Introns within ribosomal protein genes regulate the production and function of yeast ribosomes.

In budding yeast, the most abundantly spliced pre-mRNAs encode ribosomal proteins (RPs). To investigate the contribution of splicing to ribosome production and function, we systematically eliminated introns from all RP genes to evaluate their impact on RNA expression, pre-rRNA processing, cell growth, and response to stress. The majority of introns were required for optimal cell fitness or growth under stress. Most introns are found in duplicated RP genes, and surprisingly, in the majority of cases, deleting the intron from one gene copy affected the expression of the other in a nonreciprocal manner. Consistently, 70% of all duplicated genes were asymmetrically expressed, and both introns and gene deletions displayed copy-specific phenotypic effects. Together, our results indicate that splicing in yeast RP genes mediates intergene regulation and implicate the expression ratio of duplicated RP genes in modulating ribosome function.

Open versus robotic partial nephrectomy in obese patients: a multi-institutional propensity score-matched analysis (UroCCR 43-Robese study).

INTRODUCTION: There is limited evidence on the outcomes of robotic partial nephrectomy (RPN) and open partial nephrectomy (OPN) in obese patients (BMI ≥ 30 kg/m2). In this study, we aimed to compare perioperative and oncological outcomes of RPN and OPN. METHODS: We relied on data from patients who underwent PN from 2009 to 2017 at 16 departments of urology participating in the UroCCR network, which were collected prospectively. In an effort to adjust for potential confounders, a propensity-score matching was performed. Perioperative outcomes were compared between OPN and RPN patients. Disease-free survival (DFS) and overall survival (OS) were estimated using the Kaplan-Meier method and compared using the log-rank test. RESULTS: Overall, 1277 obese patients (932 robotic and 345 open were included. After propensity score matching, 166 OPN and 166 RPN individuals were considered for the study purposes; no statistically significant difference among baseline demographic or tumor-specific characteristics was present. A higher overall complication rate and major complications rate were recorded in the OPN group (37 vs. 25%, p = 0.01 and 21 vs. 10%, p = 0.007; respectively). The length of stay was also significantly longer in the OPN group, before and after propensity-score matching (p < 0.001). There were no significant differences in Warm ischemia time (p = 0.66), absolute change in eGFR (p = 0.45) and positive surgical margins (p = 0.12). At a median postoperative follow-up period of 24 (8-40) months, DFS and OS were similar in the two groups (all p > 0.05). CONCLUSIONS: In this study, RPN was associated with better perioperative outcomes (improvement of major complications rate and LOS) than OPN. The oncological outcomes were found to be similar between the two approaches.

Reovirus µ2 protein modulates host cell alternative splicing by reducing protein levels of U5 snRNP core components.

Mammalian orthoreovirus (MRV) is a double-stranded RNA virus from the Reoviridae family presenting a promising activity as an oncolytic virus. Recent studies have underlined MRV's ability to alter cellular alternative splicing (AS) during infection, with a limited understanding of the mechanisms at play. In this study, we investigated how MRV modulates AS. Using a combination of cell biology and reverse genetics experiments, we demonstrated that the M1 gene segment, encoding the µ2 protein, is the primary determinant of MRV's ability to alter AS, and that the amino acid at position 208 in µ2 is critical to induce these changes. Moreover, we showed that the expression of µ2 by itself is sufficient to trigger AS changes, and its ability to enter the nucleus is not required for all these changes. Moreover, we identified core components of the U5 snRNP (i.e. EFTUD2, PRPF8, and SNRNP200) as interactors of µ2 that are required for MRV modulation of AS. Finally, these U5 snRNP components are reduced at the protein level by both MRV infection and µ2 expression. Our findings identify the reduction of U5 snRNP components levels as a new mechanism by which viruses alter cellular AS.

Impact of radiation therapy on artificial urinary sphincter implantation in male patients: A multicenter study.

