Regulation of Epithelial-Mesenchymal Transitions by Alternative Splicing: Potential New Area for Cancer Therapeutics.

The epithelial-mesenchymal transition (EMT) is a complicated biological process in which cells with epithelial phenotype are transformed into mesenchymal cells with loss of cell polarity and cell-cell adhesion and gain of the ability to migrate. EMT and the reverse mesenchymal-epithelial transitions (METs) are present during cancer progression and metastasis. Using the dynamic switch between EMT and MET, tumour cells can migrate to neighbouring organs or metastasize in the distance and develop resistance to traditional chemotherapy and targeted drug treatments. Growing evidence shows that reversing or inhibiting EMT may be an advantageous approach for suppressing the migration of tumour cells or distant metastasis. Among different levels of modulation of EMT, alternative splicing (AS) plays an important role. An in-depth understanding of the role of AS and EMT in cancer is not only helpful to better understand the occurrence and regulation of EMT in cancer progression, but also may provide new therapeutic strategies. This review will present and discuss various splice variants and splicing factors that have been shown to play a crucial role in EMT.

Dysregulation of Spliceosomes Complex Induces Retinitis Pigmentosa-Like Characteristics in sf3b4-Depleted Zebrafish.

The SF3B4 gene encodes a highly conserved protein that plays a critical role in mRNA splicing. Mutations in this gene are known to cause Nager syndrome, a rare craniofacial disorder. Although SF3B4 expression is detected in the optic vesicle before it is detected in the limb and somite, the role of SF3B4 in the eye is not well understood. This study investigated the function of sf3b4 in the retina by performing transcriptome profiles, immunostaining, and behavioral analysis of sf3b4-/- mutant zebrafish. Results from this study suggest that dysregulation of the spliceosome complex affects not only craniofacial development but also retinogenesis. Zebrafish lacking functional sf3b4 displayed characteristics similar to retinitis pigmentosa (RP), marked by severe retinal pigment epithelium defects and rod degeneration. Pathway analysis revealed altered retinol metabolism and retinoic acid signaling in the sf3b4-/- mutants. Supplementation of retinoic acid rescued key cellular phenotypes observed in the sf3b4-/- mutants, offering potential therapeutic strategies for RP in the future. In conclusion, this study sheds light on the previously unknown role of SF3B4 in retinogenesis and provides insights into the underlying mechanisms of RP.

Global Shift in Alternative Splicing and Therapeutic Susceptibilities in Leukemia Driven by METTL3 Overexpression.

SUMMARY: Mutations in splicing factors are commonly observed in chronic lymphocytic leukemia (CLL); however, other mechanisms can also contribute to the dysregulation of alternative splicing. One example is the overexpression of the m6A RNA methyltransferase METTL3, that by depositing the epitranscriptomic mark in spliceosome transcripts leads to aberrant splicing, but at the same time creates vulnerability to METTL3 inhibitors. See related article by Wu et al., p. 228 (8) .

Comparative Analysis of Environment-Responsive Alternative Splicing in the Inflorescences of Cultivated and Wild Tomato Species.

Cultivated tomato (Solanum lycopersicum) is bred for fruit production in optimized environments, in contrast to harsh environments where their ancestral relatives thrive. The process of domestication and breeding has profound impacts on the phenotypic plasticity of plant development and the stress response. Notably, the alternative splicing (AS) of precursor message RNA (pre-mRNA), which is one of the major factors contributing to transcriptome complexity, is responsive to developmental cues and environmental change. To determine a possible association between AS events and phenotypic plasticity, we investigated environment-responsive AS events in the inflorescences of cultivated tomato and its ancestral relatives S. pimpinellifolium. Despite that similar AS frequencies were detected in the cultivated tomato variety Moneymaker and two S. pimpinellifolium accessions under the same growth conditions, 528 genes including splicing factors showed differential splicing in the inflorescences of plants grown in open fields and plastic greenhouses in the Moneymaker variety. In contrast, the two S. pimpinellifolium accessions, LA1589 and LA1781, had 298 and 268 genes showing differential splicing, respectively. Moreover, seven heat responsive genes showed opposite expression patterns in response to changing growth conditions between Moneymaker and its ancestral relatives. Accordingly, there were eight differentially expressed splice variants from genes involved in heat response in Moneymaker. Our results reveal distinctive features of AS events in the inflorescences between cultivated tomato and its ancestral relatives, and show that AS regulation in response to environmental changes is genotype dependent.

