Recruitment of the m6A/m6Am demethylase FTO to target RNAs by the telomeric zinc finger protein ZBTB48.

BACKGROUND: N6-methyladenosine (m6A), the most abundant internal modification on eukaryotic mRNA, and N6, 2'-O-dimethyladenosine (m6Am), are epitranscriptomic marks that function in multiple aspects of posttranscriptional regulation. Fat mass and obesity-associated protein (FTO) can remove both m6A and m6Am; however, little is known about how FTO achieves its substrate selectivity. RESULTS: Here, we demonstrate that ZBTB48, a C2H2-zinc finger protein that functions in telomere maintenance, associates with FTO and binds both mRNA and the telomere-associated regulatory RNA TERRA to regulate the functional interactions of FTO with target transcripts. Specifically, depletion of ZBTB48 affects targeting of FTO to sites of m6A/m6Am modification, changes cellular m6A/m6Am levels and, consequently, alters decay rates of target RNAs. ZBTB48 ablation also accelerates growth of HCT-116 colorectal cancer cells and modulates FTO-dependent regulation of Metastasis-associated protein 1 (MTA1) transcripts by controlling the binding to MTA1 mRNA of the m6A reader IGF2BP2. CONCLUSIONS: Our findings thus uncover a previously unknown mechanism of posttranscriptional regulation in which ZBTB48 co-ordinates RNA-binding of the m6A/m6Am demethylase FTO to control expression of its target RNAs.

Establishment of orthotopic osteosarcoma animal models in immunocompetent rats through muti-rounds of in-vivo selection.

Immunodeficient murine models are usually used as the preclinical models of osteosarcoma. Such models do not effectively simulate the process of tumorigenesis and metastasis. Establishing a suitable animal model for understanding the mechanism of osteosarcoma and the clinical translation is indispensable. The UMR-106 cell suspension was injected into the marrow cavity of Balb/C nude mice. Tumor masses were harvested from nude mice and sectioned. The tumor fragments were transplanted into the marrow cavities of SD rats immunosuppressed with cyclosporine A. Through muti-rounds selection in SD rats, we constructed orthotopic osteosarcoma animal models using rats with intact immune systems. The primary tumor cells were cultured in-vitro to obtain the immune-tolerant cell line. VX2 tumor fragments were transplanted into the distal femur and parosteal radius of New Zealand white rabbit to construct orthotopic osteosarcoma animal models in rabbits. The rate of tumor formation in SD rats (P1 generation) was 30%. After four rounds of selection and six rounds of acclimatization in SD rats with intact immune systems, we obtained immune-tolerant cell lines and established the orthotopic osteosarcoma model of the distal femur in SD rats. Micro-CT images confirmed tumor-driven osteolysis and the bone destruction process. Moreover, the orthotopic model was also established in New Zealand white rabbits by implanting VX2 tumor fragments into rabbit radii and femurs. We constructed orthotopic osteosarcoma animal models in rats with intact immune systems through muti-rounds in-vivo selection and the rabbit osteosarcoma model.

Targeting nanoplatform synergistic glutathione depletion-enhanced chemodynamic, microwave dynamic, and selective-microwave thermal to treat lung cancer bone metastasis.

Once bone metastasis occurs in lung cancer, the efficiency of treatment can be greatly reduced. Current mainstream treatments are focused on inhibiting cancer cell growth and preventing bone destruction. Microwave ablation (MWA) has been used to treat bone tumors. However, MWA may damage the surrounding normal tissues. Therefore, it could be beneficial to develop a nanocarrier combined with microwave to treat bone metastasis. Herein, a microwave-responsive nanoplatform (MgFe2O4@ZOL) was constructed. MgFe2O4@ZOL NPs release the cargos of Fe3+, Mg2+ and zoledronic acid (ZOL) in the acidic tumor microenvironment (TME). Fe3+ can deplete intracellular glutathione (GSH) and catalyze H2O2 to generate •OH, resulting in chemodynamic therapy (CDT). In addition, the microwave can significantly enhance the production of reactive oxygen species (ROS), thereby enabling the effective implementation of microwave dynamic therapy (MDT). Moreover, Mg2+ and ZOL promote osteoblast differentiation. In addition, MgFe2O4@ZOL NPs could target and selectively heat tumor tissue and enhance the effect of microwave thermal therapy (MTT). Both in vitro and in vivo experiments revealed that synergistic targeting, GSH depletion-enhanced CDT, MDT, and selective MTT exhibited significant antitumor efficacy and bone repair. This multimodal combination therapy provides a promising strategy for the treatment of bone metastasis in lung cancer patients.

