hnRNPM protects against the dsRNA-mediated interferon response by repressing LINE-associated cryptic splicing.

RNA splicing is pivotal in post-transcriptional gene regulation, yet the exponential expansion of intron length in humans poses a challenge for accurate splicing. Here, we identify hnRNPM as an essential RNA-binding protein that suppresses cryptic splicing through binding to deep introns, maintaining human transcriptome integrity. Long interspersed nuclear elements (LINEs) in introns harbor numerous pseudo splice sites. hnRNPM preferentially binds at intronic LINEs to repress pseudo splice site usage for cryptic splicing. Remarkably, cryptic exons can generate long dsRNAs through base-pairing of inverted ALU transposable elements interspersed among LINEs and consequently trigger an interferon response, a well-known antiviral defense mechanism. Significantly, hnRNPM-deficient tumors show upregulated interferon-associated pathways and elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity by repressing cryptic splicing and suggest that targeting hnRNPM in tumors may be used to trigger an inflammatory immune response, thereby boosting cancer surveillance.

Human platelet lysate enhances in vivo activity of CAR-Vδ2 T cells by reducing cellular senescence and apoptosis.

BACKGROUND AIMS: Vγ9Vδ2 T cells are an attractive cell platform for the off-the-shelf cancer immunotherapy as the result of their lack of alloreactivity and inherent multi-pronged cytotoxicity, which could be further amplified with chimeric antigen receptors (CARs). In this study, we sought to enhance the in vivo longevity of CAR-Vδ2 T cells by modulating ex vivo manufacturing conditions and selecting an optimal CAR costimulatory domain. METHODS: Specifically, we compared the anti-tumor activity of Vδ2 T cells expressing anti-CD19 CARs with costimulatory endodomains derived from CD28, 4-1BB or CD27 and generated in either standard fetal bovine serum (FBS)- or human platelet lysate (HPL)-supplemented medium. RESULTS: We found that HPL supported greater expansion of CAR-Vδ2 T cells with comparable in vitro cytotoxicity and cytokine secretion to FBS-expanded CAR-Vδ2 T cells. HPL-expanded CAR-Vδ2 T cells showed enhanced in vivo anti-tumor activity with longer T-cell persistence compared with FBS counterparts, with 4-1BB costimulated CAR showing the greatest activity. Mechanistically, HPL-expanded CAR Vδ2 T cells exhibited reduced apoptosis and senescence transcriptional pathways compared to FBS-expanded CAR-Vδ2 T cells and increased telomerase activity. CONCLUSIONS: This study supports enhancement of therapeutic potency of CAR-Vδ2 T cells through a manufacturing improvement.

Synergistic monitoring of PM2.5 and CO2 based on active and passive remote sensing fusion during the 2022 Beijing Winter Olympics.

Green and low-carbon are the keywords of the 2022 Beijing Winter Olympic Games (WOG) and the core of sustainable development. Beijing's P M 2.5 and C O 2 emissions attracted worldwide attention during WOG. However, the complex emission sources and frequently changing weather patterns make it impossible for a single monitoring approach to meet the high-resolution, full-coverage monitoring requirements. Therefore, we proposed an active-passive remote sensing fusion method to address this issue. The haze layer height (HLH) was first retrieved from vertical aerosol profiles measured by our high-spectral-resolution lidar located near Olympic venues, which provides new insights into the nonuniform boundary layer and the residual aerosol aloft above it. Second, we developed a bootstrap aggregating (bagging) method that assimilates the lidar-based HLH, satellite-based AOD, and meteorological data to estimate the hourly P M 2.5 with 1 km resolution. The P M 2.5 at Beijing region, Bird's Nest, and Yanqing venues during WOG was 23.00±18.33, 22.91±19.48, and 16.33±10.49µg/m 3, respectively. Third, we also derived the C O 2 enhancements, C O 2 spatial gradients resulting from human activities, and annual growth rate (AGR) to estimate the performance of carbon emission management in Beijing. Based on the top-down method, the results showed an average C O 2 enhancement of 1.62 ppm with an annual decline rate of 2.92 ppm. Finally, we compared the monitoring data with six other international cities. The results demonstrated that Beijing has the largest P M 2.5 annual decline rate of 7.43µg/m 3, while the C O 2 AGR is 1.46 ppm and keeps rising, indicating Beijing is still on its way to carbon peaking and needs to strive for carbon neutrality.

