Meet the authors: The Ji lab.

We talk to the Ji lab about their paper, "RNA Pol II preferentially regulates ribosomal protein expression by trapping disassociated subunits" (in this issue), lessons from their scientific journey so far, and what inspires them along their scientific paths.

RNA Pol II preferentially regulates ribosomal protein expression by trapping disassociated subunits.

RNA polymerase II (RNA Pol II) has been recognized as a passively regulated multi-subunit holoenzyme. However, the extent to which RNA Pol II subunits might be important beyond the RNA Pol II complex remains unclear. Here, fractions containing disassociated RPB3 (dRPB3) were identified by size exclusion chromatography in various cells. Through a unique strategy, i.e., "specific degradation of disassociated subunits (SDDS)," we demonstrated that dRPB3 functions as a regulatory component of RNA Pol II to enable the preferential control of 3' end processing of ribosomal protein genes directly through its N-terminal domain. Machine learning analysis of large-scale genomic features revealed that the little elongation complex (LEC) helps to specialize the functions of dRPB3. Mechanistically, dRPB3 facilitates CBC-PCF11 axis activity to increase the efficiency of 3' end processing. Furthermore, RPB3 is dynamically regulated during development and diseases. These findings suggest that RNA Pol II gains specific regulatory functions by trapping disassociated subunits in mammalian cells.

Targeted protein degradation reveals RNA Pol II heterogeneity and functional diversity.

RNA polymerase II (RNA Pol II) subunits are thought to be involved in various transcription-associated processes, but it is unclear whether they play different regulatory roles in modulating gene expression. Here, we performed nascent and mature transcript sequencing after the acute degradation of 12 mammalian RNA Pol II subunits and profiled their genomic binding sites and protein interactomes to dissect their molecular functions. We found that RNA Pol II subunits contribute differently to RNA Pol II cellular localization and transcription processes and preferentially regulate RNA processing (such as RNA splicing and 3' end maturation). Genes sensitive to the depletion of different RNA Pol II subunits tend to be involved in diverse biological functions and show different RNA half-lives. Sequences, associated protein factors, and RNA structures are correlated with RNA Pol II subunit-mediated differential gene expression. These findings collectively suggest that the heterogeneity of RNA Pol II and different genes appear to depend on some of the subunits.

Peptide-Functionalized Nanoinhibitor Restrains Brain Tumor Growth by Abrogating Mesenchymal-Epithelial Transition Factor (MET) Signaling.

Malignant gliomas are the most common primary brain tumors and are associated with aggressive growth, high morbidity, and mortality. Aberrant mesenchymal-epithelial transition factor (MET) activation occurs in approximately 30% of glioma patients and correlates with poor prognosis, elevated invasion, and increased drug resistance. Therefore, MET has emerged as an attractive target for glioma therapy. In this study, we developed a novel nanoinhibitor by conjugating MET-targeting cMBP peptides on the G4 dendrimer. Compared to the binding affinity of the free peptide ( KD = 3.96 × 10-7 M), the binding affinity of the nanoinhibitor to MET increased 3 orders of magnitude to 1.32 × 10-10 M. This nanoinhibitor efficiently reduced the proliferation and invasion of human glioblastoma U87MG cells in vitro by blocking MET signaling with remarkably attenuated levels of phosphorylated MET ( pMET) and its downstream signaling proteins, such as pAKT and pERK1/2. Although no obvious therapeutic effect was observed after treatment with free cBMP peptide, in vivo T2-weighted magnetic resonance imaging (MRI) showed a significant delay in tumor growth after intravenous injection of the nanoinhibitor. The medium survival in mouse models was extended by 59%, which is similar to the effects of PF-04217903, a small molecule MET inhibitor currently in clinical trials. Immunoblotting studies of tumor homogenate verified that the nanoinhibitor restrained glioma growth by blocking MET downstream signaling. pMET and its downstream proteins pAKT and pERK1/2, which are involved in the survival and invasion of cancer cells, decreased in the nanoinhibitor-treated group by 44.2%, 62.2%, and 32.3%, respectively, compared with those in the control group. In summary, we developed a peptide-functionalized MET nanoinhibitor that showed extremely high binding affinity to MET and effectively inhibited glioma growth by blocking MET downstream signaling. To the best of our knowledge, this is the first report of therapeutic inhibition of glioma growth by blocking MET signaling with a novel nanoinhibitor. Compared to antibodies and chemical inhibitors in clinical trials, the nanoinhibitor blocks MET signaling and provides a new approach for the treatment of glioma with the advantages of high efficiency, affordability, and, most importantly, potentially reduced drug resistance.