Long noncoding RNA X-inactive specific transcript (lncRNA XIST) inhibits hepatic insulin resistance by competitively binding microRNA-182-5p.

BACKGROUND: What is highlighted in this study refers to the role and molecular mechanism of long noncoding RNA (lncRNA) X-inactive specific transcript (XIST) in cells with insulin resistance (IR). METHODS: In this study, LX-2 cells were applied to establish IR model in vitro. The expressions of lncRNA XIST, phosphoenolpyruvate carboxykinase (PEPCK,) and glucose-6-phosphatase (G6Pase) were quantified by quantitative reverse transcription polymerase chain reaction. The 2-deoxy-d-glucose-6-phosphate (2-DG6P) level was detected utilizing 2-deoxy-d-glucose (2-DG) uptake measurement kit. Western blot was adopted to measure the protein expressions of insulin-like growth factor-1 receptor (IGF-1R), G6Pase, PEPCK, and phosphatidylinositol 3-kinase (PI3K)/Akt pathway-related genes. StarBase was used to predict the targeting relationship between lncRNA XIST or IGF-1R with miR-182-5p, the results of which were verified by dual-luciferase reporter, RNA pull-down, and RNA immunoprecipitation assays. Rescue experiments were conducted to investigate the effect of miR-182-5p on IR cells. Next, low-expressed lncRNA XIST and high-expressed miR-182-5p were observed in IR cells. RESULTS: Upregulation of lncRNA XIST increased IGF-1R and 2-DG6P levels, decreased G6Pase and PEPCK expressions, and promoted PI3K/Akt pathway activation in IR cells. LncRNA XIST sponged miR-182-5p which targeted IGF-1R. MiR-182-5p mimic reversed the above effects of lncRNA XIST overexpression on IR cells. CONCLUSIONS: In conclusion, lncRNA XIST/miR-182-5p axis alleviates hepatic IR in vitro via IGF-1R/PI3K/Akt signaling pathway, which could be the promising therapeutic target.

Premetastatic Niche Mimicking Bone-On-A-Chip: A Microfluidic Platform to Study Bone Metastasis in Cancer Patients.

Primary cancer modulates the bone microenvironment to sow the seeds of dormancy and metastasis in tumor cells, leading to multiple organ metastasis and death. In this study, 3D printing and bone-on-a-chip (BOC) are combined to develop a BOC platform that mimics the pre-metastatic niches (PMNs) and facilitates elucidation of the interactions between bone-resident cells and metastatic tumor cells under the influence of primary cancer. Photocrosslinkable gelatin methacrylate (GelMA) is used as a 3D culturing hydrogel to encapsulate cells, and circulate tumor culture medium (CM) adjacent to the hydrogel to verify the critical role of mesenchymal stem cells (MSCs) and osteoclasts (RAW264.7s). Three niches: the dormancy niche, the perivascular niche, and the "vicious cycle" niche, are devised to recapitulate bone metastasis in one chip with high cell viability and excellent nutrient exchange. With respect to tumor dormancy and reactivation, the invadopodia formation of A549 lung cancer cells in communication with MSCs and RAW264.7 via the cortactin pathway is researched. As a proof of concept, the functionality and practicality of the platform are demonstrated by analyzing the invadopodia formation and the influence of various cells, and the establishment of the dynamic niches paves the way to understanding PMN formation and related drug discovery.

