In Vivo Electron Paramagnetic Resonance Molecular Profiling of Tumor Microenvironment upon Tumor Progression to Malignancy in an Animal Model of Breast Cancer.

PURPOSE: Hypoxia and acidosis are recognized tumor microenvironment (TME) biomarkers of cancer progression. Alterations in cancer redox status and metabolism are also associated with elevated levels of intracellular glutathione (GSH) and interstitial inorganic phosphate (Pi). This study aims to evaluate the capability of these biomarkers to discriminate between stages and inform on a switch to malignancy. PROCEDURES: These studies were performed using MMTV-PyMT( +) female transgenic mice that spontaneously develop breast cancer and emulate human tumor staging. In vivo assessment of oxygen concentration (pO2), extracellular acidity (pHe), Pi, and GSH was performed using L-band electron paramagnetic resonance spectroscopy and multifunctional trityl and GSH-sensitive nitroxide probes. RESULTS: Profiling of the TME showed significant deviation of measured biomarkers upon tumor progression from pre-malignancy (pre-S4) to the malignant stage (S4). For the combined marker, HOP: (pHe × pO2)/Pi, a value > 186 indicated that the tumors were pre-malignant in 85% of the mammary glands analyzed, and when < 186, they were malignant 42% of the time. For GSH, a value < 3 mM indicated that the tumors were pre-malignant 74% of the time, and when > 3 mM, they were malignant 80% of the time. The only marker that markedly deviated as early as stage 1 (S1) from its value in pre-S1 was elevated Pi, followed by a decrease of pHe and pO2 and increase in GSH at later stages. CONCLUSION: Molecular TME profiling informs on alteration of tumor redox and metabolism during tumor staging. Early elevation of interstitial Pi at S1 may reflect tumor metabolic alterations that demand elevated phosphorus supply in accordance with the high rate growth hypothesis. These metabolic changes are supported by the following decrease of pHe due to a high tumor reliance on glycolysis and increase of intracellular GSH, a major intracellular redox buffer. The appreciable decrease in TME pO2 was observed only at malignant S4, apparently as a consequence of tumor mass growth and corresponding decrease in perfusion efficacy and increase in oxygen consumption as the tumor cells proliferate.

Training doctoral students in critical thinking and experimental design using problem-based learning.

BACKGROUND: Traditionally, doctoral student education in the biomedical sciences relies on didactic coursework to build a foundation of scientific knowledge and an apprenticeship model of training in the laboratory of an established investigator. Recent recommendations for revision of graduate training include the utilization of graduate student competencies to assess progress and the introduction of novel curricula focused on development of skills, rather than accumulation of facts. Evidence demonstrates that active learning approaches are effective. Several facets of active learning are components of problem-based learning (PBL), which is a teaching modality where student learning is self-directed toward solving problems in a relevant context. These concepts were combined and incorporated in creating a new introductory graduate course designed to develop scientific skills (student competencies) in matriculating doctoral students using a PBL format. METHODS: Evaluation of course effectiveness was measured using the principals of the Kirkpatrick Four Level Model of Evaluation. At the end of each course offering, students completed evaluation surveys on the course and instructors to assess their perceptions of training effectiveness. Pre- and post-tests assessing students' proficiency in experimental design were used to measure student learning. RESULTS: The analysis of the outcomes of the course suggests the training is effective in improving experimental design. The course was well received by the students as measured by student evaluations (Kirkpatrick Model Level 1). Improved scores on post-tests indicate that the students learned from the experience (Kirkpatrick Model Level 2). A template is provided for the implementation of similar courses at other institutions. CONCLUSIONS: This problem-based learning course appears effective in training newly matriculated graduate students in the required skills for designing experiments to test specific hypotheses, enhancing student preparation prior to initiation of their dissertation research.

Biocompatible Monophosphonated Trityl Spin Probe, HOPE71, for In Vivo Measurement of pO2, pH, and [Pi] by Electron Paramagnetic Resonance Spectroscopy.