A Ratiometric SERS Probe for Imaging the Macrophage Phenotypes in Live Mice with Epilepsy and Brain Tumor.

Macrophage performs multiple functions such as pathogen phagocytosis, antigen presentation, and tissue remodeling by polarizing toward a spectrum of phenotypes. Dynamic imaging of macrophage phenotypes is critical for evaluating disease progression and the therapeutic response of drug candidates. However, current technologies cannot identify macrophage phenotypes in vivo. Herein, a surface-enhanced Raman scattering nanoprobe, AH1, which enables the accurate determination of physiological pH with high sensitivity and tissue penetration depth through ratiometric Raman signals is developed. Due to the phenotype-dependent metabolic reprogramming, AH1 can effectively identify macrophage subpopulations by measuring the acidity levels in phagosomes. After intravenous administration, AH1 not only visualizes the spatial distribution of macrophage phenotypes in brain tumors and epileptic regions of mouse models, but also reveals the repolarization of macrophages in brain lesions after drug intervention. This work provides a new tool for dynamically monitoring the disease-associated immune microenvironment and evaluating the efficacy of immune-therapeutics in vivo.

Guiding Epilepsy Surgery with an LRP1-Targeted SPECT/SERRS Dual-Mode Imaging Probe.

Accurate identification of the resectable epileptic lesion is a precondition of operative intervention to drug-resistant epilepsy (DRE) patients. However, even when multiple diagnostic modalities are combined, epileptic foci cannot be accurately identified in ∼30% of DRE patients. Inflammation-associated low-density lipoprotein receptor-related protein-1 (LRP1) has been validated to be a surrogate target for imaging epileptic foci. Here, we reported an LRP1-targeted dual-mode probe that is capable of providing comprehensive epilepsy information preoperatively with SPECT imaging while intraoperatively delineating epileptic margins in a sensitive high-contrast manner with surface-enhanced resonance Raman scattering (SERRS) imaging. Notably, a novel and universal strategy for constructing self-assembled monolayer (SAM)-based Raman reporters was proposed for boosting the sensitivity, stability, reproducibility, and quantifiability of the SERRS signal. The probe showed high efficacy to penetrate the blood-brain barrier. SPECT imaging showed the probe could delineate the epileptic foci clearly with a high target-to-background ratio (4.11 ± 0.71, 2 h). Further, with the assistance of the probe, attenuated seizure frequency in the epileptic mouse models was achieved by using SPECT together with Raman images before and during operation, respectively. Overall, this work highlights a new strategy to develop a SPECT/SERRS dual-mode probe for comprehensive epilepsy surgery that can overcome the brain shift by the co-registration of preoperative SPECT and SERRS intraoperative images.

Emergence of Responsive Surface-Enhanced Raman Scattering Probes for Imaging Tumor-Associated Metabolites.

As a core hallmark of cancer, metabolic reprogramming alters the metabolic networks of cancer cells to meet their insatiable appetite for energy and nutrient. Tumor-associated metabolites, the products of metabolic reprogramming, are valuable in evaluating tumor occurrence and progress timely and accurately because their concentration variations usually happen earlier than the aberrances demonstrated in tissue structure and function. As an optical spectroscopic technique, surface-enhanced Raman scattering (SERS) offers advantages in imaging tumor-associated metabolites, including ultrahigh sensitivity, high specificity, multiplexing capacity, and uncompromised signal intensity. This review first highlights recent advances in the development of stimuli-responsive SERS probes. Then the mechanisms leading to the responsive SERS signal triggered by tumor metabolites are summarized. Furthermore, biomedical applications of these responsive SERS probes, such as the image-guided tumor surgery and liquid biopsy examination for tumor molecular typing, are summarized. Finally, the challenges and prospects of the responsive SERS probes for clinical translation are also discussed.

Direct injection ultra-performance liquid chromatography-tandem mass spectrometry for the high-throughput determination of 11 illicit drugs and metabolites in wastewater.