Twenty years of EMT: A state of the field from TEMTIA X.

TEMTIA X, the tenth symposium organized by the EMT international Association (TEMTIA) took place in Paris on November 7th-10th, 2022. Similarly to the previous meetings, it reviewed most recent aspects of the epithelial-mesenchymal transition, a cellular process involved during distinct stages of development, but also during wound healing and fibrosis to some level. EMT steps are likewise typically described with various extents during tumor cell progression and metastasis. The meeting emphasized the intermediate stages involved in the process and their potential physiological or pathological importance, taking advantage of the expansion of molecular methods at single cell level. It also introduced new descriptions of EMT occurrences during early embryogenesis. In addition, sessions explored how EMT reflects cell metabolism and how the process can mingle with immune response, particularly during tumor progression, providing new targets, that were discussed, among others, for cancer therapy. Finally, it introduced a new perception of EMT biological meaning based on an evolutionary perspective. The meeting integrated the TEMTIA general assembly , allowing general discussion about the future of the association, starting with the site of the next meeting, now decided to take place in Seattle (US), late 2024.

LINE-associated cryptic splicing induces dsRNA-mediated interferon response and tumor immunity.

RNA splicing plays a critical role in post-transcriptional gene regulation. Exponential expansion of intron length poses a challenge for accurate splicing. Little is known about how cells prevent inadvertent and often deleterious expression of intronic elements due to cryptic splicing. In this study, we identify hnRNPM as an essential RNA binding protein that suppresses cryptic splicing through binding to deep introns, preserving transcriptome integrity. Long interspersed nuclear elements (LINEs) harbor large amounts of pseudo splice sites in introns. hnRNPM preferentially binds at intronic LINEs and represses LINE-containing pseudo splice site usage for cryptic splicing. Remarkably, a subgroup of the cryptic exons can form long dsRNAs through base-pairing of inverted Alu transposable elements scattered in between LINEs and trigger interferon immune response, a well-known antiviral defense mechanism. Notably, these interferon-associated pathways are found to be upregulated in hnRNPM-deficient tumors, which also exhibit elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity. Targeting hnRNPM in tumors may be used to trigger an inflammatory immune response thereby boosting cancer surveillance.

Feasibility and preclinical efficacy of CD7-unedited CD7 CAR T cells for T cell malignancies.

Chimeric antigen receptor (CAR)-mediated targeting of T lineage antigens for the therapy of blood malignancies is frequently complicated by self-targeting of CAR T cells or their excessive differentiation driven by constant CAR signaling. Expression of CARs targeting CD7, a pan-T cell antigen highly expressed in T cell malignancies and some myeloid leukemias, produces robust fratricide and often requires additional mitigation strategies, such as CD7 gene editing. In this study, we show fratricide of CD7 CAR T cells can be fully prevented using ibrutinib and dasatinib, the pharmacologic inhibitors of key CAR/CD3ζ signaling kinases. Supplementation with ibrutinib and dasatinib rescued the ex vivo expansion of unedited CD7 CAR T cells and allowed regaining full CAR-mediated cytotoxicity in vitro and in vivo on withdrawal of the inhibitors. The unedited CD7 CAR T cells persisted long term and mediated sustained anti-leukemic activity in two mouse xenograft models of human T cell acute lymphoblastic leukemia (T-ALL) by self-selecting for CD7-, fratricide-resistant CD7 CAR T cells that were transcriptionally similar to control CD7-edited CD7 CAR T cells. Finally, we showed feasibility of cGMP manufacturing of unedited autologous CD7 CAR T cells for patients with CD7+ malignancies and initiated a phase I clinical trial (ClinicalTrials.gov: NCT03690011) using this approach. These results indicate pharmacologic inhibition of CAR signaling enables generating functional CD7 CAR T cells without additional engineering.

Alternatively spliced CSF3R isoforms in SRSF2 P95H mutated myeloid neoplasms.