Multifunctional 3D-printed scaffolds eradiate orthotopic osteosarcoma and promote osteogenesis via microwave thermo-chemotherapy combined with immunotherapy.

Tumor recurrence and a lack of bone-tissue integration are two critical concerns in the surgical treatment of osteosarcoma. Thus, an advanced multifunctional therapeutic platform capable of simultaneously eliminating residual tumor cells and promoting bone regeneration is urgently needed for efficient osteosarcoma treatment. Herein, to thoroughly eliminate tumors and simultaneously promote bone regeneration, an intelligent multifunctional therapeutic scaffold has been engineered by integrating microwave-responsive zeolitic imidazolate framework 8 (ZIF-8) nanomaterials loaded with a chemotherapeutic drug and an immune checkpoint inhibitor onto 3D-printed titanium scaffolds. The constructed scaffold features distinct microwave-thermal sensitization and tumor microenvironment-responsive characteristics, which can induce tumor immunogenic death by microwave hyperthermia and chemotherapy. Orthotopic implantation of the nanocomposite scaffold results in an enhanced immune response against osteosarcoma that may effectively inhibit tumor recurrence through synergistic immunotherapy. During long-term implantation, the zinc ions released from the degradation of ZIF-8 can induce the osteogenic differentiation of stem cells. The porous structure and mechanical properties of the 3D-printed titanium scaffolds provide a structural microenvironment for bone regeneration. This study provides a paradigm for the design of multifunctional microwave-responsive composite scaffolds for use as a therapy for osteosarcoma, which could lead to improved strategies for the treatment of the disease.

Comparing the Accuracy of Seven Scoring Systems in Predicting Survival of Lung Cancer Patients With Spinal Metastases: An External Validation From Two Centers.

STUDY DESIGN: Retrospective case series. OBJECTIVE: To investigate the accuracy of seven scoring systems for the prediction of survival in lung cancer patients with spinal metastases (SPM). SUMMARY OF BACKGROUND DATA: Although survival scoring systems have been developed for surgical decision-making, the reliability and validity of these models are unclear for specific cancer types. As the prevalence of patients with lung cancer increases, it is imperative to determine the accuracy of these models for lung cancer patients with SPM. MATERIALS AND METHODS: This is a retrospective study of a cohort of lung cancer patients with SPM who underwent spine surgery between 2019 and 2021 at two centers. The optimal area under the curve (AUC) was calculated to evaluate the accuracy of seven candidate scoring systems at 3, 6, and 12 months. Calibration and decision curve analysis was used for further validation. RESULTS: A total of 166 patients (mean age: 58.98±10.94; 105 males and 61 females) with SPM were included. The median postoperative survival was 12.87±0.93 months. The modified Bauer score, revised Tokuhashi score, Linden score, Tomita score, the Skeletal Oncology Research Group nomogram, and the New England Spinal Metastasis Score in prediction survival at 3, 6, and 12 months showed a slightly weaker AUC (range 0.464-0.659). The AUC of the Katagiri-New score in predicting 1-year survival for lung cancer patients was the highest (0.708; range 0.619-0.798). The decision curve analysis showed that the Katagiri-New score led to a greater net benefit than the strategies of changing management for all patients or none of the patients. CONCLUSIONS: This study suggests that the most commonly used models have limitations in predicting survival in patients undergoing spinal surgery for metastatic lung cancer and underestimate survival. In this sample of lung cancer patients, the Katagiri-New Scoring system score had the best performance in predicting 1-year survival. LEVEL OF EVIDENCE: 4.

External validation of the SORG machine learning algorithms for predicting 90-day and 1-year survival of patients with lung cancer-derived spine metastases: a recent bi-center cohort from China.