The measurement of illicit drugs in wastewater is increasingly being adopted as a method for objective monitoring of population-level illicit drug use. This work describes the first small-volume direct-injection ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the simultaneous determination of 11 illicit drugs and metabolites in wastewater. The method required an injection volume of only 30 µL of wastewater sample, the limits of detection (LOD) ranged from 0.4 ng/L to 2 ng/L and the lower limits of quantitation (LLOQ) ranged from 1 ng/L to 5 ng/L. Application of the method to real wastewater samples collected from wastewater treatment plants revealed morphine in all samples, together with other illicit drugs (methamphetamine, codeine, ketamine, and nor-ketamine) in some samples. Small-volume direct injection showed great potential as an efficient method for the high-throughput determination of illicit drugs in wastewater.

Intelligent SERS Navigation System Guiding Brain Tumor Surgery by Intraoperatively Delineating the Metabolic Acidosis.

Surgeons face challenges in intraoperatively defining margin of brain tumors due to its infiltrative nature. Extracellular acidosis caused by metabolic reprogramming of cancer cells is a reliable marker for tumor infiltrative regions. Although the acidic margin-guided surgery shows promise in improving surgical prognosis, its clinical transition is delayed by having the exogenous probes approved by the drug supervision authority. Here, an intelligent surface-enhanced Raman scattering (SERS) navigation system delineating glioma acidic margins without administration of exogenous probes is reported. With assistance of this system, the metabolites at the tumor cutting edges can be nondestructively transferred within a water droplet to a SERS chip with pH sensitivity. Homemade deep learning model automatically processes the Raman spectra collected from the SERS chip and delineates the pH map of tumor resection bed with increased speed. Acidity correlated cancer cell density and proliferation level are demonstrated in tumor cutting edges of animal models and excised tissues from glioma patients. The overall survival of animal models post the SERS system guided surgery is significantly increased in comparison to the conventional strategy used in clinical practice. This SERS system holds the promise in accelerating clinical transition of acidic margin-guided surgery for solid tumors with infiltrative nature.

CTCF DNA-binding domain undergoes dynamic and selective protein-protein interactions.

CTCF is a predominant insulator protein required for three-dimensional chromatin organization. However, the roles of its insulation of enhancers in a 3D nuclear organization have not been fully explained. Here, we found that the CTCF DNA-binding domain (DBD) forms dynamic self-interacting clusters. Strikingly, CTCF DBD clusters were found to incorporate other insulator proteins but are not coenriched with transcriptional activators in the nucleus. This property is not observed in other domains of CTCF or the DBDs of other transcription factors. Moreover, endogenous CTCF shows a phenotype consistent with the DBD by forming small protein clusters and interacting with CTCF motif arrays that have fewer transcriptional activators bound. Our results reveal an interesting phenomenon in which CTCF DBD interacts with insulator proteins and selectively localizes to nuclear positions with lower concentrations of transcriptional activators, providing insights into the insulation function of CTCF.

Imaging epileptic foci in mouse models via a low-density lipoprotein receptor-related protein-1 targeting strategy.

BACKGROUND: In the setting of drug-resistant epilepsy (DRE), the success of surgery depends on the ability to accurately locate the epileptic foci to be resected or disconnected. However, the epileptic foci in a considerable percentage of the DRE patients cannot be adequately localised. This warrants the need for a reliable imaging strategy to identify the "concealed" epileptic regions. METHODS: Brain specimens from DRE patients and kainate-induced epileptic mouse models were immuno-stained to evaluate the integrity of the blood-brain barrier (BBB). The expression of low-density lipoprotein receptor-related protein-1 (LRP1) in the epileptic region of DRE patients and kainate models was studied by immunofluorescence. A micellar-based LRP1-targeted paramagnetic probe (Gd3+-LP) was developed and its ability to define the epileptic foci was investigated by magnetic resonance imaging (MRI). FINDINGS: The integrity of the BBB in the epileptic region of DRE patients and kainate mouse models were demonstrated. LRP1 expression levels in the epileptic foci of DRE patients and kainate models were 1.70-2.38 and 2.32-3.97 folds higher than in the control brain tissues, respectively. In vivo MRI demonstrated that Gd3+-LP offered 1.68 times higher (P < 0.05) T1-weighted intensity enhancement in the ipsilateral hippocampus of chronic kainite models than the control probe without LRP1 specificity. INTERPRETATION: The expression of LRP1 is up-regulated in vascular endothelium, activated glia in both DRE patients and kainate models. LRP1-targeted imaging strategy may provide an alternative strategy to define the "concealed" epileptic foci by overcoming the intact BBB. FUNDING: This work was supported by the National Natural Science Foundation, Shanghai Science and Technology Committee, Shanghai Municipal Science and Technology, Shanghai Municipal Health and Family Planning Commission and the National Postdoctoral Program for Innovative Talents.

