCF) to the SF3B2-binding genomic regions. SF3B2 also modulates CTCF transcriptional activity. Our findings indicate that SF3B2 has a dual function in both transcription and RNA stability, leading to head and neck squamous cell carcinoma progression.

The role of Mediator and Little Elongation Complex in transcription termination.

Mediator is a coregulatory complex that regulates transcription of Pol II-dependent genes. Previously, we showed that human Mediator subunit MED26 plays a role in the recruitment of Super Elongation Complex (SEC) or Little Elongation Complex (LEC) to regulate the expression of certain genes. MED26 plays a role in recruiting SEC to protein-coding genes including c-myc and LEC to small nuclear RNA (snRNA) genes. However, how MED26 engages SEC or LEC to regulate distinct genes is unclear. Here, we provide evidence that MED26 recruits LEC to modulate transcription termination of non-polyadenylated transcripts including snRNAs and mRNAs encoding replication-dependent histone (RDH) at Cajal bodies. Our findings indicate that LEC recruited by MED26 promotes efficient transcription termination by Pol II through interaction with CBC-ARS2 and NELF/DSIF, and promotes 3' end processing by enhancing recruitment of Integrator or Heat Labile Factor to snRNA or RDH genes, respectively.

Gain-of-Function MN1 Truncation Variants Cause a Recognizable Syndrome with Craniofacial and Brain Abnormalities.

MN1 was originally identified as a tumor-suppressor gene. Knockout mouse studies have suggested that Mn1 is associated with craniofacial development. However, no MN1-related phenotypes have been established in humans. Here, we report on three individuals who have de novo MN1 variants that lead to a protein lacking the carboxyl (C) terminus and who presented with severe developmental delay, craniofacial abnormalities with specific facial features, and structural abnormalities in the brain. An in vitro study revealed that the deletion of the C-terminal region led to increased protein stability, an inhibitory effect on cell proliferation, and enhanced MN1 aggregation in nuclei compared to what occurred in the wild type, suggesting that a gain-of-function mechanism is involved in this disease. Considering that C-terminal deletion increases the fraction of intrinsically disordered regions of MN1, it is possible that altered phase separation could be involved in the mechanism underlying the disease. Our data indicate that MN1 participates in transcriptional regulation of target genes through interaction with the transcription factors PBX1, PKNOX1, and ZBTB24 and that mutant MN1 impairs the binding with ZBTB24 and RING1, which is an E3 ubiquitin ligase. On the basis of our findings, we propose the model that C-terminal deletion interferes with MN1's interaction molecules related to the ubiquitin-mediated proteasome pathway, including RING1, and increases the amount of the mutant protein; this increase leads to the dysregulation of MN1 target genes by inhibiting rapid MN1 protein turnover.

Association between third molar agenesis patterns and agenesis of other teeth in a Japanese orthodontic population.

The purpose of this study was to investigate the patterns of third molar agenesis and incidence of agenesis of other permanent teeth in a Japanese orthodontic patient group. A total of 417 Japanese subjects (134 males and 283 females) with agenesis of one or more third molars were divided into four groups according to the agenesis pattern, and 874 other Japanese subjects (302 males and 572 females) without third molar agenesis were assigned to a control group. Panoramic radiographs and medical and dental records were used to examine for tooth agenesis. The Chi-square test and odds ratio were used to make statistical comparisons. The prevalence of third molar agenesis worked out at 32.3% with no statistically significant gender difference. A high prevalence rate of agenesis of third molars, unilateral or bilateral, could be considered characteristic of the Japanese orthodontic population. Significant increases in occurrence of oligodontia, and unilateral or bilateral agenesis of other teeth, including maxillary lateral incisors and maxillary and mandibular second premolars, were observed in all or almost all of the third molar agenesis groups, compared with the controls. A significantly increased prevalence rate of mandibular lateral incisor agenesis was observed in almost all of the third molar agenesis groups. The Japanese patients with third molar agenesis had a significantly increased occurrence of oligodontia, and unilateral or bilateral agenesis of maxillary and mandibular lateral incisors and second premolars, except for bilateral agenesis of mandibular lateral incisors.