BACKGROUND CONTEXT: The survival prediction of lung cancer-derived spinal metastases is often underestimated by several scores. The SORG machine learning (ML) algorithm is considered a promising tool to predict the risk of 90-day and 1-year mortality in patients with spinal metastases, but not been externally validated for lung cancer. PURPOSE: This study aimed to externally validate the SORG ML algorithms on lung cancer-derived spinal metastases patients from two large-volume, tertiary medical centers between 2018 and 2021. STUDY DESIGN/SETTING: Retrospective, cohort study. PATIENT SAMPLE: Patients aged 18 years or older at two tertiary medical centers in China are treated surgically for spinal metastasis. OUTCOME MEASURES: Mortality within 90 days of surgery, mortality within 1 year of surgery. METHODS: The baseline characteristics were compared between the development cohort and our validation cohort. Discrimination (receiver operating curve), calibration (calibration plot, intercept, and slope), the overall performance (Brier score), and decision curve analysis was used to assess the overall performance of the SORG ML algorithms. RESULTS: This study included 150 patients with lung cancer-derived spinal metastases from two medical centers in China. Ninety-day and 1-year mortality rates were 12.9% (19/147) and 51.3% (60/117), respectively. Lung Cancer with targeted therapies had the lowest Hazard Ratio (HR=0.490), showing an optimal protecting factor. The AUC of the SORG ML algorithm for 90-day mortality prediction in lung cancer-derived spinal metastases is 0.714. While the AUC for 1-year mortality prediction is 0.832 (95CI%, 0.758-0.906). The algorithm for 1-year mortality was well-calibrated with an intercept of 0.13 and a calibration slope of 1.00. However, the 90-day mortality prediction was underestimated with an intercept of 0.60 and a slope of 0.37. The SORG ML algorithms for 1-year mortality showed a greater net benefit than the "treats all or no patients" strategies. CONCLUSIONS: In the latest cohort of lung cancer-derived spinal metastases in China, the SORG algorithms for predicting 1-year mortality performed well on external validation. However, 90-day mortality was underestimated. The algorithm should be further validated by single primary tumor-derived metastasis treated with the latest comprehensive treatment in diverse populations.

Kinematic alterations of the ankle in subjects with generalized joint hypermobility compared with the controls: A cross-sectional study.

INTRODUCTION: Generalized joint hypermobility (GJH) is a hereditary connective tissue disease in which the range of motion (ROM) of multiple joints exceeds the normal range, and the ROM varies with age, gender, and ethnicity. At present, the six-degree-of-freedom (6-DOF) of ankle kinematics among people with GJH have not been studied. To investigate the kinematic characteristics in the ankle during treadmill gait of university students with generalized joint hypermobility compared to normal participants. We hypothesized that compared to the participants in the control group, those with GJH would exhibit kinematic characteristics of poorer active motion stability in the ankle during treadmill gait. METHODS: Healthy university student volunteers aged 18-24 (excluding those with a history of ankle trauma, etc.) were recruited and divided into a control group (50 volunteers) and a GJH group (Beighton score ≥4, 50 volunteers). Data of the 6-DOF kinematics of ankle was collected using a 3D gait analysis system. Variables were evaluated using independent t-tests and Wilcoxon signed-rank tests. RESULTS: In the proximal/distal parameter, proximal displacement was significantly increased in the GJH group compared with the control group during 4-9% and 96-97% of the gait phase (loading response and terminal swing phase), with an increase of (0.1-0.2 cm, p < .05). Regarding the proximal/distal, internal/external, plantarflexion/dorsiflexion, and anterior/posterior parameters, the participants with GJH exhibited greater ROM than those in the control group throughout the gait cycle (0.24 ± 0.22 cm vs. 0.19 ± 0.15 cm, p = 0.047, 5.56 ± 2.90° vs. 4.48 ± 3.30°, p = .020, 23.05 ± 5.75° vs. 20.36 ± 4.91°, p < .001, 0.65 ± 0.30 cm vs. 0.55 ± 0.27 cm, p = .018). However, ROM of inversion/eversion translation was found to be decreased in the GJH group compared to the control group (8.92 ± 1.59° vs. 9.47 ± 1.37°, p = .009). In addition, there was no statistical difference between the GJH group and the control group in ROM of medial/lateral translation (0.05 ± 0.06 cm vs. 0.04 ± 0.05 cm, p = .131). CONCLUSION: Our results confirm that our hypothesis is not valid. Although there were a few differences in each gait parameter of the ankle between the GJH group and the control group, the difference was not significant. These results indicate that the presence of GJH has less effect on ankle kinematics and enhance our knowledge of the relationship between GJH and 6-DOF of ankle kinematics.

