Experimental evidence for the functional importance and adaptive advantage of A-to-I RNA editing in fungi.

Adenosine-to-inosine (A-to-I) editing is the most prevalent type of RNA editing in animals, and it occurs in fungi specifically during sexual reproduction. However, it is debatable whether A-to-I RNA editing is adaptive. Deciphering the functional importance of individual editing sites is essential for the mechanistic understanding of the adaptive advantages of RNA editing. Here, by performing gene deletion for 17 genes with conserved missense editing (CME) sites and engineering underedited (ue) and overedited (oe) mutants for 10 CME sites using site-specific mutagenesis at the native locus in Fusarium graminearum, we demonstrated that two CME sites in CME5 and CME11 genes are functionally important for sexual reproduction. Although the overedited mutant was normal in sexual reproduction, the underedited mutant of CME5 had severe defects in ascus and ascospore formation like the deletion mutant, suggesting that the CME site of CME5 is co-opted for sexual development. The preediting residue of Cme5 is evolutionarily conserved across diverse classes of Ascomycota, while the postediting one is rarely hardwired into the genome, implying that editing at this site leads to higher fitness than a genomic A-to-G mutation. More importantly, mutants expressing only the underedited or the overedited allele of CME11 are defective in ascosporogenesis, while those expressing both alleles displayed normal phenotypes, indicating that concurrently expressing edited and unedited versions of Cme11 is more advantageous than either. Our study provides convincing experimental evidence for the long-suspected adaptive advantages of RNA editing in fungi and likely in animals.

UCHL3 induces radiation resistance and acquisition of mesenchymal phenotypes by deubiquitinating POLD4 in glioma stem cells.

BACKGROUND: The high degree of intratumoral genomic heterogeneity is a major obstacle for glioblastoma (GBM) tumors, one of the most lethal human malignancies, and is thought to influence conventional therapeutic outcomes negatively. The proneural-to-mesenchymal transition (PMT) of glioma stem cells (GSCs) confers resistance to radiation therapy in glioblastoma patients. POLD4 is associated with cancer progression, while the mechanisms underlying PMT and tumor radiation resistance have remained elusive. METHOD: Expression and prognosis of the POLD family were analyzed in TCGA, the Chinese Glioma Genome Atlas (CGGA) and GEO datasets. Tumorsphere formation and in vitro limiting dilution assay were performed to investigate the effect of UCHL3-POLD4 on GSC self-renewal. Apoptosis, TUNEL, cell cycle phase distribution, modification of the Single Cell Gel Electrophoresis (Comet), γ-H2AX immunofluorescence, and colony formation assays were conducted to evaluate the influence of UCHL3-POLD4 on GSC in ionizing radiation. Coimmunoprecipitation and GST pull-down assays were performed to identify POLD4 protein interactors. In vivo, intracranial xenograft mouse models were used to investigate the molecular effect of UCHL3, POLD4 or TCID on GCS. RESULT: We determined that POLD4 was considerably upregulated in MES-GSCs and was associated with a meagre prognosis. Ubiquitin carboxyl terminal hydrolase L3 (UCHL3), a DUB enzyme in the UCH protease family, is a bona fide deubiquitinase of POLD4 in GSCs. UCHL3 interacted with, depolyubiquitinated, and stabilized POLD4. Both in vitro and in vivo assays indicated that targeted depletion of the UCHL3-POLD4 axis reduced GSC self-renewal and tumorigenic capacity and resistance to IR treatment by impairing homologous recombination (HR) and nonhomologous end joining (NHEJ). Additionally, we proved that the UCHL3 inhibitor TCID induced POLD4 degradation and can significantly enhance the therapeutic effect of IR in a gsc-derived in situ xenograft model. CONCLUSION: These findings reveal a new signaling axis for GSC PMT regulation and highlight UCHL3-POLD4 as a potential therapeutic target in GBM. TCID, targeted for reducing the deubiquitinase activity of UCHL3, exhibited significant synergy against MES GSCs in combination with radiation.

Tumor neoantigens derived from RNA editing events show significant clinical relevance in melanoma patients treated with immunotherapy.