Hypoxia, acidosis, and elevated inorganic phosphate concentration are characteristics of the tumor microenvironment in solid tumors. There are a number of methods for measuring each parameter individually in vivo, but the only method to date for noninvasive measurement of all three variables simultaneously in vivo is electron paramagnetic spectroscopy paired with a monophosphonated trityl radical, pTAM/HOPE. While HOPE has been successfully used for in vivo studies upon intratissue injection, it cannot be delivered intravenously due to systemic toxicity and albumin binding, which causes significant signal loss. Therefore, we present HOPE71, a monophosphonated trityl radical derived from the very biocompatible trityl probe, Ox071. Here, we describe a straightforward synthesis of HOPE71 starting with Ox071 and report its EPR sensitivities to pO2, pH, and [Pi] with X-band and L-band EPR spectroscopy. We also confirm that HOPE71 lacks albumin binding, shows low cytotoxicity, and has systemic tolerance. Finally, we demonstrate its ability to profile the tumor microenvironment in vivo in a mouse model of breast cancer.

Multi-Omics Immune Interaction Networks in Lung Cancer Tumorigenesis, Proliferation, and Survival.

There are currently no effective biomarkers for prognosis and optimal treatment selection to improve non-small cell lung cancer (NSCLC) survival outcomes. This study further validated a seven-gene panel for diagnosis and prognosis of NSCLC using RNA sequencing and proteomic profiles of patient tumors. Within the seven-gene panel, ZNF71 expression combined with dendritic cell activities defined NSCLC patient subgroups (n = 966) with distinct survival outcomes (p = 0.04, Kaplan-Meier analysis). ZNF71 expression was significantly associated with the activities of natural killer cells (p = 0.014) and natural killer T cells (p = 0.003) in NSCLC patient tumors (n = 1016) using Chi-squared tests. Overexpression of ZNF71 resulted in decreased expression of multiple components of the intracellular intrinsic and innate immune systems, including dsRNA and dsDNA sensors. Multi-omics networks of ZNF71 and the intracellular intrinsic and innate immune systems were computed as relevant to NSCLC tumorigenesis, proliferation, and survival using patient clinical information and in-vitro CRISPR-Cas9/RNAi screening data. From these networks, pan-sensitive and pan-resistant genes to 21 NCCN-recommended drugs for treating NSCLC were selected. Based on the gene associations with patient survival and in-vitro CRISPR-Cas9, RNAi, and drug screening data, MEK1/2 inhibitors PD-198306 and U-0126, VEGFR inhibitor ZM-306416, and IGF-1R inhibitor PQ-401 were discovered as potential targeted therapy that may also induce an immune response for treating NSCLC.

Large-scale synthesis of a monophosphonated tetrathiatriarylmethyl spin probe for concurrent in vivo measurement of pO2, pH and inorganic phosphate by EPR.

Low-field electron paramagnetic resonance spectroscopy paired with pTAM, a mono-phosphonated triarylmethyl radical, is an unmatched technique for concurrent and non-invasive measurement of oxygen concentration, pH, and inorganic phosphate concentration for in vivo investigations. However, the prior reported synthesis is limited by its low yield and poor scalability, making wide-spread application of pTAM unfeasible. Here, we report a new strategy for the synthesis of pTAM with significantly greater yields demonstrated on a large scale. We also present a standalone application with user-friendly interface for automatic spectrum fitting and extraction of pO2, pH, and [Pi] values. Finally, we confirm that pTAM remains in the extracellular space and has low cytotoxicity appropriate for local injection.

Molecular Analysis of ZNF71 KRAB in Non-Small-Cell Lung Cancer.