Alternatively spliced colony stimulating factor 3 receptor (CSF3R) isoforms Class III and Class IV are observed in myelodysplastic syndromes (MDS), but their roles in disease remain unclear. We report that the MDS-associated splicing factor SRSF2 affects the expression of Class III and Class IV isoforms and perturbs granulopoiesis. Add-back of the Class IV isoform in Csf3r-null mouse progenitor cells increased granulocyte progenitors with impaired neutrophil differentiation, while add-back of the Class III produced dysmorphic neutrophils in fewer numbers. These CSF3R isoforms were elevated in patients with myeloid neoplasms harboring SRSF2 mutations. Using in vitro splicing assays, we confirmed increased Class III and Class IV transcripts when SRSF2 P95 mutations were co-expressed with the CSF3R minigene in K562 cells. Since SRSF2 regulates splicing partly by recognizing exonic splicing enhancer (ESE) sequences on pre-mRNA, deletion of either ESE motifs within CSF3R exon 17 decreased Class IV transcript levels without affecting Class III. CD34+ cells expressing SRSF2 P95H showed impaired neutrophil differentiation in response to G-CSF and was accompanied by increased levels of Class IV. Our findings suggest that SRSF2 P95H promotes Class IV splicing by binding to key ESE sequences in CSF3R exon 17, and that SRSF2, when mutated, contributes to dysgranulopoiesis.

Optical-feedback cavity ring-down spectroscopy for NO2 extinction coefficient measurement using a continuous wave laser diode.

Optical-feedback (OF) cavity ring-down spectroscopy consisting of a linear cavity is developed by employing a continuous wave laser diode (LD) with multi-longitudinal modes. Due to the OF effect caused by the cavity output laser back into the LD, the laser frequency is locked, and the intracavity laser intensity is enhanced. We use different concentrations of NO2 gases to test the apparatus, and the results show good agreement with theoretical values. Owing to the compactness of the laser source and high detection accuracy, the device can be used for detection of low-concentration absorbent gases in the environmental monitoring field.

Regulation of Alternative Splicing during Epithelial-Mesenchymal Transition.

Alternative splicing is an essential mechanism of gene regulation, giving rise to remarkable protein diversity in higher eukaryotes. Epithelial-mesenchymal transition (EMT) is a developmental process that plays an essential role in metazoan embryogenesis. Recent studies have revealed that alternative splicing serves as a fundamental layer of regulation that governs cells to undergo EMT. In this review, we summarize recent findings on the functional impact of alternative splicing in EMT and EMT-associated activities. We then discuss the regulatory mechanisms that control alternative splicing changes during EMT.

Methods for Characterization of Alternative RNA Splicing.

Quantification of alternative splicing to detect the abundance of differentially spliced isoforms of a gene in total RNA can be accomplished via RT-PCR using both quantitative real-time and semiquantitative PCR methods. These methods require careful PCR primer design to ensure specific detection of particular splice isoforms. We will also describe analysis of alternative splicing using a splicing "minigene" in mammalian cell tissue culture to facilitate investigation of the regulation of alternative splicing of a particular exon of interest.

The bone microenvironment invigorates metastatic seeds for further dissemination.

Metastasis has been considered as the terminal step of tumor progression. However, recent genomic studies suggest that many metastases are initiated by further spread of other metastases. Nevertheless, the corresponding pre-clinical models are lacking, and underlying mechanisms are elusive. Using several approaches, including parabiosis and an evolving barcode system, we demonstrated that the bone microenvironment facilitates breast and prostate cancer cells to further metastasize and establish multi-organ secondary metastases. We uncovered that this metastasis-promoting effect is driven by epigenetic reprogramming that confers stem cell-like properties on cancer cells disseminated from bone lesions. Furthermore, we discovered that enhanced EZH2 activity mediates the increased stemness and metastasis capacity. The same findings also apply to single cell-derived populations, indicating mechanisms distinct from clonal selection. Taken together, our work revealed an unappreciated role of the bone microenvironment in metastasis evolution and elucidated an epigenomic reprogramming process driving terminal-stage, multi-organ metastases.