This study aimed to investigate the clinical significance of RNA editing (RE) and RNA editing derived (RED-) neoantigens in melanoma patients treated with immunotherapy. Vardict and VEP were used to identify the somatic mutations. RE events were identified by Reditools2 and filtered by the custom pipeline. miRTar2GO was implemented to predict the RE whether located in miRNA targets within the 3' UTR region. NetMHCpan and NetCTLpan were used to identify and characterize RED-neoantigens. In total, 7116 RE events were identified, most of which were A-to-I events. Using our custom pipeline, 631 RED-neoantigens were identified that show a significantly greater peptide-MHC affinity, and facilitate epitope processing and presentation than wild-type peptides. The OS of the patients with high RED-neoantigens burden was significantly longer ( P  = 0.035), and a significantly higher RED-neoantigens burden was observed in responders ( P  = 0.048). The area under the curve of the RED-neoantigen was 0.831 of OS. Then, we validated the reliability of RED-neoantigens in predicting the prognosis in an independent cohort and found that patients with high RED-neoantigens exhibited a longer OS ( P  = 0.008). To our knowledge, this is the first study to systematically assess the clinical relevance of RED-neoantigens in melanoma patients treated with immunotherapy.

CRISPR-assisted detection of RNA-protein interactions in living cells.

We have developed CRISPR-assisted RNA-protein interaction detection method (CARPID), which leverages CRISPR-CasRx-based RNA targeting and proximity labeling to identify binding proteins of specific long non-coding RNAs (lncRNAs) in the native cellular context. We applied CARPID to the nuclear lncRNA XIST, and it captured a list of known interacting proteins and multiple previously uncharacterized binding proteins. We generalized CARPID to explore binders of the lncRNAs DANCR and MALAT1, revealing the method's wide applicability in identifying RNA-binding proteins.

Directed Evolution of an Enhanced POU Reprogramming Factor for Cell Fate Engineering.

Transcription factor-driven cell fate engineering in pluripotency induction, transdifferentiation, and forward reprogramming requires efficiency, speed, and maturity for widespread adoption and clinical translation. Here, we used Oct4, Sox2, Klf4, and c-Myc driven pluripotency reprogramming to evaluate methods for enhancing and tailoring cell fate transitions, through directed evolution with iterative screening of pooled mutant libraries and phenotypic selection. We identified an artificially evolved and enhanced POU factor (ePOU) that substantially outperforms wild-type Oct4 in terms of reprogramming speed and efficiency. In contrast to Oct4, not only can ePOU induce pluripotency with Sox2 alone, but it can also do so in the absence of Sox2 in a three-factor ePOU/Klf4/c-Myc cocktail. Biochemical assays combined with genome-wide analyses showed that ePOU possesses a new preference to dimerize on palindromic DNA elements. Yet, the moderate capacity of Oct4 to function as a pioneer factor, its preference to bind octamer DNA and its capability to dimerize with Sox2 and Sox17 proteins remain unchanged in ePOU. Compared with Oct4, ePOU is thermodynamically stabilized and persists longer in reprogramming cells. In consequence, ePOU: 1) differentially activates several genes hitherto not implicated in reprogramming, 2) reveals an unappreciated role of thyrotropin-releasing hormone signaling, and 3) binds a distinct class of retrotransposons. Collectively, these features enable ePOU to accelerate the establishment of the pluripotency network. This demonstrates that the phenotypic selection of novel factor variants from mammalian cells with desired properties is key to advancing cell fate conversions with artificially evolved biomolecules.

K+ Efflux Antiporters 4, 5, and 6 Mediate pH and K+ Homeostasis in Endomembrane Compartments.