AIMS: To evaluate the impact of an history of radiation therapy on the outcomes of artificial urinary sphincter (AUS) implantation in male patients. METHODS: The charts of all patients who underwent AUS implantation for stress urinary incontinence (SUI) after prostate surgery in thirteen centers between 2004 and 2020 were retrospectively reviewed. We excluded patients with neurogenic SUI. Continence rates and incidence of complications, revision and cuff erosion were evaluated. The outcomes in irradiated men were compared to those of non irradiated men. RESULTS: A total of 1277 patients who had an AUS met the inclusion criteria with a median age of 70 years, of which 437 had an history of prior radiotherapy. There was no difference in comorbidities. In irradiated patients, postoperative social continence, urethral atrophy and infection rates were respectively 75.6%, 2.4% and 9.5% and 76.8%, 5.4%, and 5.8% in nonirradiated men (respectively, p = 0.799, p = 0.128, p = 0.148). There were more urethral erosion in irradiated male patients. After a mean follow up of 36.8 months, the explantation free survival was poorer in irradiated patients (p = 0.001). CONCLUSION: These data suggest that pelvic radiotherapy before AUS adversely affect device survival with and increased greater occurrence of infection-erosion and therefore of explantation.

Hypoxia leads to significant changes in alternative splicing and elevated expression of CLK splice factor kinases in PC3 prostate cancer cells.

BACKGROUND: Mounting evidence suggests that one of the ways that cells adapt to hypoxia is through alternative splicing. The aim of this study was firstly to examine the effect of hypoxia on the alternative splicing of cancer associated genes using the prostate cancer cell line PC3 as a model. Secondly, the effect of hypoxia on the expression of several regulators of splicing was examined. METHODS: PC3 cells were grown in 1% oxygen in a hypoxic chamber for 48 h, RNA extracted and sent for high throughput PCR analysis at the RNomics platform at the University of Sherbrooke, Canada. Genes whose exon inclusion rate PSI (ψ) changed significantly were identified, and their altered exon inclusion rates verified by RT-PCR in three cell lines. The expression of splice factors and splice factor kinases in response to hypoxia was examined by qPCR and western blotting. The splice factor kinase CLK1 was inhibited with the benzothiazole TG003. RESULTS: In PC3 cells the exon inclusion rate PSI (ψ) was seen to change by > 25% in 12 cancer-associated genes; MBP, APAF1, PUF60, SYNE2, CDC42BPA, FGFR10P, BTN2A2, UTRN, RAP1GDS1, PTPN13, TTC23 and CASP9 (caspase 9). The expression of the splice factors SRSF1, SRSF2, SRSF3, SAM68, HuR, hnRNPA1, and of the splice factor kinases SRPK1 and CLK1 increased significantly in hypoxia. We also observed that the splice factor kinase CLK3, but not CLK2 and CLK4, was also induced in hypoxic DU145 prostate, HT29 colon and MCF7 breast cancer cell lines. Lastly, we show that the inhibition of CLK1 in PC3 cells with the benzothiazole TG003 increased expression of the anti-apoptotic isoform caspase 9b. CONCLUSIONS: Significant changes in alternative splicing of cancer associated genes occur in prostate cancer cells in hypoxic conditions. The expression of several splice factors and splice factor kinases increases during hypoxia, in particular the Cdc-like splice factor kinases CLK1 and CLK3. We suggest that in hypoxia the elevated expression of these regulators of splicing helps cells adapt through alternative splicing of key cancer-associated genes. We suggest that the CLK splice factor kinases could be targeted in cancers in which hypoxia contributes to resistance to therapy.

Splicing arrays reveal novel RBM10 targets, including SMN2 pre-mRNA.