Alternative splicing analysis showed the splicing factor polypyrimidine tract-binding protein 1 as a potential target in acute myeloid leukemia therapy.

Alternative splicing (AS) is a universal post-transcriptional regulation process in cells, and increasing evidences have validated its crucial role in tumors. We collected AS event, gene expression, and clinical data of 178 AML patients from The Cancer Genome Atlas (TCGA) project. More than 1,000 AS events were found associated with overall survival (OS), and alternate promoter (AP) events were the most significant. The expression of the KIAA0930 transcript was the most significantly different AS event selected from AP events and significantly correlated with the expression of the splicing factor (SF) polypyrimidine tract-binding protein 1 (PTBP1). Then, the roles of PTBP1 on AS of the KIAA0930 and the proliferation of AML cells were confirmed. KIAA0930 variant 1 (KIAA0930-1) was upregulated and variant 2 (KIAA0930-2) downregulated with knockdown PTBP1 expression of AML cells by specific shRNA. A low level of PTBP1 can decrease the proliferation ability of AML cells. In conclusion, the results showed that PTBP1 might be a potential target for AML therapy.

YTHDC1 is downregulated by the YY1/HDAC2 complex and controls the sensitivity of ccRCC to sunitinib by targeting the ANXA1-MAPK pathway.

BACKGROUND: Tyrosine kinase inhibitors (TKIs) such as sunitinib are multitarget antiangiogenic agents in clear cell renal cell carcinoma (ccRCC). They are widely used in the treatment of advanced/metastatic renal cancer. However, resistance to TKIs is common in the clinic, particularly after long-term treatment. YTHDC1 is the main nuclear reader protein that binds with m6A to regulate the splicing, export and stability of mRNA. However, the specific role and corresponding mechanism of YTHDC1 in renal cancer cells are still unclear. METHODS: The Cancer Genome Atlas (TCGA) dataset was used to study the expression of YTHDC1 in ccRCC. Cell counting kit-8 (CCK-8), wound healing, Transwell and xenograft assays were applied to explore the biological function of YTHDC1 in ccRCC. Western blot, quantitative real time PCR (RT‒qPCR), RNA immunoprecipitation PCR (RIP-qPCR), methylated RIP-qPCR (MeRIP-qPCR) and RNA sequencing (RNA-seq) analyses were applied to study the YY1/HDAC2/YTHDC1/ANXA1 axis in renal cancer cells. The CCK-8 assay and xenograft assay were used to study the role of YTHDC1 in determining the sensitivity of ccRCC to sunitinib. RESULTS: Our results demonstrated that YTHDC1 is downregulated in ccRCC tissues compared with normal tissues. Low expression of YTHDC1 is associated with a poor prognosis in patients with ccRCC. Subsequently, we showed that YTHDC1 inhibits the progression of renal cancer cells via downregulation of the ANXA1/MAPK pathways. Moreover, we also showed that the YTHDC1/ANXA1 axis modulates the sensitivity of tyrosine kinase inhibitors. We then revealed that HDAC2 inhibitors resensitize ccRCC to tyrosine kinase inhibitors through the YY1/HDAC2 complex. We have identified a novel YY1/HDAC2/YTHDC1/ANXA1 axis modulating the progression and chemosensitivity of ccRCC. CONCLUSION: We identified a novel YY1/HDAC2/YTHDC1/ANXA1 axis modulating the progression and chemosensitivity of ccRCC.

OsPPR939, a nad5 splicing factor, is essential for plant growth and pollen development in rice.