Regulation of alternative polyadenylation by the C2H2-zinc-finger protein Sp1.

Alternative polyadenylation (APA) enhances gene regulatory potential by increasing the diversity of mRNA transcripts. 3' UTR shortening through APA correlates with enhanced cellular proliferation and is a widespread phenomenon in tumor cells. Here, we show that the ubiquitously expressed transcription factor Sp1 binds RNA in vivo and is a common repressor of distal poly(A) site usage. RNA sequencing identified 2,344 genes (36% of the total mapped mRNA transcripts) with lengthened 3' UTRs upon Sp1 depletion. Sp1 preferentially binds the 3' UTRs of such lengthened transcripts and inhibits cleavage at distal sites by interacting with the subunits of the core cleavage and polyadenylation (CPA) machinery. The 3' UTR lengths of Sp1 target genes in breast cancer patient RNA-seq data correlate with Sp1 expression levels, implicating Sp1-mediated APA regulation in modulating tumorigenic properties. Taken together, our findings provide insights into the mechanism for dynamic APA regulation by unraveling a previously unknown function of the DNA-binding transcription factor Sp1.

Frequently expressed glypican-3 as a promising novel therapeutic target for osteosarcomas.

Osteosarcoma (OS) is the most common bone malignancy without a reliable therapeutic target. Glypican-3 (GPC3) mutation and upregulation have been detected in multidrug resistant OS, and anti-GPC3 immunotherapy can effectively suppress the growth of organoids. Further profiling of GPC3 mutations and expression patterns in OS is of clinical significance. To address these issues, fresh OS specimens were collected from 24 patients for cancer-targeted next-generation sequencing (NGS) and three-dimensional patient-derived organoid (PDO) culture. A tumor microarray was prepared using 37 archived OS specimens. Immunohistochemical (IHC) staining was performed on OS specimens and microarrays to profile GPC3 and CD133 expression as well as intratumoral distribution patterns. RT-PCR was conducted to semiquantify GPC3 and CD133 expression levels in the OS tissues. Anti-GPC3 immunotherapy was performed on OS organoids with or without GPC3 expression and its efficacy was analyzed using multiple experimental approaches. No OS cases with GPC3 mutations were found, except for the positive control (OS-08). IHC staining revealed GPC3 expression in 73.77% (45/61) of OSs in weak (+; 29/45), moderate (++; 8/45), and strong (+++; 8/45) immunolabeling densities. The intratumoral distribution of GPC3-positive cells was variable in the focal (+; 10%-30%; 8/45), partial (++; 31%-70%; 22/45), and the most positive patterns (+++; >71%; 15/45), which coincided with CD133 immunolabeling (P = 9.89 × 10-10 ). The anti-GPC3 antibody efficiently inhibits Wnt/ß-catenin signaling and induces apoptosis in GPC3-positive PDOs and PDXs, as opposed to GPC3-negative PDOs and PDXs. The high frequency of GPC3 and CD133 co-expression and the effectiveness of anti-wild-type GPC3-Ab therapy in GPC3-positive OS models suggest that GPC3 is a novel prognostic parameter and a promising therapeutic target for osteosarcoma.

The effects of combined microwave ablation and open surgery for the treatment of lung cancer-derived thoracolumbar metastases.