BACKGROUND: RBM10 is an RNA binding protein involved in message stabilization and alternative splicing regulation. The objective of the research described herein was to identify novel targets of RBM10-regulated splicing. To accomplish this, we downregulated RBM10 in human cell lines, using small interfering RNAs, then monitored alternative splicing, using a reverse transcription-PCR screening platform. RESULTS: RBM10 knockdown (KD) provoked alterations in splicing events in 10-20% of the pre-mRNAs, most of which had not been previously identified as RBM10 targets. Hierarchical clustering of the genes affected by RBM10 KD revealed good conservation of alternative exon inclusion or exclusion across cell lines. Pathway annotation showed RAS signaling to be most affected by RBM10 KD. Of particular interest was the finding that splicing of SMN pre-mRNA, encoding the survival of motor neuron (SMN) protein, was influenced by RBM10 KD. Inhibition of RBM10 resulted in preferential expression of the full-length, exon 7 retaining, SMN transcript in four cancer cell lines and one normal skin fibroblast cell line. SMN protein is expressed from two genes, SMN1 and SMN2, but the SMN1 gene is homozygously disrupted in people with spinal muscular atrophy; as a consequence, all of the SMN that is expressed in people with this disease is from the SMN2 gene. Expression analyses using primary fibroblasts from control, carrier and spinal muscle atrophy donors demonstrated that RBM10 KD resulted in preferential expression of the full-length, exon 7 retaining, SMN2 transcript. At the protein level, upregulation of the full-length SMN2 was also observed. Re-expression of RBM10, in a stable RBM10 KD cancer cell line, correlated with a reversion of the KD effect, demonstrating specificity. CONCLUSION: Our work has not only expanded the number of pre-mRNA targets for RBM10, but identified RBM10 as a novel regulator of SMN2 alternative inclusion.

Global profiling of alternative RNA splicing events provides insights into molecular differences between various types of hepatocellular carcinoma.

BACKGROUND: Dysregulations in alternative splicing (AS) patterns have been associated with many human diseases including cancer. In the present study, alterations to the global RNA splicing landscape of cellular genes were investigated in a large-scale screen from 377 liver tissue samples using high-throughput RNA sequencing data. RESULTS: Our study identifies modifications in the AS patterns of transcripts encoded by more than 2500 genes such as tumor suppressor genes, transcription factors, and kinases. These findings provide insights into the molecular differences between various types of hepatocellular carcinoma (HCC). Our analysis allowed the identification of 761 unique transcripts for which AS is misregulated in HBV-associated HCC, while 68 are unique to HCV-associated HCC, 54 to HBV&HCV-associated HCC, and 299 to virus-free HCC. Moreover, we demonstrate that the expression pattern of the RNA splicing factor hnRNPC in HCC tissues significantly correlates with patient survival. We also show that the expression of the HBx protein from HBV leads to modifications in the AS profiles of cellular genes. Finally, using RNA interference and a reverse transcription-PCR screening platform, we examined the implications of cellular proteins involved in the splicing of transcripts involved in apoptosis and demonstrate the potential contribution of these proteins in AS control. CONCLUSIONS: This study provides the first comprehensive portrait of global changes in the RNA splicing signatures that occur in hepatocellular carcinoma. Moreover, these data allowed us to identify unique signatures of genes for which AS is misregulated in the different types of HCC.

Tumor microenvironment-associated modifications of alternative splicing.

Pre-mRNA alternative splicing is modified in cancer, but the origin and specificity of these changes remain unclear. Here, we probed ovarian tumors to identify cancer-associated splicing isoforms and define the mechanism by which splicing is modified in cancer cells. Using high-throughput quantitative PCR, we monitored the expression of splice variants in laser-dissected tissues from ovarian tumors. Surprisingly, changes in alternative splicing were not limited to the tumor tissues but were also found in the tumor microenvironment. Changes in the tumor-associated splicing events were found to be regulated by splicing factors that are differentially expressed in cancer tissues. Overall, ∼20% of the alternative splicing events affected by the down-regulation of the splicing factors QKI and RBFOX2 were altered in the microenvironment of ovarian tumors. Together, our results indicate that the tumor microenvironment undergoes specific changes in alternative splicing orchestrated by a limited number of splicing factors.

Mode competition and anomalous cooling in a multimode phonon laser.

We study mode competition in a multimode "phonon laser" comprised of an optical cavity employing a highly reflective membrane as the output coupler. Mechanical gain is provided by the intracavity radiation pressure, to which many mechanical modes are coupled. We calculate the gain and find that strong oscillation in one mode suppresses the gain in other modes. For sufficiently strong oscillation, the gain of the other modes actually switches sign and becomes damping, a process we call "anomalous cooling." We demonstrate that mode competition leads to single-mode operation and find excellent agreement with our theory, including anomalous cooling.

Cavity-enhanced measurements for determining dielectric-membrane thickness and complex index of refraction.