KEY MESSAGE: P-subfamily PPR protein OsPPR939, which can be phosphorylated by OsS6K1, regulates plant growth and pollen development by involving in the splicing of mitochondrial nad5 introns 1, 2, and 3. In land plants, pentatricopeptide repeat (PPR) proteins play key roles in mitochondrial group II intron splicing, but how these nucleus-encoded proteins are imported into mitochondria is unknown. To date, a few PPR proteins have been characterized in rice (Oryza sativa). Here, we demonstrate that the mitochondrion-localized P-subfamily PPR protein OsPPR939 is required for the splicing of nad5 introns 1, 2, and 3 in rice. Complete knockout or partial disruption of OsPPR939 function resulted in different degrees of growth retardation and pollen sterility. The dramatically reduced splicing efficiency of these introns in osppr939-4 and osppr939-5 led to reduced mitochondrial complex I abundance and activity and enhanced expression of alternative respiratory pathway genes. Complementation with OsPPR939 rescued the defective plant morphology of osppr939-4 and restored its decreased splicing efficiency of nad5 introns 1, 2, and 3. Therefore, OsPPR939 plays crucial roles in plant growth and pollen development by splicing mitochondrial nad5 introns 1, 2, and 3. More importantly, the 12th amino acid Ser in the N-terminal targeting sequence of OsPPR939 is phosphorylated by OsS6K1, and truncated OsPPR939 with a non-phosphorylatable S12A mutation in its presequence could not be imported into mitochondria, suggesting that phosphorylation of this amino acid plays an important role in the mitochondrial import of OsPPR939. To our knowledge, the 12th residue Ser on OsPPR939 is the first experimentally proven phosphorylation site in PPR proteins. Our results provide a basis for investigating the regulatory mechanism of PPR proteins at the post-translational level.

[Screening of drugs that selectively inhibit uveal melanoma cells with SF3B1 mutations].

OBJECTIVE: To screen compounds that can selectively inhibit uveal melanoma cells with splicing factor 3B subunit 1 (SF3B1) mutations in comparison with isogenic SF3B1 wild-type counterparts in a cell model of SF3B1 mutant allele knockout. METHODS: Principal component analysis was used to analyze transcriptome alternative splicing in TCGA cohorts of uveal melanoma with wild-type SF3B1 and SF3B1 mutations, and abnormal alternative splicing events derived from SF3B1 mutations were identified. The SF3B1 mutant allele in Mel202 cells was knocked out using CRISPR-Cas9 technology, and Sanger sequencing was used to verify the edited sequence. MTT and colony formation assays were used to assess the proliferation of Mel202 and Mut-KO cells. RT-PCR agarose electrophoresis combined with Sanger sequencing was used to determine alternative splicing events in Mel202 and Mut-KO cells. MTT assay was performed to screen the compounds that showed selective inhibitory effect against Mel202 cells with SF3B1 mutation. RESULTS: Specific knockout of SF3B1 mutant allele in Mel202 cells obviously promoted the cell proliferation and caused changes in alternative splicing of ZDHHC16 and DYNLL1 transcripts. The screening data showed that 13 compounds had selective inhibitory activity against Mel202 cells with SF3B1 mutation (Fold change≥2), and among them, tetrandrine and lapatinib showed good dose-effect curves. CONCLUSION: This study provides a cell screening model for identification of potential individualized treatment drugs for patients with uveal melanoma with SF3B1 mutation.

Sex-dimorphic Rostro-caudal Patterns of 5'-AMP-activated Protein Kinase Activation and Glucoregulatory Transmitter Marker Protein Expression in the Ventrolateral Ventromedial Hypothalamic Nucleus (VMNvl) in Hypoglycemic Male and Female Rats: Impact of Estradiol.

The ventromedial hypothalamic nucleus-ventrolateral part (VMNvl) is an estradiol-sensitive structure that controls sex-specific behavior. Electrical reactivity of VMNvl neurons to hypoglycemia infers that cellular energy stability is monitored there. Current research investigated the hypothesis that estradiol elicits sex-dimorphic patterns of VMNvl metabolic sensor activation and gluco-regulatory neurotransmission during hypoglycemia. Rostral-, middle-, and caudal-VMNvl tissue was separately micropunch-dissected from letrozole (Lz)- or vehicle-injected male and estradiol- or vehicle-implanted ovariectomized (OVX) female rats for Western blot analysis of total and phosphorylated 5'-AMP-activated protein kinase (AMPK) protein expression and gluco-stimulatory [neuronal nitric oxide synthase (nNOS); steroidogenic factor-1 (SF1) or -inhibitory (glutamate decarboxylase65/67 (GAD)] transmitter marker proteins after sc insulin (INS) or vehicle injection. In both sexes, hypoglycemic up-regulation of phosphoAMPK was estradiol-dependent in rostral and middle, but not caudal VMNvl. AMPK activity remained elevated after recovery from hypoglycemia over the rostro-caudal VMNvl in female, but only in the rostral segment in male. In each sex, hypoglycemia correspondingly augmented or suppressed nNOS profiles in rostral and middle versus caudal VMNvl; these segmental responses persisted longer in female. Rostral and middle segment SF1 protein was inhibited by estradiol-independent mechanisms in hypoglycemic males, but increased by estradiol-reliant mechanisms in female. After INS injection, GAD expression was inhibited in the male rostral VMNvl without estradiol involvement, but this hormone was required for broader suppression of this profile in the female. Neuroanatomical variability of VMNvl metabolic transmitter reactivity to hypoglycemia underscores the existence of functionally different subgroups in that structure. The regional distribution and estradiol sensitivity of hypoglycemia-sensitive VMNvl neurons of each neurochemical phenotype evidently vary between sexes.