KEA4, KEA5, and KEA6 are members of the Arabidopsis (Arabidopsis thaliana) K+ efflux antiporter (KEA) family that share high sequence similarity but whose function remains unknown. Here, we show their gene expression pattern, subcellular localization, and physiological function in Arabidopsis. KEA4, KEA5, and KEA6 had similar tissue expression patterns, and the three KEA proteins localized to the Golgi, the trans-Golgi network, and the prevacuolar compartment/multivesicular bodies, suggesting overlapping roles of these proteins in the endomembrane system. Phenotypic analyses of single, double, and triple mutants confirmed functional redundancy. The triple mutant kea4 kea5 kea6 had small rosettes, short seedlings, and was sensitive to low K+ availability and to the sodicity imposed by high salinity. Also, the kea4 kea5 kea6 mutant plants had a reduced luminal pH in the Golgi, trans-Golgi network, prevacuolar compartment, and vacuole, in accordance with the K/H exchange activity of KEA proteins. Genetic analysis indicated that KEA4, KEA5, and KEA6 as well as endosomal Na+/H+exchanger5 (NHX5) and NHX6 acted coordinately to facilitate endosomal pH homeostasis and salt tolerance. Neither cancelling nor overexpressing the vacuolar antiporters NHX1 and NHX2 in the kea4 kea5 kea6 mutant background altered the salt-sensitive phenotype. The NHX1 and NHX2 proteins in the kea4 kea5 kea6 mutant background could not suppress the acidity of the endomembrane system but brought the vacuolar pH close to wild-type values. Together, these data signify that KEA4, KEA5, and KEA6 are endosomal K+ transporters functioning in maintaining pH and ion homeostasis in the endomembrane network.

Identification of 3 subpopulations of tumor-infiltrating immune cells for malignant transformation of low-grade glioma.

BACKGROUND: Tumor-infiltrating immune cells (TIICs) are highly relevant to clinical outcome of glioma. However, previous studies cannot account for the diverse functions that make up the immune response in malignant transformation (MT) from low-grade glioma (LGG) to high-grade glioma (HGG). METHODS: Transcriptome level, genomic profiles and its relationship with clinical practice were obtained from TCGA and CGGA database. The "Cell type Identification By Estimating Relative Subsets Of RNA Transcripts (CIBERSORT)" algorithm was used to estimate the fraction of 22 immune cell types. We divided the TCGA and CGGA set into an experiment set (n = 174) and a validation set (n = 74) by random number table method. Univariate and multivariate analyses were performed to evaluate the 22 TIICs' value for MT in LGG. ROC curve was plotted to calculate area under curve (AUC) and cut-off value. RESULTS: Heterogeneity between TIICs exists in both intra- and inter-groups. Several TIICs are notably associated with tumor grade, molecular subtypes and survival. T follicular helper (TFH) cells, activated NK Cells and M0 macrophages were screened out to be independent predictors for MT in LGG and formed an immune risk score (IRS) (AUC = 0.732, p < 0.001, 95% CI 0.657-0.808 cut-off value = 0.191). In addition, the IRS model was validated by validation group, Immunohistochemistry (IHC) and functional enrichment analyses. CONCLUSIONS: The proposed IRS model provides promising novel signatures for predicting MT from LGG to HGG and may bring a better design of glioma immunotherapy studies in years to come.

Na+ ,K+ /H+ antiporters regulate the pH of endoplasmic reticulum and auxin-mediated development.

AtNHX5 and AtNHX6 are endosomal Na+ ,K+ /H+ antiporters that are critical for growth and development in Arabidopsis, but the mechanism behind their action remains unknown. Here, we report that AtNHX5 and AtNHX6, functioning as H+ leak, control auxin homeostasis and auxin-mediated development. We found that nhx5 nhx6 exhibited growth variations of auxin-related defects. We further showed that nhx5 nhx6 was affected in auxin homeostasis. Genetic analysis showed that AtNHX5 and AtNHX6 were required for the function of the endoplasmic reticulum (ER)-localized auxin transporter PIN5. Although AtNHX5 and AtNHX6 were colocalized with PIN5 at ER, they did not interact directly. Instead, the conserved acidic residues in AtNHX5 and AtNHX6, which are essential for exchange activity, were required for PIN5 function. AtNHX5 and AtNHX6 regulated the pH in ER. Overall, AtNHX5 and AtNHX6 may regulate auxin transport across the ER via the pH gradient created by their transport activity. H+ -leak pathway provides a fine-tuning mechanism that controls cellular auxin fluxes.

E2F1-regulated USP5 contributes to the tumorigenic capacity of glioma stem cells through the maintenance of OCT4 stability.