The material properties of silicon nitride (SiN) play an important role in the performance of SiN membranes used in optomechanical applications. An optimum design of a subwavelength high-contrast grating requires accurate knowledge of the membrane thickness and index of refraction, and its performance is ultimately limited by material absorption. Here we describe a cavity-enhanced method to measure the thickness and complex index of refraction of dielectric membranes with small, but nonzero, absorption coefficients. By determining Brewster's angle and an angle at which reflection is minimized by means of destructive interference, both the real part of the index of refraction and the sample thickness can be measured. A comparison of the losses in the empty cavity and the cavity containing the dielectric sample provides a measurement of the absorption.

The Impact of Histological Variants on Oncological Outcomes After Surgical Resection of a Nonmetastatic Renal Cell Carcinoma with Tumor Thrombus: A Multi-institutional Study.

Background: There is no definitive evidence of the prognosis impact of histological variants (HVs) in patients who undergo surgical resection of a nonmetastatic renal cell carcinoma (nm-RCC) with venous tumor thrombus (TT). Objective: To investigate the impact of HVs on the prognosis of patients with nm-RCC with TT after radical surgery. Design setting and participants: Patients who underwent radical nephrectomy with the removal of the venous TT for an nm-RCC were included in a retrospective study. Outcome measurements and statistical analysis: Three groups were identified: clear cell (ccRCC), papillary (pRCC), and chromophobe (chRCC) RCC. The primary outcome measures (disease-free and overall survival [OS]) were assessed using the Kaplan-Meier method and compared using the log-rank test. Univariate and multivariate Cox proportional hazard models were used to study the impact of HVs on survival. Results and limitations: A total of 873 patients were included. The histological subtypes were distributed as follows: ccRCC in 780 cases, pRCC in 58 cases, and chRCC in 35 cases. At the time of data analysis, 612 patients were recurrence free and 228 had died. A survival analysis revealed significant differences in both OS and recurrence-free survival across histological subtypes, with the poorest outcomes observed in pRCC patients (p < 0.05). In a multivariable analysis, pRCC was independently associated with worse disease-free survival and OS (hazard ratio [HR]: 1.71; p = 0.01 and HR: 1.24; p = 0.04), while chRCC was associated with more favorable outcomes than ccRCC (HR: 0.05; p < 0.001 and HR: 0.02; p < 0.001). A limitation of the study is its retrospective nature. Conclusions: In this multicentric series, HVs appeared to impact the medium-term oncological prognosis of kidney cancer with TT. Patient summary: This study investigated the differences in oncological outcomes among histological variants (clear cell, papillary, and chromophobe) in a cohort of nonmetastatic renal cell carcinoma patients with venous tumor thrombus extension. We observed that these histological variants within this specific subgroup exhibit distinct outcomes, with papillary renal cell carcinoma being associated with the worst prognosis.

Can Simplified PADUA Renal (SPARE) Nephrometry scoring system help predict renal function outcomes after robot-assisted partial nephrectomy? (UroCCR study 93).

BACKGROUND: The SPARE Nephrometry Score (NS) is described as easier to implement than the RENAL and PADUA NSs, currently more widely used. Our objective was to compare the accuracy of SPARE NS in predicting renal function outcomes following RAPN. METHODS: A multicentric retrospective study was conducted using French kidney cancer network (UroCCR, NCT03293563) database. All patients included had RAPN for cT1 renal tumors between May 2010 and March 2021. SPARE was compared to RENAL, PADUA and Tumor Size to predict postoperative acute kidney injury (AKI), chronic kidney disease (CKD) upstaging, de novo CKD at 3-6 months follow-up and Trifecta failure. The ability of the different NSs and tumor size to predict renal function outcomes was evaluated using uni- and multivariate logistic regression models. RESULTS: According to our study criteria, 1171 patients were included. Mean preoperative tumor size and estimated glomerular filtration rate (eGFR) were 3.4±1.4 cm and 85.8 mL/min/1.73 m2. In total, 266 (22.7%), 87 (7.4%), 94 (8%), and 624 (53.3%) patients had AKI, de novo CKD, CKD upstaging, and Trifecta failure, respectively. In multivariate analysis, all three NSs and tumor size were independent predictors of AKI, CKD de novo, CKD upgrade and Trifecta failure. There was no significant difference between all three NS and tumor sizes in predicting renal function outcomes. CONCLUSIONS: SPARE Score seems to be a valid alternative to predict renal function outcomes after RAPN. Nevertheless, in our study, tumor size was as accurate as NSs in predicting postoperative outcomes and, therefore, seems to be the logical choice for surgical decisions.