RNA editing in cancer impacts mRNA abundance in immune response pathways.

BACKGROUND: RNA editing generates modifications to the RNA sequences, thereby increasing protein diversity and shaping various layers of gene regulation. Recent studies have revealed global shifts in editing levels across many cancer types, as well as a few specific mechanisms implicating individual sites in tumorigenesis or metastasis. However, most tumor-associated sites, predominantly in noncoding regions, have unknown functional relevance. RESULTS: Here, we carry out integrative analysis of RNA editing profiles between epithelial and mesenchymal tumors, since epithelial-mesenchymal transition is a key paradigm for metastasis. We identify distinct editing patterns between epithelial and mesenchymal tumors in seven cancer types using TCGA data, an observation further supported by single-cell RNA sequencing data and ADAR perturbation experiments in cell culture. Through computational analyses and experimental validations, we show that differential editing sites between epithelial and mesenchymal phenotypes function by regulating mRNA abundance of their respective genes. Our analysis of RNA-binding proteins reveals ILF3 as a potential regulator of this process, supported by experimental validations. Consistent with the known roles of ILF3 in immune response, epithelial-mesenchymal differential editing sites are enriched in genes involved in immune and viral processes. The strongest target of editing-dependent ILF3 regulation is the transcript encoding PKR, a crucial player in immune and viral response. CONCLUSIONS: Our study reports widespread differences in RNA editing between epithelial and mesenchymal tumors and a novel mechanism of editing-dependent regulation of mRNA abundance. It reveals the broad impact of RNA editing in cancer and its relevance to cancer-related immune pathways.

A combinatorially regulated RNA splicing signature predicts breast cancer EMT states and patient survival.

During breast cancer metastasis, the developmental process epithelial-mesenchymal transition (EMT) is abnormally activated. Transcriptional regulatory networks controlling EMT are well-studied; however, alternative RNA splicing also plays a critical regulatory role during this process. A comprehensive understanding of alternative splicing (AS) and the RNA binding proteins (RBPs) that regulate it during EMT and their impact on breast cancer remains largely unknown. In this study, we annotated AS in the breast cancer TCGA data set and identified an AS signature that is capable of distinguishing epithelial and mesenchymal states of the tumors. This AS signature contains 25 AS events, among which nine showed increased exon inclusion and 16 showed exon skipping during EMT. This AS signature accurately assigns the EMT status of cells in the CCLE data set and robustly predicts patient survival. We further developed an effective computational method using bipartite networks to identify RBP-AS networks during EMT. This network analysis revealed the complexity of RBP regulation and nominated previously unknown RBPs that regulate EMT-associated AS events. This study highlights the importance of global AS regulation during EMT in cancer progression and paves the way for further investigation into RNA regulation in EMT and metastasis.

The RNA-binding protein AKAP8 suppresses tumor metastasis by antagonizing EMT-associated alternative splicing.

Alternative splicing has been shown to causally contribute to the epithelial-mesenchymal transition (EMT) and tumor metastasis. However, the scope of splicing factors that govern alternative splicing in these processes remains largely unexplored. Here we report the identification of A-Kinase Anchor Protein (AKAP8) as a splicing regulatory factor that impedes EMT and breast cancer metastasis. AKAP8 not only is capable of inhibiting splicing activity of the EMT-promoting splicing regulator hnRNPM through protein-protein interaction, it also directly binds to RNA and alters splicing outcomes. Genome-wide analysis shows that AKAP8 promotes an epithelial cell state splicing program. Experimental manipulation of an AKAP8 splicing target CLSTN1 revealed that splice isoform switching of CLSTN1 is crucial for EMT. Moreover, AKAP8 expression and the alternative splicing of CLSTN1 predict breast cancer patient survival. Together, our work demonstrates the essentiality of RNA metabolism that impinges on metastatic breast cancer.

Performance estimation of space-borne high-spectral-resolution lidar for cloud and aerosol optical properties at 532 nm.