Genome-wide analyses of chromatin interactions after the loss of Pol I, Pol II, and Pol III.

BACKGROUND: The relationship between transcription and the 3D chromatin structure is debated. Multiple studies have shown that transcription affects global Cohesin binding and 3D genome structures. However, several other studies have indicated that inhibited transcription does not alter chromatin conformations. RESULTS: We provide the most comprehensive evidence to date to demonstrate that transcription plays a relatively modest role in organizing the local, small-scale chromatin structures in mammalian cells. We show degraded Pol I, Pol II, and Pol III proteins in mESCs cause few or no changes in large-scale 3D chromatin structures, selected RNA polymerases with a high abundance of binding sites or active promoter-associated interactions appear to be relatively more affected after the degradation, transcription inhibition alters local, small loop domains, as indicated by high-resolution chromatin interaction maps, and loops with bound Pol II but without Cohesin or CTCF are identified and found to be largely unchanged after transcription inhibition. Interestingly, Pol II depletion for a longer time significantly affects the chromatin accessibility and Cohesin occupancy, suggesting that RNA polymerases are capable of affecting the 3D genome indirectly. These direct and indirect effects explain the previous inconsistent findings on the influence of transcription inhibition on the 3D genome. CONCLUSIONS: We conclude that Pol I, Pol II, and Pol III loss alters local, small-scale chromatin interactions in mammalian cells, suggesting that the 3D chromatin structures are pre-established and relatively stable.

A pH ratiometrically responsive surface enhanced resonance Raman scattering probe for tumor acidic margin delineation and image-guided surgery.

Surgery remains the mainstay for most solid tumor treatments. However, surgeons face challenges in intra-operatively identifying invasive tumor margins due to their infiltrative nature. Incomplete excision usually leads to early recurrence, while aggressive resection may injure adjacent functional tissues. Herein, we report a pH responsive ratiometric surface-enhanced Raman scattering (SERRS) probe that determined physiological pHs with a high sensitivity and tissue penetration depth via an innovative mechanism named spatial orientation induced intramolecular energy transfer (SOIET). Due to the positive correlation between tumor acidity and malignancy, an acidic margin-guided surgery strategy was implemented in live animal models by intra-operatively assessing tissue pH/malignancy of the suspicious tissues in tumor cutting edges. This surgery remarkably extended the survival of animal models and minimized their post-surgical complications, showing promise in precisely identifying invasive tumor boundaries and achieving a balance between maximum tumor debulking and minimal functional impairment.

Surface-Enhanced Resonance Raman Scattering-Guided Brain Tumor Surgery Showing Prognostic Benefit in Rat Models.

Glioma is the most frequent form of malignant brain tumors. Surgical debulking is a major strategy for glioma treatment. However, there is a great challenge for the neurosurgeons to intraoperatively identify the true margins of glioma because of its infiltrative nature. Tumor residues or microscopic satellite foci left in the resection bed are the main reasons leading to early recurrence as well as poor prognosis. In this study, a surface-enhanced resonance Raman scattering (SERRS) probe was developed to intraoperatively guide glioma resection. In this probe, molecular reporters with absorptive maxima at the near-infrared wavelength range were covalently functionalized on the surface of gold nanostars. This SERRS probe demonstrated an ultrahigh sensitivity with a detection limit of 5.0 pM in aqueous solution. By the development of glioma xenografts in a mouse dorsal skin window chamber, extravasation of this probe from leaky tumor vasculature as functions of time and distance to tumor boundary was investigated. Importantly, the invasive margin of the tumor xenograft was demarcated by this probe with a high signal-to-background ratio. Preoperative magnetic resonance imaging (MRI) first defined the position of orthotopic glioma xenografts in the brain of rat models, and the craniotomy plan was designed. The brain tumor was then excised intraoperatively step-by-step with the assistance of a handheld Raman scanner till the Raman signals of the probe completely disappeared in the resection bed. Notably, longitudinal MRI showed that SERRS-guided surgery significantly reduced the tumor recurrence rate and improved the overall survival of rat models compared with the white light-guided surgery. Overall, this work demonstrates the prognostic benefit of SERRS-guided glioma surgery in animal models. Because delineation of tumor-invasive margins is a common challenge faced by the surgeons, this SERRS probe with a picomolar detection limit holds the promise in improving the surgical outcome of different types of infiltrated tumors.