Mesenchymal glioma stem cells (MES GSCs) are a subpopulation of cells in glioblastoma (GBM) that contribute to a worse prognosis owing to their highly aggressive nature and resistance to radiation therapy. Here, OCT4 is characterized as a critical factor in sustaining the stemness phenotype of MES GSC. We find that OCT4 is expressed intensively in MES GSC and is intimately associated with poor prognosis, moreover, OCT4 depletion leads to diminished invasive capacity and impairment of the stem phenotype in MES GSC. Subsequently, we demonstrated that USP5 is a deubiquitinating enzyme which directly interacts with OCT4 and preserves OCT4 stability through its deubiquitination. USP5 was additionally proven to be aberrantly over-expressed in MES GSCs, and its depletion resulted in a noticeable diminution of OCT4 and consequently a reduced self-renewal and tumorigenic capacity of MES GSCs, which can be substantially restored by ectopic expression of OCT4. In addition, we detected the dominant molecule that regulates USP5 transcription, E2F1, with dual luciferase reporter gene analysis. In combination, targeting the E2F1-USP5-OCT4 axis is a potentially emerging strategy for the therapy of GBM.

The highly conserved RNA-binding specificity of nucleocapsid protein facilitates the identification of drugs with broad anti-coronavirus activity.

The binding of SARS-CoV-2 nucleocapsid (N) protein to both the 5'- and 3'-ends of genomic RNA has different implications arising from its binding to the central region during virion assembly. However, the mechanism underlying selective binding remains unknown. Herein, we performed the high-throughput RNA-SELEX (HTR-SELEX) to determine the RNA-binding specificity of the N proteins of various SARS-CoV-2 variants as well as other ß-coronaviruses and showed that N proteins could bind two unrelated sequences, both of which were highly conserved across all variants and species. Interestingly, both sequences are virtually absent from the human transcriptome; however, they exhibit a highly enriched, mutually complementary distribution in the coronavirus genome, highlighting their varied functions in genome packaging. Our results provide mechanistic insights into viral genome packaging, thereby increasing the feasibility of developing drugs with broad-spectrum anti-coronavirus activity by targeting RNA binding by N proteins.

An atlas of the binding specificities of transcription factors in Pseudomonas aeruginosa directs prediction of novel regulators in virulence.

A high-throughput systematic evolution of ligands by exponential enrichment assay was applied to 371 putative TFs in Pseudomonas aeruginosa, which resulted in the robust enrichment of 199 unique sequence motifs describing the binding specificities of 182 TFs. By scanning the genome, we predicted in total 33,709 significant interactions between TFs and their target loci, which were more than 11-fold enriched in the intergenic regions but depleted in the gene body regions. To further explore and delineate the physiological and pathogenic roles of TFs in P. aeruginosa, we constructed regulatory networks for nine major virulence-associated pathways and found that 51 TFs were potentially significantly associated with these virulence pathways, 32 of which had not been characterized before, and some were even involved in multiple pathways. These results will significantly facilitate future studies on transcriptional regulation in P. aeruginosa and other relevant pathogens, and accelerate to discover effective treatment and prevention strategies for the associated infectious diseases.

A compendium of DNA-binding specificities of transcription factors in Pseudomonas syringae.

Pseudomonas syringae is a Gram-negative and model pathogenic bacterium that causes plant diseases worldwide. Here, we set out to identify binding motifs for all 301 annotated transcription factors (TFs) of P. syringae using HT-SELEX. We successfully identify binding motifs for 100 TFs. We map functional interactions between the TFs and their targets in virulence-associated pathways, and validate many of these interactions and functions using additional methods such as ChIP-seq, electrophoretic mobility shift assay (EMSA), RT-qPCR, and reporter assays. Our work identifies 25 virulence-associated master regulators, 14 of which had not been characterized as TFs before.

Arabidopsis NHX5 and NHX6 regulate PIN6-mediated auxin homeostasis and growth.