Patient stratification in myelodysplastic syndromes: how a puzzle may become a map.

Heterogeneity is the disease-defining epithet of myelodysplastic syndromes (MDS), a clonal disorder of hematopoietic stem and progenitor cells. During the last decade, significant progress has been made to better understand the diversity of clinical, molecular, cellular, and immunological factors that are bound to the prognosis and outcomes of patients with MDS. Despite the rapid generation of all of this biological information, how to implement it has fallen short. Redefining clinical tools to use this new information remains a challenge. The holistic integration of novel, high-impact individual risk parameters such as patient-reported outcomes or mutational and immunological data into conventional risk stratification systems may further refine patient subgroups, improve predictive power for survival, and provide a next-generation classification and prognosis system for patients with MDS. Dichotomic treatment strategies in patients with MDS according to their patient and disease profiles highlight the importance of precise risk stratification, which may be complemented by the definition of granular cohorts of patients with myeloid neoplasms and a druggable target (ie, IDH1/2 mutations) across conventional blast thresholds.

Sex-Dimorphic Behavioral Alterations and Altered Neurogenesis in U12 Intron Splicing-Defective Zrsr1 Mutant Mice.

Mutant mice with respect to the splicing factor Zrsr1 present altered spermatogenesis and infertility. To investigate whether Zrsr1 is involved in the homeostatic control that the hypothalamus exerts over reproductive functions, we first analyzed both differential gene and isoform expression and alternative splicing alterations in Zrsr1 mutant (Zrsr1mu) hypothalamus; second, we analyzed the spontaneous and social behavior of Zrsr1mu mice; and third, we analyzed adult cell proliferation and survival in the Zrsr1mu hypothalamus. The Zrsr1mu hypothalamus showed altered expression of genes and isoforms related to the glutathione metabolic process, synaptonemal complex assembly, mRNA transport, and altered splicing events involving the enrichment of U12-type intron retention (IR). Furthermore, increased IR in U12-containing genes related with the prolactin, progesterone, and gonadotropin-releasing hormone (GnRH) reproductive signaling pathway was observed. This was associated with a hyperactive phenotype in both males and females, with an anxious phenotype in females, and with increased social interaction in males, instead of the classical aggressive behavior. In addition, Zrsr1mu females but not males exhibited reduced cell proliferation in both the hypothalamus and the subventricular zone. Overall, these results suggest that Zrsr1 expression and function are relevant to organization of the hypothalamic cell network controlling behavior.

Therapeutic Applications of Targeted Alternative Splicing to Cancer Treatment.

A growing body of studies has documented the pathological influence of impaired alternative splicing (AS) events on numerous diseases, including cancer. In addition, the generation of alternatively spliced isoforms is frequently noted to result in drug resistance in many cancer therapies. To gain comprehensive insights into the impacts of AS events on cancer biology and therapeutic developments, this paper highlights recent findings regarding the therapeutic routes of targeting alternative-spliced isoforms and splicing regulators to treatment strategies for distinct cancers.

Modulation of SF1 Neuron Activity Coordinately Regulates Both Feeding Behavior and Associated Emotional States.