High-throughput quantification of splicing isoforms.

Most human messenger RNAs (mRNAs) are alternatively spliced and many exhibit disease-specific splicing patterns. However, the contribution of most splicing events to the development and maintenance of human diseases remains unclear. As the contribution of alternative splicing events to diagnosis and prognosis is becoming increasingly recognized, it becomes important to develop precise methods to quantify the abundance of these isoforms in clinical samples. Here we present a pipeline for real-time PCR annotation of splicing events (RASE) that allows accurate identification of a large number of splicing isoforms in human tissues. The RASE automatically designed specific primer pairs for 81% of all alternative splicing events in the NCBI build 36 database. Experimentally, the majority of the RASE designed primers resulted in isoform-specific amplification suitable for quantification in human cell lines or in formalin-fixed, paraffin-embedded (FFPE) RNA extract. Using this pipeline it is now possible to rapidly identify splicing isoform signatures in different types of human tissues or to validate complete sets of data generated by microarray expression profiling and deep sequencing techniques.

Detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its first variants in fourplex real-time quantitative reverse transcription-PCR assays.

The early diagnosis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections is required to identify and isolate contagious patients to prevent further transmission of SARS-CoV-2. In this study, we present a multitarget real-time TaqMan reverse transcription PCR (rRT-PCR) assay for the quantitative detection of SARS-CoV-2 and some of its circulating variants harboring mutations that give the virus a selective advantage. Seven different primer-probe sets that included probes containing locked nucleic acid (LNA) nucleotides were designed to amplify specific wild-type and mutant sequences in Orf1ab, Envelope (E), Spike (S), and Nucleocapsid (N) genes. Furthermore, a newly developed primer-probe set targeted human ß2-microglobulin (B2M) as a highly sensitive internal control for RT efficacy. All singleplex and fourplex assays detected ≤ 14 copies/reaction of quantified synthetic RNA transcripts, with a linear amplification range of nine logarithmic orders. Primer-probe sets for detection of SARS-CoV-2 exhibited no false-positive amplifications with other common respiratory pathogens, including human coronaviruses NL63, 229E, OC43, and HKU-1. Fourplex assays were evaluated using 160 clinical samples positive for SARS-CoV-2. Results showed that SARS-CoV-2 viral RNA was detected in all samples, including viral strains harboring mutations in the Spike coding sequence that became dominant in the pandemic. Given the emergence of SARS-CoV-2 variants and their rapid spread in some populations, fourplex rRT-PCR assay containing four primer-probe sets represents a reliable approach to allow quicker detection of circulating relevant variants in a single reaction.

Active Surveillance for Biopsy Proven Renal Oncocytomas: Outcomes and Feasibility.

OBJECTIVES: To report the outcomes and feasibility of active surveillance (AS) of biopsy-proven renal oncocytomas. METHODS: Multicentric retrospective study (2010-2016) in 6 academic centers that included patients with biopsy-proven renal oncocytomas who were allocated to AS (imperative or elective indication) with a follow-up ≥1 year. Imaging was performed at least once a year, by CT-scan or ultrasound or MRI. Conversion to active treatment (surgical excision or ablative treatment) was at the discretion of the urologist. The primary endpoint was renal tumor growth (cm/year). Secondary outcomes included accuracy of biopsy, incidence, and reason to change AS to active treatment. RESULTS: Eighty-nine patients were included: Median age 67 years (26-89) and median tumor size 26 mm [15-90] on diagnosis. During a mean follow-up of 43 months'' (median 36 [12-180]), mean tumor growth was 0.24 cm/year. No predictive factors (demographical, radiological or histologic) of tumor growth could be identified. Conversion from AS to active treatment occurred in 24 patients (27%) (13 surgical excisions, 11 ablative procedures), in a median time of 45 (12-76) months'' after diagnosis. Tumor growth was the main indication to convert AS to active treatment (58%) with 8% of the patients opting to discontinue AS. No patient had metastatic progression nor disease-specific death. The correlation between biopsy and surgical specimen was 92%. CONCLUSION: Active surveillance for biopsy-proven renal oncocytomas was oncologically safe and patient adherence was high. No predictive factor for tumor growth could be identified but the tumor growth rate was low, and biopsy efficacy was high.