Catalytic activity of MsbA reconstituted in nanodisc particles is modulated by remote interactions with the bilayer.

ATP-binding cassette (ABC) transporters couple hydrolysis of ATP with vectorial transport across the cell membrane. We have reconstituted ABC transporter MsbA in nanodiscs of various sizes and lipid compositions to test whether ATPase activity is modulated by the properties of the bilayer. ATP hydrolysis rates, Michaelis-Menten parameters, and dissociation constants of substrate analog ATP-γ-S demonstrated that physicochemical properties of the bilayer modulated binding and ATPase activity. This is remarkable when considering that the catalytic unit is located ~50Šfrom the transmembrane region. Our results validated the use of nanodiscs as an effective tool to reconstitute MsbA in an active catalytic state, and highlighted the close relationship between otherwise distant transmembrane and ATPase modules.

Maxillary first molar agenesis and other dental anomalies.

OBJECTIVE: To explore the association of maxillary first molar agenesis with other dental anomalies in Japanese orthodontic patients. MATERIALS AND METHODS: A total of 32 subjects with one or two congenitally missing maxillary first molars (group M) were selected and divided into group 1M (12 subjects with one maxillary first molar missing) and group 2M (20 subjects with two maxillary first molars missing). As controls, 32 sex-matched subjects without agenesis of maxillary first molars were collected (group C). Panoramic and periapical radiographs, cephalograms, study models, intraoral photographs, and anamnestic data were used to identify anomalies of permanent teeth. Chi-square, Fisher's exact, Kruskal-Wallis, and Steel-Dwass tests were used to make statistical comparisons. RESULTS: The prevalence rates of tooth agenesis other than the maxillary first molars and advanced tooth agenesis, with third molars excluded, were significantly higher in group 2M than in group C. The absence of second premolars was most common. The prevalence rate of third molar agenesis was significantly higher in groups 1M and 2M than in group C. The occurrence of symmetrical agenesis of the mandibular third molars was particularly notable in group 2M as compared to group 1M, in which maxillary third molar agenesis was predominant. There was no significant association between maxillary first molar agenesis and other dental anomalies, except for agenesis of teeth other than maxillary first molars. CONCLUSION: Agenesis of maxillary first molars is associated with a higher prevalence of other permanent tooth agenesis and advanced tooth agenesis.

Non-denaturing solubilization of inclusion bodies.

It has been a conventional notion that cytoplasmic recombinant expression leads to either soluble protein or inclusion bodies. In the latter case, it was always assumed that proteins in inclusion bodies (IBs) are more or less unfolded and hence require complete denaturing condition for solubilization, which uses strong detergents, urea or guanidine hydrochloride. However, we often observe distribution of expressed proteins in both soluble and insoluble fractions. In such expression, IBs are often loose and of flocculate morphology. We believe that such distribution is due to association of near native structures of the expressed proteins, which cause either aggregation into insoluble fractions or unstable soluble proteins. In our experience, although not reported by others, interleukin-1alpha, interferon-gamma, tumor necrosis factors, fibroblast growth factors, His-tagged fyn kinase and many other proteins showed such behavior. If this occurs, we have experienced problems of instability, low yield and insolubility whether purification is done from the soluble fraction or by refolding of IBs. Arginine has shown great promise in non-denaturating solubilization of some of these proteins we have tested.

Utilization of Arg-elution method for FLAG-tag based chromatography.

FLAG-tag is one of the commonly used purification technologies for recombinant proteins. An antibody, M2, specifically binds to the FLAG-tag whether it is attached to N- or C-terminus of proteins to be purified. The bound proteins are generally eluted by competition with a large excess of free FLAG peptide. This requires synthetic FLAG peptide and also removal of bound FLAG peptide for M2 column regeneration. We have shown before that arginine at mild pH can effectively dissociate protein-protein or protein-ligand interactions, e.g. in Protein-A, antigen and dye-affinity chromatography. We have tested here elution of FLAG-fused proteins by arginine for columns of M2-immobilized resin using several proteins in comparison with competitive elution by FLAG peptide or low pH glycine buffer. Active and folded proteins were successfully and effectively eluted using 0.5-1M arginine at pH 3.5-4.4, as reported in this paper.