Our previous study found that zinc finger protein 71 (ZNF71) mRNA expression was associated with chemosensitivity and its protein expression was prognostic of non-small-cell lung cancer (NSCLC). The Krüppel associated box (KRAB) transcriptional repression domain is commonly present in human zinc finger proteins, which are linked to imprinting, silencing of repetitive elements, proliferation, apoptosis, and cancer. This study revealed that ZNF71 KRAB had a significantly higher expression than the ZNF71 KRAB-less isoform in NSCLC tumors (n = 197) and cell lines (n = 117). Patients with higher ZNF71 KRAB expression had a significantly worse survival outcome than patients with lower ZNF71 KRAB expression (log-rank p = 0.04; hazard ratio (HR): 1.686 [1.026, 2.771]), whereas ZNF71 overall and KRAB-less expression levels were not prognostic in the same patient cohort. ZNF71 KRAB expression was associated with epithelial-to-mesenchymal transition (EMT) in both patient tumors and cell lines. ZNF71 KRAB was overexpressed in NSCLC cell lines resistant to docetaxel and paclitaxel treatment compared to chemo-sensitive cell lines, consistent with its association with poor prognosis in patients. Therefore, ZNF71 KRAB isoform is a more effective prognostic factor than ZNF71 overall and KRAB-less expression for NSCLC. Functional analysis using CRISPR-Cas9 and RNA interference (RNAi) screening data indicated that a knockdown/knockout of ZNF71 did not significantly affect NSCLC cell proliferation in vitro.

Hypoxia-Inducible Factor α Subunits Regulate Tie2-Expressing Macrophages That Influence Tumor Oxygen and Perfusion in Murine Breast Cancer.

Tie2-expressing monocytes/macrophages (TEMs) are a distinct subset of proangiogenic monocytes selectively recruited to tumors in breast cancer. Because of the hypoxic nature of solid tumors, we investigated if oxygen, via hypoxia-inducible transcription factors HIF-1α and HIF-2α, regulates TEM function in the hypoxic tumor microenvironment. We orthotopically implanted PyMT breast tumor cells into the mammary fat pads of syngeneic LysMcre, HIF-1α fl/fl /LysMcre, or HIF-2α fl/fl /LysMcre mice and evaluated the tumor TEM population. There was no difference in the percentage of tumor macrophages among the mouse groups. In contrast, HIF-1α fl/fl /LysMcre mice had a significantly smaller percentage of tumor TEMs compared with control and HIF-2α fl/fl /LysMcre mice. Proangiogenic TEMs in macrophage HIF-2α-deficient tumors presented significantly more CD31+ microvessel density but exacerbated hypoxia and tissue necrosis. Reduced numbers of proangiogenic TEMs in macrophage HIF-1α-deficient tumors presented significantly less microvessel density but tumor vessels that were more functional as lectin injection revealed more perfusion, and functional electron paramagnetic resonance analysis revealed more oxygen in those tumors. Macrophage HIF-1α-deficient tumors also responded significantly to chemotherapy. These data introduce a previously undescribed and counterintuitive prohypoxia role for proangiogenic TEMs in breast cancer which is, in part, suppressed by HIF-2α.

Synthesis, Characterization, and Application of a Highly Hydrophilic Triarylmethyl Radical for Biomedical EPR.

Stable tetrathiatriarylmethyl radicals have significantly contributed to the recent progress in biomedical electron paramagnetic resonance (EPR) due to their unmatched stability in biological media and long relaxation times. However, the lipophilic core of the most commonly used structure (Finland trityl) is responsible for its interaction with plasma biomacromolecules, such as albumin, and self-aggregation at high concentrations and/or low pH. While Finland trityl is generally considered inert toward many reactive radical species, we report that sulfite anion radical efficiently substitutes the three carboxyl moieties of Finland trityl with a high rate constant of 3.53 × 108 M-1 s-1, leading to a trisulfonated Finland trityl radical. This newly synthesized highly hydrophilic trityl radical shows an ultranarrow linewidth (ΔBpp = 24 mG), a lower affinity for albumin than Finland trityl, and a high aqueous solubility even at acidic pH. Therefore, this new tetrathiatriarylmethyl radical can be considered as a superior spin probe in comparison to the widely used Finland trityl. One of its potential applications was demonstrated by in vivo mapping oxygen in a mouse model of breast cancer. Moreover, we showed that one of the three sulfo groups can be easily substituted with S-, N-, and P-nucleophiles, opening access to various monofunctionalized sulfonated trityl radicals.