NHX5 and NHX6, endosomal Na+,K+/H+ antiporters in Arabidopsis thaliana, play a vital role in growth and development. Our previous study has shown that NHX5 and NHX6 function as H+ leak to regulate auxin-mediated growth in Arabidopsis. In this report, we investigated the function of NHX5 and NHX6 in controlling PIN6-mediated auxin homeostasis and growth in Arabidopsis. Phenotypic analyses found that NHX5 and NHX6 were critical for the function of PIN6, an auxin transporter. We further showed that PIN6 depended on NHX5 and NHX6 in regulating auxin homeostasis. NHX5 and NHX6 were colocalized with PIN6, but they did not interact physically. The conserved acidic residues that are vital for the activity of NHX5 and NHX6 were critical for PIN6 function. Together, NHX5 and NHX6 may regulate PIN6 function by their transport activity.

Ubiquitin-Specific Protease 3 Promotes Glioblastoma Cell Invasion and Epithelial-Mesenchymal Transition via Stabilizing Snail.

Epithelial-mesenchymal transition (EMT) represents one of the most important events in the invasion of glioblastomas (GBM); therefore, better understanding of mechanisms that govern EMT is crucial for the treatment of GBMs. In this study, we report that the deubiquitinase ubiquitin-specific protease 3 (USP3) is significantly upregulated in GBMs and correlates with a shorter median overall and relapse-free survival. Silencing of USP3 attenuates the migration and invasion abilities of GBM cells in vitro and tumor growth in an orthotopic xenograft mouse model. Mechanistically, we identify USP3 as a bona fide deubiquitinase for Snail, a master transcription factor that promotes EMT, in GBM cells. USP3 interacts directly with Snail and stabilizes Snail via deubiquitination. Ectopic expression of Snail could largely rescue the inhibitory effects of USP3 depletion on migration, invasion, and tumor growth of GBM cells. In addition, we found that USP3 strongly correlates with Snail expression in primary human GBM samples. Overall, our findings reveal a critical USP3-Snail signaling axis in EMT and invasion, and provide an effective therapeutic approach against GBM. IMPLICATIONS: Our study establishes USP3-mediated Snail stabilization as an important mechanism underlying GBM invasion and progression, and provides a rationale for potential therapeutic interventions in the treatment of GBM.

USP9X deubiquitinates ALDH1A3 and maintains mesenchymal identity in glioblastoma stem cells.

The mesenchymal (MES) subtype of glioblastoma (GBM) stem cells (GSCs) represents a subpopulation of cancer cells that are notorious for their highly aggressive nature and resistance to conventional therapy. Aldehyde dehydrogenase 1A3 (ALDH1A3) has been recently suggested as a key determinant for the maintenance of MES features of GSCs. However, the mechanisms underpinning aberrant ALDH1A3 expression remain elusive. Here, we identified ubiquitin-specific protease 9X (USP9X) as a bona fide deubiquitinase of ALDH1A3 in MES GSCs. USP9X interacted with, depolyubiquitylated, and stabilized ALDH1A3. Moreover, we showed that FACS-sorted USP9Xhi cells were enriched for MES GSCs with high ALDH1A3 activity and potent tumorigenic capacity. Depletion of USP9X markedly downregulated ALDH1A3, resulting in a loss of self-renewal and tumorigenic capacity of MES GSCs, which could be largely rescued by ectopic expression of ALDH1A3. Furthermore, we demonstrated that the USP9X inhibitor WP1130 induced ALDH1A3 degradation and showed marked therapeutic efficacy in MES GSC-derived orthotopic xenograft models. Additionally, USP9X strongly correlated with ALDH1A3 expression in primary human GBM samples and had a prognostic value for patients with the MES subgroup. Collectively, our findings unveil USP9X as a key deubiquitinase for ALDH1A3 protein stabilization and a potential target for GSC-directed therapy.

Transcriptomic view of survival during early seedling growth of the extremophyte Haloxylon ammodendron.