PRMT5 inhibition disrupts splicing and stemness in glioblastoma.

Glioblastoma (GBM) is a deadly cancer in which cancer stem cells (CSCs) sustain tumor growth and contribute to therapeutic resistance. Protein arginine methyltransferase 5 (PRMT5) has recently emerged as a promising target in GBM. Using two orthogonal-acting inhibitors of PRMT5 (GSK591 or LLY-283), we show that pharmacological inhibition of PRMT5 suppresses the growth of a cohort of 46 patient-derived GBM stem cell cultures, with the proneural subtype showing greater sensitivity. We show that PRMT5 inhibition causes widespread disruption of splicing across the transcriptome, particularly affecting cell cycle gene products. We identify a GBM splicing signature that correlates with the degree of response to PRMT5 inhibition. Importantly, we demonstrate that LLY-283 is brain-penetrant and significantly prolongs the survival of mice with orthotopic patient-derived xenografts. Collectively, our findings provide a rationale for the clinical development of brain penetrant PRMT5 inhibitors as treatment for GBM.

Respiratory tract samples collected from patients in a region of Quebec, Canada, indicate the absence of early circulation of SARS-CoV-2 infection.

Background: The first documented case of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in Quebec was confirmed on February 27, 2020. Retracing the first cases that occur within a geographical region may provide insight regarding the evolution and spread of SARS-CoV-2 in that region because the spread of undiagnosed cases may facilitate the initial community amplification of the virus. Methods: We performed a retrospective analysis of respiratory tract samples collected for influenza testing in a region of Quebec, Canada, to look for evidence of early circulation of SARS-CoV-2. Frozen nucleic acid extracts initially collected for influenza testing between January 1 and February 20, 2020, were tested for SARS-CoV-2 using a reverse transcription-polymerase chain reaction assay. Results: During the study period, 1,440 of 2,121 (67.9%) nucleic acid extracts from individual patients were available for retrospective testing. None of the samples tested positive for SARS-CoV-2. Conclusions: The results suggest that SARS-CoV-2 was not circulating within the region before February 20, 2020, because many samples, representing more than two-thirds of all samples tested for influenza during early 2020, were tested. Further studies using a similar methodology to determine the date of onset of SARS-CoV-2 in different countries and geographic areas could enhance our understanding of the current pandemic.

Historique: Le premier cas démontré d'infection par le syndrome respiratoire aigu sévère à coronavirus 2 (SARS-CoV-2) au Québec a été confirmé le 27 février 2020. Le retraçage du premier cas survenu dans une région géographique peut donner un aperçu de l'évolution et de la propagation du virus SARS-CoV-2 dans cette région, car la transmission des cas non diagnostiqués peut favoriser l'amplification initiale du virus dans la communauté. Méthodologie: Les chercheurs ont procédé à l'analyse rétrospective des échantillons respiratoires prélevés pour le dépistage de la grippe dans une région du Québec, au Canada, afin de trouver des preuves de circulation précoce du virus SARS-CoV-2D. Les extraits d'acide nucléique congelés entre le 1er janvier et le 20 février 2020 ont été soumis au dépistage du virus SARS-CoV-2 au moyen de l'amplification en chaîne par polymérase après transcriptase inverse. Résultats: Pendant la période de l'étude, 1 440 des 2 121 extraits d'acide nucléique (67,9 %) provenant de patients différents étaient disponibles en vue de tests rétrospectifs. Aucun n'a été positif au virus SARS-CoV-2. Conclusions: D'après les résultats, le virus SARS-CoV-2 n'était pas en circulation dans la région avant le 20 février 2020, car de nombreux échantillons, représentant plus des deux tiers de tous ceux ayant servi au dépistage de la grippe au début de l'année 2020, ont été soumis au dépistage. D'autres études faisant appel à une méthodologie semblable pour déterminer la date d'apparition du virus SARS-CoV-2 dans divers pays et diverses régions géographiques pourraient permettre de mieux comprendre la pandémie en cours.