Circulating extracellular vesicle content reveals de novo DNA methyltransferase expression as a molecular method to predict septic shock.

Extracellular vesicles (EVs) are mRNA-containing cell fragments shed into circulation during pathophysiological events. DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) regulate gene expression by modifying DNA methylation and altering transcription. Sepsis is a systemic insult resulting in vascular dysfunction, which can lead to shock and death. We analysed plasma from ICU patients for circulating EV numbers, defined as particles isolated from 1 mL plasma at 21,000xg, and DNMTs mRNA content as prognostic markers of septic shock. Compared to plasma from critically ill patients with or without sepsis, plasma from septic shock patients contained more EVs per mL, expressed as total DNMTs mRNAs over 5 days, and more individual DNMT mRNAs at each day. A comparison of EV-DNMT1 (maintenance methylation) with EV-DNMT3A+DNMT3B (de novo methylation) expression correlated highly with severity, and EVs from septic shock patients carried more total DNMT mRNAs and more DNMT3A+DNMT3B mRNAs than control or sepsis EVs. Total plasma EVs also correlated with sepsis severity. EV-DNMT mRNAs load, when coupled with total plasma EV number, may be a novel method to diagnose septic shock upon ICU admittance and offer opportunities to more precisely intervene with standard therapy or other targeted interventions to regulate EV release and/or specific DNMT activity.

In Vitro Expansion of Bone Marrow Derived Mesenchymal Stem Cells Alters DNA Double Strand Break Repair of Etoposide Induced DNA Damage.

Mesenchymal stem cells (MSCs) are of interest for use in diverse cellular therapies. Ex vivo expansion of MSCs intended for transplantation must result in generation of cells that maintain fidelity of critical functions. Previous investigations have identified genetic and phenotypic alterations of MSCs with in vitro passage, but little is known regarding how culturing influences the ability of MSCs to repair double strand DNA breaks (DSBs), the most severe of DNA lesions. To investigate the response to DSB stress with passage in vitro, primary human MSCs were exposed to etoposide (VP16) at various passages with subsequent evaluation of cellular damage responses and DNA repair. Passage number did not affect susceptibility to VP16 or the incidence and repair kinetics of DSBs. Nonhomologous end joining (NHEJ) transcripts showed little alteration with VP16 exposure or passage; however, homologous recombination (HR) transcripts were reduced following VP16 exposure with this decrease amplified as MSCs were passaged in vitro. Functional evaluations of NHEJ and HR showed that MSCs were unable to activate NHEJ repair following VP16 stress in cells after successive passage. These results indicate that ex vivo expansion of MSCs alters their ability to perform DSB repair, a necessary function for cells intended for transplantation.

GPKOW is essential for pre-mRNA splicing in vitro and suppresses splicing defect caused by dominant-negative DHX16 mutation in vivo.

Human GPKOW [G-patch (glycine-rich) domain and KOW (Kyrpides, Ouzounis and Woese) domain] protein contains a G-patch domain and two KOW domains, and is a homologue of Arabidopsis MOS2 and Saccharomyces Spp2 protein. GPKOW is found in the human spliceosome, but its role in pre-mRNA splicing remains to be elucidated. In this report, we showed that GPKOW interacted directly with the DHX16/hPRP2 and with RNA. Immuno-depletion of GPKOW from HeLa nuclear extracts resulted in an inactive spliceosome that still bound DHX16. Adding back recombinant GPKOW restored splicing to the depleted extract. In vivo, overexpression of GPKOW partially suppressed the splicing defect observed in dominant-negative DHX16 mutant expressing cells. Mutations at the G-patch domain greatly diminished the GPKOW-DHX16 interaction; however, the mutant was active in splicing and was able to suppress splicing defect. Mutations at the KOW1 domain slightly altered the GPKOW-RNA interaction, but the mutant was less functional in vitro and in vivo. Our results indicated that GPKOW can functionally impact DHX16 but that interaction between the proteins is not required for this activity.