OBJECTIVE: To investigate the clinical effects of microwave ablation (MWA) in addition to open surgery for the treatment of lung cancer-derived thoracolumbar metastases. METHODS: This was a single-institution, retrospective, cohort study. From January 2019 to December 2020, a total of 47 patients with lung cancer-derived thoracolumbar metastases underwent posterior spinal canal decompression and fixation surgery in our hospital. Two independent surgical teams treated these patients. One group underwent open surgery combined with MWA therapy, while the other had open surgery only (control). The pre- and post-operative visual analog scale (VAS) scores and the overall survival (OS) were compared between the MWA and control groups. The Frankel Grade classification was applied for the evaluation of the post-surgical spinal cord function. Improvement was defined as an increase of at least one rank from the pre-operative scores. Each patient was evaluated pre- and post-operatively at 48 h, 1 month, and 3-month intervals. Data on surgical-related complications were recorded. RESULTS: Thirty men and 17 women were included, with an average age of 57.9 ± 11.4 years (range, 26-81 years). Twenty-eight patients underwent MWA and were in the MWA group, and 19 patients were included in the control group. Post-operatively all patients were followed up regularly; the median follow-up time was 12 months (range, 3-24 months), and their median OS was 14 months. Patients in the MWA group had a lower VAS score than those in the control group at the 48-h (1.75 ± 1.01 vs 2.47 ± 0.96, P = 0.01) and 1-month (1.79 ± 0.92 vs 2.53 ± 1.35, P = 0.048) check-ups. At the 3-month evaluation, the VAS score differences between the two groups were not significant (P = 0.133). After surgery, spinal cord function improvement was not significantly different between the MWA and control groups (P = 0.515). MWA therapy combined with open surgery was not associated with increased OS compared with the control group (P = 0.492). CONCLUSION: MWA can be an effective and safe pain-relief method but may not extend the OS of patients with lung cancer.

Clinical guidelines for microwave ablation of spinal metastases.

Spinal metastases are the most common source of morbidity in patients with cancer. Recently, microwave ablation has produced satisfactory results in the management of spinal metastases. However, there is still controversy in terms of clinical treatment, such as indication, power, time, and temperature. To standardize the application of microwave ablation technology and reduce the risk of surgical-related complications in spinal metastases, in this report, we aimed to summarize the current evidence and clinical experience of microwave ablation and developed a clinical guideline, initiated by the Musculoskeletal Tumor Group of the Committee for Minimally Invasive Therapy in Oncology of the Chinese Anti-Cancer Association. The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach was used in to rate the quality of evidence and the strength of recommendations, and the Reporting Items for Practice Guidelines in Healthcare (RIGHT) checklist was strictly followed to report the guideline. Finally, 15 evidence-based recommendations were formulated based on the 15 most concerned clinical questions among orthopedic surgeons, oncologists, and interventional radiologists in China. This guideline aims to promote the science-based normalization of microwave ablation for the treatment of spinal metastases.

Identification of GPC3 mutation and upregulation in a multidrug resistant osteosarcoma and its spheroids as therapeutic target.