Cloud and aerosol contribute with great uncertainty in Earth's radiative budget. There is an urgent need for global 3-D observation of these atmospheric constituents. High-spectral-resolution Lidar (HSRL) can obtain vertical atmosphere profile with high accuracy, hence several space-borne HSRLs are planned to launch in few years. However, as far as we know, the performance evaluation of space-borne HSRL has not been reported yet. In this paper, we present the characteristics of a new designed space-borne HSRL for aerosol and cloud optical property profiling (ACHSRL), which is part of the Aerosol & Carbon Detection Lidar (ACDL) developed in China. The ACHSRL is essentially similar to the famous Cloud-Aerosol Lidar with Orthogonal Polarization (CALIOP), which is on board the Cloud-Aerosol Lidar and Infrared Pathfinder Satellite Observation (CALIPSO). Moreover, the ACHSRL employs an iodine absorption filter as the spectral discriminator. The atmospheric optical properties data observed by CALIOP is used to estimate the performance of ACHSRL. We chose the level 2 profile data (version 4.10) in South Japan in June 2015 to compare the detection uncertainty of ACHSRL and CALIOP. The simulation calculates the uncertainties of ACHSRL and makes a statistic analysis. The analysis result demonstrates that 73.63% of the backscatter coefficient uncertainties are below 40% for ACHSRL. By contrast, the number is 30.72% for CALIOP. As for absolute extinction coefficient errors, the statistics shows that 76.01% of the extinction coefficient uncertainties are lower than 0.2 km-1 for ACHSRL, while that for CALIOP are 56.97%. The assessment shows that ACHSRL could measure the particulate optical properties with better accuracy and compared with CALIOP. The estimation in this study reveals that the next generation space-borne HSRLs have a promising future.

A high-throughput screen identifies small molecule modulators of alternative splicing by targeting RNA G-quadruplexes.

RNA secondary structures have been increasingly recognized to play an important regulatory role in post-transcriptional gene regulation. We recently showed that RNA G-quadruplexes, which serve as cis-elements to recruit splicing factors, play a critical role in regulating alternative splicing during the epithelial-mesenchymal transition. In this study, we performed a high-throughput screen using a dual-color splicing reporter to identify chemical compounds capable of regulating G-quadruplex-dependent alternative splicing. We identify emetine and its analog cephaeline as small molecules that disrupt RNA G-quadruplexes, resulting in inhibition of G-quadruplex-dependent alternative splicing. Transcriptome analysis reveals that emetine globally regulates alternative splicing, including splicing of variable exons that contain splice site-proximal G-quadruplexes. Our data suggest the use of emetine and cephaeline for investigating mechanisms of G-quadruplex-associated alternative splicing.

CD44 splice isoform switching determines breast cancer stem cell state.

Although changes in alternative splicing have been observed in cancer, their functional contributions still remain largely unclear. Here we report that splice isoforms of the cancer stem cell (CSC) marker CD44 exhibit strikingly opposite functions in breast cancer. Bioinformatic annotation in patient breast cancer in The Cancer Genome Atlas (TCGA) database reveals that the CD44 standard splice isoform (CD44s) positively associates with the CSC gene signatures, whereas the CD44 variant splice isoforms (CD44v) exhibit an inverse association. We show that CD44s is the predominant isoform expressed in breast CSCs. Elimination of the CD44s isoform impairs CSC traits. Conversely, manipulating the splicing regulator ESRP1 to shift alternative splicing from CD44v to CD44s leads to an induction of CSC properties. We further demonstrate that CD44s activates the PDGFRß/Stat3 cascade to promote CSC traits. These results reveal CD44 isoform specificity in CSC and non-CSC states and suggest that alternative splicing provides functional gene versatility that is essential for distinct cancer cell states and thus cancer phenotypes.

DPYSL3 modulates mitosis, migration, and epithelial-to-mesenchymal transition in claudin-low breast cancer.