Detection of Alternatively Spliced or Processed RNAs in Cancer Using Oligonucleotide Microarray.

Deregulation of gene expression plays a pivotal role in tumorigenesis, so the ability to detect RNA alterations is of great value in cancer diagnosis and management. DNA microarrays have been used to measure changes in mRNA or microRNA level, but less often the change of RNA isoforms. Here we appraise the utilization of microarray in detecting alternatively processed RNAs, which have alternative splice forms, retained introns, or altered 3' untranslated regions. We cover the methodology and focus on cancer studies. Recent development in parallel or deep sequencing used in transcriptome analysis is also discussed.

Bone marrow osteoblast vulnerability to chemotherapy.

Osteoblasts are a major component of the bone marrow microenvironment, which provide support for hematopoietic cell development. Functional disruption of any element of the bone marrow niche, including osteoblasts, can potentially impair hematopoiesis. We have studied the effect of two widely used drugs with different mechanisms of action, etoposide (VP16) and melphalan, on murine osteoblasts at distinct stages of maturation. VP16 and melphalan delayed maturation of preosteoblasts and altered CXCL12 protein levels, a key regulator of hematopoietic cell homing to the bone marrow. Sublethal concentrations of VP16 and melphalan also decreased the levels of several transcripts which contribute to the composition of the extracellular matrix (ECM) including osteopontin (OPN), osteocalcin (OCN), and collagen 1A1 (Col1a1). The impact of chemotherapy on message and protein levels for some targets was not always aligned, suggesting differential responses at the transcription and translation or protein stability levels. As one of the main functions of a mature osteoblast is to synthesize ECM of a defined composition, disruption of the ratio of its components may be one mechanism by which chemotherapy affects the ability of osteoblasts to support hematopoietic recovery coincident with altered marrow architecture. Collectively, these observations suggest that the osteoblast compartment of the marrow hematopoietic niche is vulnerable to functional dysregulation by damage imposed by agents frequently used in clinical settings. Understanding the mechanistic underpinning of chemotherapy-induced changes on the hematopoietic support capacity of the marrow microenvironment may contribute to improved strategies to optimize patient recovery post-transplantation.

Bone marrow osteoblast damage by chemotherapeutic agents.

Hematopoietic reconstitution, following bone marrow or stem cell transplantation, requires a microenvironment niche capable of supporting both immature progenitors and stem cells with the capacity to differentiate and expand. Osteoblasts comprise one important component of this niche. We determined that treatment of human primary osteoblasts (HOB) with melphalan or VP-16 resulted in increased phospho-Smad2, consistent with increased TGF-ß1 activity. This increase was coincident with reduced HOB capacity to support immature B lineage cell chemotaxis and adherence. The supportive deficit was not limited to committed progenitor cells, as human embryonic stem cells (hESC) or human CD34+ bone marrow cells co-cultured with HOB pre-exposed to melphalan, VP-16 or rTGF-ß1 had profiles distinct from the same populations co-cultured with untreated HOB. Functional support deficits were downstream of changes in HOB gene expression profiles following chemotherapy exposure. Melphalan and VP-16 induced damage of HOB suggests vulnerability of this critical niche to therapeutic agents frequently utilized in pre-transplant regimens and suggests that dose escalated chemotherapy may contribute to post-transplantation hematopoietic deficits by damaging structural components of this supportive niche.

Melphalan exposure induces an interleukin-6 deficit in bone marrow stromal cells and osteoblasts.