Seedling establishment in an extreme environment requires an integrated genomic and physiological response to survive multiple abiotic stresses. The extremophyte, Haloxylon ammodendron is a pioneer species capable of colonizing temperate desert sand dunes. We investigated the induced and basal transcriptomes in H. ammodendron under water-deficit stress during early seedling establishment. We find that not only drought-responsive genes, but multiple genes in pathways associated with salt, osmotic, cold, UV, and high-light stresses were induced, suggesting an altered regulatory stress response system. Additionally, H. ammodendron exhibited enhanced biotic stress tolerance by down-regulation of genes that were generally up-regulated during pathogen entry in susceptible plants. By comparing the H. ammodendron basal transcriptome to six closely related transcriptomes in Amaranthaceae, we detected enriched basal level transcripts in H. ammodendron that shows preadaptation to abiotic stress and pathogens. We found transcripts that were generally maintained at low levels and some induced only under abiotic stress in the stress-sensitive model, Arabidopsis thaliana to be highly expressed under basal conditions in the Amaranthaceae transcriptomes including H. ammodendron. H. ammodendron shows coordinated expression of genes that regulate stress tolerance and seedling development resource allocation to support survival against multiple stresses in a sand dune dominated temperate desert environment.

A novel AtKEA gene family, homolog of bacterial K+/H+ antiporters, plays potential roles in K+ homeostasis and osmotic adjustment in Arabidopsis.

AtKEAs, homologs of bacterial KefB/KefC, are predicted to encode K(+)/H(+) antiporters in Arabidopsis. The AtKEA family contains six genes forming two subgroups in the cladogram: AtKEA1-3 and AtKEA4-6. AtKEA1 and AtKEA2 have a long N-terminal domain; the full-length AtKEA1 was inactive in yeast. The transport activity was analyzed by expressing the AtKEA genes in yeast mutants lacking multiple ion carriers. AtKEAs conferred resistance to high K(+) and hygromycin B but not to salt and Li(+) stress. AtKEAs expressed in both the shoot and root of Arabidopsis. The expression of AtKEA1, -3 and -4 was enhanced under low K(+) stress, whereas AtKEA2 and AtKEA5 were induced by sorbitol and ABA treatments. However, osmotic induction of AtKEA2 and AtKEA5 was not observed in aba2-3 mutants, suggesting an ABA regulated mechanism for their osmotic response. AtKEAs' expression may not be regulated by the SOS pathway since their expression was not affected in sos mutants. The GFP tagging analysis showed that AtKEAs distributed diversely in yeast. The Golgi localization of AtKEA3 was demonstrated by both the stably transformed seedlings and the transient expression in protoplasts. Overall, AtKEAs expressed and localized diversely, and may play roles in K(+) homeostasis and osmotic adjustment in Arabidopsis.

MicroRNA-7 regulates glioblastoma cell invasion via targeting focal adhesion kinase expression.

BACKGROUND: Invasion growth is the most characteristic biological phenotype of glioblastoma, but the molecular mechanism in glioma cell invasion is poorly understood. Recent data have showed that microRNA plays an essential role in tumor invasion. Our study aimed to explore the mechanism of miR-7 involved in the control of glioblastoma cell invasion. METHODS: Glioma cell invasion was evaluated by transwell and scratch assays after up-regulation of miR-7 using miR-7 mimics in U87 and U251 cells. Luciferase reporter assay was used to determine focal adhesion kinase (FAK) as a target of miR-7. The levels of miR-7, matrix metalloproteinases (MMP)-2 and MMP-9 mRNA were detected by PCR assay, and the levels of FAK, MMP-2, MMP-9, total and phosphorylation serine/threonine kinase (AKT), and extracellular signal-regulated kinase (ERK) 1/2 were measured by Western blotting analysis. RESULTS: Over-expression of miR-7 inhibited the invasion and migration activity of U87 and U251 cells. And up-regulation of miR-7 reduced FAK protein expression, Further, luciferase reporter assay showed that miR-7 modulated FAK expression directly by binding 3'UTR of FAK mRNA. In addition, miR-7 repressed p-ERK1/2 and p-AKT level, MMP-2 and MMP-9 expression. Finally, the inverse relationship between FAK and miR-7 expression was certificated in human glioma tissues. CONCLUSION: To our knowledge, these data indicate for the first time that miR-7 directly regulates cell invasion by targeting FAK in glioblastoma and that miR-7 could be a potential therapeutic target for glioblastoma intervention.