BACKGROUND: Drug resistance and the lack of molecular therapeutic target are the main challenges in the management of osteosarcomas (OSs). Identification of novel genetic alteration(s) related with OS recurrence and chemotherapeutic resistance would be of scientific and clinical significance. METHODS: To identify potential genetic alterations related with OS recurrence and chemotherapeutic resistance, the biopsies of a 20-year-old male osteosarcoma patient were collected at primary site (p-OS) and from its metastatic tumor (m-OS) formed after 5 months of adjuvant chemotherapy. Both OS specimens were subjected to cancer-targeted next generation sequencing (NGS) and their cell suspensions were cultured under three-dimensional condition to establish spheroid therapeutic model. Transcript-oriented Sanger sequencing for GPC3, the detected mutated gene, was performed on RNA samples of p-OS and m-OS tissues and spheroids. The effects of anti-GPC3 antibody and its combination with cisplatin on m-OS spheroids were elucidated. RESULTS: NGS revealed 4 mutations (GPC3, SOX10, MDM4 and MAPK8) and 6 amplifications (MDM2, CDK4, CCND3, RUNX2, GLI1 and FRS2) in p-OS, and 3 mutations (GPC3, SOX10 and EGF) and 10 amplifications (CDK4, CCND3, MDM2, RUNX2, GLI1, FRS2, CARD11, RAC1, SLC16A7 and PMS2) in m-OS. Among those alterations, the mutation abundance of GPC3 was the highest (56.49%) in p-OS and showed 1.54 times increase in m-OS. GPC3 transcript-oriented Sanger sequencing confirmed the mutation at 1046 in Exon 4, and immunohistochemical staining showed increased GPC3 production in m-OS tissues and its spheroids. EdU cell proliferation and Calcein/PI cell viability assays revealed that of the anti-OS first line drugs (doxorubicin, cisplatin, methotrexate, ifosfamide and carboplatin), 10 µM carboplatin exerted the best inhibitory effects on the p-OS but not the m-OS spheroids. 2 µg/mL anti-GPC3 antibody effectively committed m-OS spheroids to death by itself (76.43%) or in combination with cisplatin (92.93%). CONCLUSION: This study demonstrates increased abundance and up-regulated expression of mutant GPC3 in metastatic osteosarcoma and its spheroids with multidrug resistance. As GPC3-targeting therapy has been used to treat hepatocellular carcinomas and it is also effective to OS PDSs, GPC3 would be a novel prognostic parameter and therapeutic target of osteosarcomas.

RPRD1A and RPRD1B are human RNA polymerase II C-terminal domain scaffolds for Ser5 dephosphorylation.

The RNA polymerase II (RNAPII) C-terminal domain (CTD) heptapeptide repeats (1-YSPTSPS-7) undergo dynamic phosphorylation and dephosphorylation during the transcription cycle to recruit factors that regulate transcription, RNA processing and chromatin modification. We show here that RPRD1A and RPRD1B form homodimers and heterodimers through their coiled-coil domains and interact preferentially via CTD-interaction domains (CIDs) with RNAPII CTD repeats phosphorylated at S2 and S7. Crystal structures of the RPRD1A, RPRD1B and RPRD2 CIDs, alone and in complex with RNAPII CTD phosphoisoforms, elucidate the molecular basis of CTD recognition. In an example of cross-talk between different CTD modifications, our data also indicate that RPRD1A and RPRD1B associate directly with RPAP2 phosphatase and, by interacting with CTD repeats where phospho-S2 and/or phospho-S7 bracket a phospho-S5 residue, serve as CTD scaffolds to coordinate the dephosphorylation of phospho-S5 by RPAP2.

Control of the RNA polymerase II phosphorylation state in promoter regions by CTD interaction domain-containing proteins RPRD1A and RPRD1B.

RNA polymerase II (RNAP II) C-terminal domain (CTD) phosphorylation is important for various transcription-related processes. Here, we identify by affinity purification and mass spectrometry three previously uncharacterized human CTD-interaction domain (CID)-containing proteins, RPRD1A, RPRD1B and RPRD2, which co-purify with RNAP II and three other RNAP II-associated proteins, RPAP2, GRINL1A and RECQL5, but not with the Mediator complex. RPRD1A and RPRD1B can accompany RNAP II from promoter regions to 3'-untranslated regions during transcription in vivo, predominantly interact with phosphorylated RNAP II, and can reduce CTD S5- and S7-phosphorylated RNAP II at target gene promoters. Thus, the RPRD proteins are likely to have multiple important roles in transcription.

A lentiviral functional proteomics approach identifies chromatin remodeling complexes important for the induction of pluripotency.