Feeding requires the integration of homeostatic drives with emotional states relevant to food procurement in potentially hostile environments. The ventromedial hypothalamus (VMH) regulates feeding and anxiety, but how these are controlled in a concerted manner remains unclear. Using pharmacogenetic, optogenetic, and calcium imaging approaches with a battery of behavioral assays, we demonstrate that VMH steroidogenic factor 1 (SF1) neurons constitute a nutritionally sensitive switch, modulating the competing motivations of feeding and avoidance of potentially dangerous environments. Acute alteration of SF1 neuronal activity alters food intake via changes in appetite and feeding-related behaviors, including locomotion, exploration, anxiety, and valence. In turn, intrinsic SF1 neuron activity is low during feeding and increases with both feeding termination and stress. Our findings identify SF1 neurons as a key part of the neurocircuitry that controls both feeding and related affective states, giving potential insights into the relationship between disordered eating and stress-associated psychological disorders in humans.

Effects and mechanisms of acupuncture and moxibustion on reproductive endocrine function in male rats with partial androgen deficiency.

OBJECTIVES: Partial androgen deficiency of the aging male (PADAM) is characterised by a deficiency in serum androgen levels. Both electroacupuncture (EA) and mild moxibustion (MM) can raise serum testosterone levels in PADAM. We investigated the mechanisms underlying the use of EA and MM in a rodent model of PADAM. METHODS: Fifty rats received cyclophosphamide injection over 5 consecutive days to induce PADAM, which was verified by comparing total testosterone (TT) and free testosterone (FT) levels with 10 non-PADAM healthy control rats (CON). Successful modelling was confirmed in 43 of 50 rats, 40 of which were randomly divided into untreated (PADAM), EA-treated (PADAM+EA), MM-treated (PADAM+MM), and androlin (AD)-treated (PADAM+AD) groups (n=10 each). EA and MM were administered at BL23 and CV4 acupuncture points for 8 weeks, and no treatment was given to rats in the PADAM and CON groups. Serum levels of luteinising hormone (LH) and follicle-stimulating hormone (FSH), mRNA expression of cytochrome P450c17 (P450c17) and 3ß-hydroxysteroid dehydrogenase 1 (3ß-HSD1), and protein levels of cytochrome P450 side chain cleavage (P450scc), 17ß-hydroxysteroid dehydrogenase 3 (17ß-HSD3) and steroidogenic factor 1 (SF-1) were evaluated after 8 weeks. RESULTS: Both EA and mild MM significantly increased serum TT and FT levels with MM displaying superiority. P450scc, 17ß-HSD3 and SF-1 protein expression, and P450c17 and 3ß-HSD1 mRNA expression, were significantly increased and serum LH and FSH levels were significantly decreased in PADAM+EA and PADAM+MM relative to PADAM rats. Moreover, serum LH and FSH levels were significantly lower and 17ß-HSD3 protein expression significantly higher in PADAM+MM relative to PADAM+EA rats. CONCLUSIONS: EA and MM at the BL23 and CV4 acupuncture points appear to be effective treatments for PADAM, and MM displays superior efficacy to EA.

SNEV(Prp19/PSO4) deficiency increases PUVA-induced senescence in mouse skin.

Senescent cells accumulate during ageing in various tissues and contribute to organismal ageing. However, factors that are involved in the induction of senescence in vivo are still not well understood. SNEV(P) (rp19/) (PSO) (4) is a multifaceted protein, known to be involved in DNA damage repair and senescence, albeit only in vitro. In this study, we used heterozygous SNEV(+/-) mice (SNEV-knockout results in early embryonic lethality) and wild-type littermate controls as a model to elucidate the role of SNEV(P) (rp19/) (PSO) (4) in DNA damage repair and senescence in vivo. We performed PUVA treatment as model system for potently inducing cellular senescence, consisting of 8-methoxypsoralen in combination with UVA on mouse skin to induce DNA damage and premature skin ageing. We show that SNEV(P) (rp19/) (PSO) (4) expression decreases during organismal ageing, while p16, a marker of ageing in vivo, increases. In response to PUVA treatment, we observed in the skin of both SNEV(P) (rp19/) (PSO) (4) and wild-type mice an increase in γ-H2AX levels, a DNA damage marker. In old SNEV(P) (rp19/) (PSO) (4) mice, this increase is accompanied by reduced epidermis thickening and increase in p16 and collagenase levels. Thus, the DNA damage response occurring in the mouse skin upon PUVA treatment is dependent on SNEV(P) (rp19/) (PSO) (4) expression and lower levels of SNEV(P) (rp19/) (PSO) (4) , as in old SNEV(+/-) mice, result in increase in cellular senescence and acceleration of premature skin ageing.