Bone marrow stromal cells (BMSC) and osteoblasts are critical components of the microenvironment that support hematopoietic recovery following bone marrow transplantation. Aggressive chemotherapy not only affects tumor cells, but also influences additional structural and functional components of the microenvironment. Successful reconstitution of hematopoiesis following stem cell or bone marrow transplantation after aggressive chemotherapy is dependent upon components of the microenvironment maintaining their supportive function. This includes secretion of soluble factors and expression of cellular adhesion molecules that impact on development of hematopoietic cells. In the current study, we investigated the effects of chemotherapy treatment on BMSC and human osteoblast (HOB) expression of interleukin-6 (IL-6) as one regulatory factor. IL-6 is a pleiotropic cytokine which has diverse effects on hematopoietic cell development. In the current study we demonstrate that exposure of BMSC or HOB to melphalan leads to decreases in IL-6 protein expression. Decreased IL-6 protein is the most pronounced following melphalan exposure compared to several other chemotherapeutic agents tested. We also observed that melphalan decreased IL-6 mRNA in both BMSC and HOB. Finally, using a model of BMSC or HOB co-cultured with myeloma cells exposed to melphalan, we observed that IL-6 protein was also decreased, consistent with treatment of adherent cells alone. Collectively, these observations are of dual significance. First, suggesting that chemotherapy induced IL-6 deficits in the bone marrow occur which may result in defective hematopoietic support of early progenitor cells. In contrast, the decrease in IL-6 protein may be a beneficial mechanism by which melphalan acts as a valuable therapeutic agent for treatment of multiple myeloma, where IL-6 present in the bone marrow acts as a proliferative factor and contributes to disease progression. Taken together, these data emphasize the responsiveness of the microenvironment to diverse stress that is important to consider in therapeutic settings.

Mechanistic control of carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) splice isoforms by the heterogeneous nuclear ribonuclear proteins hnRNP L, hnRNP A1, and hnRNP M.

Carcinoembryonic antigen-related cell adhesion molecule-1 (CEACAM1) is expressed in a variety of cell types and is implicated in carcinogenesis. Alternative splicing of CEACAM1 pre-mRNA generates two cytoplasmic domain splice variants characterized by the inclusion (L-isoform) or exclusion (S-isoform) of exon 7. Here we show that the alternative splicing of CEACAM1 pre-mRNA is regulated by novel cis elements residing in exon 7. We report the presence of three exon regulatory elements that lead to the inclusion or exclusion of exon 7 CEACAM1 mRNA in ZR75 breast cancer cells. Heterologous splicing reporter assays demonstrated that the maintenance of authentic alternative splicing mechanisms were independent of the CEACAM1 intron sequence context. We show that forced expression of these exon regulatory elements could alter CEACAM1 splicing in HEK-293 cells. Using RNA affinity chromatography, three members of the heterogeneous nuclear ribonucleoprotein family (hnRNP L, hnRNP A1, and hnRNP M) were identified. RNA immunoprecipitation of hnRNP L and hnRNP A1 revealed a binding motif located central and 3' to exon 7, respectively. Depletion of hnRNP A1 or L by RNAi in HEK-293 cells promoted exon 7 inclusion, whereas overexpression led to exclusion of the variable exon. By contrast, overexpression of hnRNP M showed exon 7 inclusion and production of CEACAM1-L mRNA. Finally, stress-induced cytoplasmic accumulation of hnRNP A1 in MDA-MB-468 cells dynamically alters the CEACAM1-S:CEACAM1:L ratio in favor of the l-isoform. Thus, we have elucidated the molecular factors that control the mechanism of splice-site recognition in the alternative splicing regulation of CEACAM1.

Regulation of CEACAM1 transcription in human breast epithelial cells.