Protein complexes and protein-protein interactions are essential for almost all cellular processes. Here, we establish a mammalian affinity purification and lentiviral expression (MAPLE) system for characterizing the subunit compositions of protein complexes. The system is flexible (i.e. multiple N- and C-terminal tags and multiple promoters), is compatible with Gateway cloning, and incorporates a reference peptide. Its major advantage is that it permits efficient and stable delivery of affinity-tagged open reading frames into most mammalian cell types. We benchmarked MAPLE with a number of human protein complexes involved in transcription, including the RNA polymerase II-associated factor, negative elongation factor, positive transcription elongation factor b, SWI/SNF, and mixed lineage leukemia complexes. In addition, MAPLE was used to identify an interaction between the reprogramming factor Klf4 and the Swi/Snf chromatin remodeling complex in mouse embryonic stem cells. We show that the SWI/SNF catalytic subunit Smarca2/Brm is up-regulated during the process of induced pluripotency and demonstrate a role for the catalytic subunits of the SWI/SNF complex during somatic cell reprogramming. Our data suggest that the transcription factor Klf4 facilitates chromatin remodeling during reprogramming.

Organization and function of APT, a subcomplex of the yeast cleavage and polyadenylation factor involved in the formation of mRNA and small nucleolar RNA 3'-ends.

Messenger RNA 3'-end formation is functionally coupled to transcription by RNA polymerase II. By tagging and purifying Ref2, a non-essential protein previously implicated in mRNA cleavage and termination, we isolated a multiprotein complex, holo-CPF, containing the yeast cleavage and polyadenylation factor (CPF) and six additional polypeptides. The latter can form a distinct complex, APT, in which Pti1, Swd2, a type I protein phosphatase (Glc7), Ssu72 (a TFIIB and RNA polymerase II-associated factor), Ref2, and Syc1 are associated with the Pta1 subunit of CPF. Systematic tagging and purification of holo-CPF subunits revealed that yeast extracts contain similar amounts of CPF and holo-CPF. By purifying holo-CPF from strains lacking Ref2 or containing truncated subunits, subcomplexes were isolated that revealed additional aspects of the architecture of APT and holo-CPF. Chromatin immunoprecipitation was used to localize Ref2, Ssu72, Pta1, and other APT subunits on small nucleolar RNA (snoRNA) genes and primarily near the polyadenylation signals of the constitutively expressed PYK1 and PMA1 genes. Use of mutant components of APT revealed that Ssu72 is important for preventing readthrough-dependent expression of downstream genes for both snoRNAs and polyadenylated transcripts. Ref2 and Pta1 similarly affect at least one snoRNA transcript.

RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach.

To physically characterize the web of interactions connecting the Saccharomyces cerevisiae proteins suspected to be RNA polymerase II (RNAPII) elongation factors, subunits of Spt4/Spt5 and Spt16/Pob3 (corresponding to human DSIF and FACT), Spt6, TFIIF (Tfg1, -2, and -3), TFIIS, Rtf1, and Elongator (Elp1, -2, -3, -4, -5, and -6) were affinity purified under conditions designed to minimize loss of associated polypeptides and then identified by mass spectrometry. Spt16/Pob3 was discovered to associate with three distinct complexes: histones; Chd1/casein kinase II (CKII); and Rtf1, Paf1, Ctr9, Cdc73, and a previously uncharacterized protein, Leo1. Rtf1 and Chd1 have previously been implicated in the control of elongation, and the sensitivity to 6-azauracil of strains lacking Paf1, Cdc73, or Leo1 suggested that these proteins are involved in elongation by RNAPII as well. Confirmation came from chromatin immunoprecipitation (ChIP) assays demonstrating that all components of this complex, including Leo1, cross-linked to the promoter, coding region, and 3' end of the ADH1 gene. In contrast, the three subunits of TFIIF cross-linked only to the promoter-containing fragment of ADH1. Spt6 interacted with the uncharacterized, essential protein Iws1 (interacts with Spt6), and Spt5 interacted either with Spt4 or with a truncated form of Spt6. ChIP on Spt6 and the novel protein Iws1 resulted in the cross-linking of both proteins to all three regions of the ADH1 gene, suggesting that Iws1 is likely an Spt6-interacting elongation factor. Spt5, Spt6, and Iws1 are phosphorylated on consensus CKII sites in vivo, conceivably by the Chd1/CKII associated with Spt16/Pob3. All the elongation factors but Elongator copurified with RNAPII.