Dissecting the expression landscape of cytochromes P450 in hepatocellular carcinoma: towards novel molecular biomarkers.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related deaths around the world. Recent advances in genomic technologies have allowed the identification of various molecular signatures in HCC tissues. For instance, differential gene expression levels of various cytochrome P450 genes (CYP450) have been reported in studies performed on limited numbers of HCC tissue samples, or focused on a small subset on CYP450s. In the present study, we monitored the expression landscape of all the members of the CYP450 family (57 genes) in more than 200 HCC tissues using RNA-Seq data from The Cancer Genome Atlas. Using stringent statistical filters and data from paired tissues, we identified significantly dysregulated CYP450 genes in HCC. Moreover, the expression level of selected CYP450s was validated by qPCR on cDNA samples from an independent cohort. Threshold values (sensitivity and specificity) based on dysregulated gene expression were also determined to allow for confident identification of HCC tissues. Finally, a global look at expression levels of the 57 members of the CYP450 family across ten different cancer types revealed specific expression signatures. Overall, this study provides useful information on the transcriptomic landscape of CYP450 genes in HCC and on new potential HCC biomarkers.

hnRNP A1/A2 and Sam68 collaborate with SRSF10 to control the alternative splicing response to oxaliplatin-mediated DNA damage.

Little is known about how RNA binding proteins cooperate to control splicing, and how stress pathways reconfigure these assemblies to alter splice site selection. We have shown previously that SRSF10 plays an important role in the Bcl-x splicing response to DNA damage elicited by oxaliplatin in 293 cells. Here, RNA affinity assays using a portion of the Bcl-x transcript required for this response led to the recovery of the SRSF10-interacting protein 14-3-3ε and the Sam68-interacting protein hnRNP A1. Although SRSF10, 14-3-3ε, hnRNP A1/A2 and Sam68 do not make major contributions to the regulation of Bcl-x splicing under normal growth conditions, upon DNA damage they become important to activate the 5' splice site of pro-apoptotic Bcl-xS. Our results indicate that DNA damage reconfigures the binding and activity of several regulatory RNA binding proteins on the Bcl-x pre-mRNA. Moreover, SRSF10, hnRNP A1/A2 and Sam68 collaborate to drive the DNA damage-induced splicing response of several transcripts that produce components implicated in apoptosis, cell-cycle control and DNA repair. Our study reveals how the circuitry of splicing factors is rewired to produce partnerships that coordinate alternative splicing across processes crucial for cell fate.

The OXR domain defines a conserved family of eukaryotic oxidation resistance proteins.

BACKGROUND: The NCOA7 gene product is an estrogen receptor associated protein that is highly similar to the human OXR1 gene product, which functions in oxidation resistance. OXR genes are conserved among all sequenced eukaryotes from yeast to humans. In this study we examine if NCOA7 has an oxidation resistance function similar to that demonstrated for OXR1. We also examine NCOA7 expression in response to oxidative stress and its subcellular localization in human cells, comparing these properties with those of OXR1. RESULTS: We find that NCOA7, like OXR1 can suppress the oxidative mutator phenotype when expressed in an E. coli strain that exhibits an oxidation specific mutator phenotype. Moreover, NCOA7's oxidation resistance function requires expression of only its carboxyl-terminal domain and is similar in this regard to OXR1. We find that, in human cells, NCOA7 is constitutively expressed and is not induced by oxidative stress and appears to localize to the nucleus following estradiol stimulation. These properties of NCOA7 are in striking contrast to those of OXR1, which is induced by oxidative stress, localizes to mitochondria, and appears to be excluded, or largely absent from nuclei. CONCLUSION: NCOA7 most likely arose from duplication. Like its homologue, OXR1, it is capable of reducing the DNA damaging effects of reactive oxygen species when expressed in bacteria, indicating the protein has an activity that can contribute to oxidation resistance. Unlike OXR1, it appears to localize to nuclei and interacts with the estrogen receptor. This raises the possibility that NCOA7 encodes the nuclear counterpart of the mitochondrial OXR1 protein and in mammalian cells it may reduce the oxidative by-products of estrogen metabolite-mediated DNA damage.