BACKGROUND: Carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) is a transmembrane protein with multiple functions in different cell types. CEACAM1 expression is frequently mis-regulated in cancer, with down-regulation reported in several tumors of epithelial origin and de novo expression of CEACAM1 in lung cancer and malignant melanoma. In this report we analyzed the regulation of CEACAM1 expression in three breast cancer cell lines that varied in CEACAM1 expression from none (MCF7) to moderate (MDA-MB-468) to high (MCF10A, comparable to normal breast). RESULTS: Using in vivo footprinting and chromatin immunoprecipitation experiments we show that the CEACAM1 proximal promoter in breast cells is bound in its active state by SP1, USF1/USF2, and IRF1/2. When down-regulated the CEACAM1 promoter remains accessible to USF2 and partially accessible to USF1. Interferon-γ up-regulates CEACAM1 mRNA by a mechanism involving further induction of IRF-1 and USF1 binding at the promoter. As predicted by this analysis, silencing of IRF1 and USF1 but not USF2 by RNAi resulted in a significant decrease in CEACAM1 protein expression in MDA-MB-468 cells. The inactive CEACAM1 promoter in MCF7 cells exhibits decreased histone acetylation at the promoter region, with no evidence of H3K9 or H3K27 trimethylation, histone modifications often linked to condensed chromatin structure. CONCLUSIONS: Our data suggest that transcription activators USF1 and IRF1 interact to modulate CEACAM1 expression and that the chromatin structure of the promoter is likely maintained in a poised state that can promote rapid induction under appropriate conditions.

Nuclear retention of unspliced pre-mRNAs by mutant DHX16/hPRP2, a spliceosomal DEAH-box protein.

Defective or imbalanced expression of spliceosomal factors has been linked to human disease; however, how a defective spliceosome affects intron-containing gene transcripts in human cells is largely unknown. DEAH-box protein DHX16 is a human orthologue of Saccharomyces cerevisiae spliceosomal protein Prp2, an RNA-dependent ATPase that activates the spliceosome before the first catalytic step of splicing. Yeast prp2 mutants accumulate unspliced RNAs from the vast majority of intron-containing genes. Here we used a genomic tiling microarray to screen transcripts from four chromosomes in human cells expressing a dominant negative DHX16 mutant and identified a number of gene transcripts that retained their introns. The mutant protein also affected gene transcripts that are sensitive to pladienolide, an SF3b inhibitor. The unspliced RNAs were retained in the nucleus, and block of nonsense-mediated decay did not affect their accumulation. Thus, a perturbation of human PRP2/DHX16 results in accumulation of unspliced transcripts, similar to the outcome in yeast prp2 mutants. The results further suggest that mutant DHX16/hPRP2 causes a defective spliceosome to retain unspliced gene transcripts in the nuclei of human cells.

Contribution of DEAH-box protein DHX16 in human pre-mRNA splicing.

Studies of mammalian splicing factors are often focused on small nuclear ribonucleoproteins or regulatory RNA-binding proteins, such as hnRNP (heterogeneous nuclear ribonucleoprotein) and SR proteins (serine/arginine-rich proteins); however, much less is known about the contribution of DExD/H-box proteins or RNA helicases in mammalian pre-mRNA splicing. The human DEAH-box protein DHX16 [also known as DBP2 (DEAD-box protein 2)], is homologous with Caenorhabditis elegans Mog-4, Schizosaccharomyces pombe Prp8 and Saccharomyces cerevisiae Prp2. In the present study, we show that DHX16 is required for pre-mRNA splicing after the formation of a pre-catalytic spliceosome. We found that anti-DHX16 antiserum inhibited the splicing reaction in vitro and the antibody immunoprecipitated pre-mRNA, splicing intermediates and spliceosomal small nuclear RNAs. Cells that expressed DHX16 that had a mutation in the helicase domain accumulated unspliced intron-containing minigene transcripts. Nuclear extracts isolated from the dominant-negative DHX16-G724N-expressing cells formed splicing complex B, but were impaired in splicing. Adding extracts containing DHX16-G724N or DHX16-S552L mutant proteins to HeLa cell nuclear extracts resulted in reduced splicing, indicating that the mutant protein directly inhibited splicing in vitro. Therefore our results show that DHX16 is needed for human pre-mRNA splicing at a step analogous to that mediated by the S. cerevisiae spliceosomal ATPase Prp2.