A Clinical Proteomic Tumor Analysis Consortium (CPTAC) proteogenomic analysis prioritized dihydropyrimidinase-like-3 (DPYSL3) as a multilevel (RNA/protein/phosphoprotein) expression outlier specific to the claudin-low (CLOW) subset of triple-negative breast cancers. A PubMed informatics tool indicated a paucity of data in the context of breast cancer, which further prioritized DPYSL3 for study. DPYSL3 knockdown in DPYSL3-positive ([Formula: see text]) CLOW cell lines demonstrated reduced proliferation, yet enhanced motility and increased expression of epithelial-to-mesenchymal transition (EMT) markers, suggesting that DPYSL3 is a multifunctional signaling modulator. Slower proliferation in DPYSL3-negative ([Formula: see text]) CLOW cells was associated with accumulation of multinucleated cells, indicating a mitotic defect that was associated with a collapse of the vimentin microfilament network and increased vimentin phosphorylation. DPYSL3 also suppressed the expression of EMT regulators SNAIL and TWIST and opposed p21 activated kinase 2 (PAK2)-dependent migration. However, these EMT regulators in turn induce DPYSL3 expression, suggesting that DPYSL3 participates in negative feedback on EMT. In conclusion, DPYSL3 expression identifies CLOW tumors that will be sensitive to approaches that promote vimentin phosphorylation during mitosis and inhibitors of PAK signaling during migration and EMT.

Coregulation of alternative splicing by hnRNPM and ESRP1 during EMT.

The epithelial-mesenchymal transition (EMT) is a fundamental developmental process that is abnormally activated in cancer metastasis. Dynamic changes in alternative splicing occur during EMT. ESRP1 and hnRNPM are splicing regulators that promote an epithelial splicing program and a mesenchymal splicing program, respectively. The functional relationships between these splicing factors in the genome scale remain elusive. Comparing alternative splicing targets of hnRNPM and ESRP1 revealed that they coregulate a set of cassette exon events, with the majority showing discordant splicing regulation. Discordant splicing events regulated by hnRNPM show a positive correlation with splicing during EMT; however, concordant events do not, indicating the role of hnRNPM in regulating alternative splicing during EMT is more complex than previously understood. Motif enrichment analysis near hnRNPM-ESRP1 coregulated exons identifies guanine-uridine rich motifs downstream from hnRNPM-repressed and ESRP1-enhanced exons, supporting a general model of competitive binding to these cis-elements to antagonize alternative splicing. The set of coregulated exons are enriched in genes associated with cell migration and cytoskeletal reorganization, which are pathways associated with EMT. Splicing levels of coregulated exons are associated with breast cancer patient survival and correlate with gene sets involved in EMT and breast cancer subtyping. This study identifies complex modes of interaction between hnRNPM and ESRP1 in regulation of splicing in disease-relevant contexts.

Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles.

BACKGROUND: The International Mouse Phenotyping Consortium is generating null allele mice for every protein-coding gene in the genome and characterizing these mice to identify gene-phenotype associations. While CRISPR/Cas9-mediated null allele production in mice is highly efficient, generation of conditional alleles has proven to be more difficult. To test the feasibility of using CRISPR/Cas9 gene editing to generate conditional knockout mice for this large-scale resource, we employed Cas9-initiated homology-driven repair (HDR) with short and long single stranded oligodeoxynucleotides (ssODNs and lssDNAs). RESULTS: Using pairs of single guide RNAs and short ssODNs to introduce loxP sites around a critical exon or exons, we obtained putative conditional allele founder mice, harboring both loxP sites, for 23 out of 30 targeted genes. LoxP sites integrated in cis in at least one mouse for 18 of 23 genes. However, loxP sites were mutagenized in 4 of the 18 in cis lines. HDR efficiency correlated with Cas9 cutting efficiency but was minimally influenced by ssODN homology arm symmetry. By contrast, using pairs of guides and single lssDNAs to introduce loxP-flanked exons, conditional allele founders were generated for all four genes targeted, although one founder was found to harbor undesired mutations within the lssDNA sequence interval. Importantly, when employing either ssODNs or lssDNAs, random integration events were detected. CONCLUSIONS: Our studies demonstrate that Cas9-mediated HDR with pairs of ssODNs can generate conditional null alleles at many loci, but reveal inefficiencies when applied at scale. In contrast, lssDNAs are amenable to high-throughput production of conditional alleles when they can be employed. Regardless of the single-stranded donor utilized, it is essential to screen for sequence errors at sites of HDR and random insertion of donor sequences into the genome.