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The spliceosome undergoes dramatic changes in a splicing cycle. Structures of B, Bact, C, C*, and intron lariat spliceosome complexes revealed mechanisms of 5'-splice site (ss) recognition, branching, and intron release, but lacked information on 3'-ss recognition, exon ligation, and exon release. Here we report a cryo-electron microscopy structure of the postcatalytic P complex at 3.3-angstrom resolution, revealing that the 3' ss is mainly recognized through non-Watson-Crick base pairing with the 5' ss and branch point. Furthermore, one or more unidentified proteins become stably associated with the P complex, securing the 3' exon and potentially regulating activity of the helicase Prp22. Prp22 binds nucleotides 15 to 21 in the 3' exon, enabling it to pull the intron-exon or ligated exons in a 3' to 5' direction to achieve 3'-ss proofreading or exon release, respectively.

Genome stability is significantly influenced by the precise coordination of chromatin complexes that facilitate the loading and eviction of histones from chromatin during replication, transcription, and DNA repair processes. In this study, we investigate the role of the Arabidopsis H3 histone chaperones ANTI-SILENCING FUNCTION 1 (ASF1) and HISTONE REGULATOR A (HIRA) in the maintenance of telomeres and 45S rDNA loci, genomic sites that are particularly susceptible to changes in the chromatin structure. We find that both ASF1 and HIRA are essential for telomere length regulation, as telomeres are significantly shorter in asf1a1b and hira mutants. However, these shorter telomeres remain localized around the nucleolus and exhibit a comparable relative H3 occupancy to the wild type. In addition to regulating telomere length, ASF1 and HIRA contribute to silencing 45S rRNA genes and affect their copy number. Besides, ASF1 supports global heterochromatin maintenance. Our findings also indicate that ASF1 transiently binds to the TELOMERE REPEAT BINDING 1 protein and the N terminus of telomerase in vivo, suggesting a physical link between the ASF1 histone chaperone and the telomere maintenance machinery., (© 2024 The Author(s). The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Backgruound: Phosphatidylinositol 3-kinase (PI3K) regulates cellular development and energy homeostasis. However, the roles of its subunits in organ development remain largely unknown., Methods: We explored the roles of PI3K catalytic subunits in steroidogenic factor 1 (SF1)-expressing cells through knockout (KO) of the p110α and p110β subunits., Results: We examined mice with a double KO of p110α and p110β in SF1-expressing cells (p110αβ KOSF1). Although these animals exhibited no significant changes in the development of the ventromedial hypothalamus, we noted pronounced hypotrophy in the adrenal cortex, testis, and ovary. Additionally, corticosterone and aldosterone levels were significantly reduced. The absence of these subunits also resulted in decreased body weight and survival rate, along with impaired glucose homeostasis, in p110αβ KOSF1 mice., Conclusion: The data demonstrate the specific roles of PI3K catalytic subunits in the development and function of SF1-expressing organs.

N6-methyladenosine (m6A) is the most abundant modification controlling RNA metabolism and cellular functions, but its roles in placental development are still poorly understood. Here, we characterized the synchronization of m6A modifications and placental functions by mapping the m6A methylome in human placentas (n = 3, each trimester), revealing that the dynamic patterns of m6A were associated with gene expression homeostasis and different biological pathways in placental development. Then, we generated trophoblast-specific knockout mice of Wtap, a critical component of methyltransferase complex, and demonstrated that Wtap was essential for trophoblast proliferation, placentation and perinatal growth. Further in vitro experiments which includes cell viability assays and series molecular binding assays demonstrated that WTAP-m6A-IGF2BP3 axis regulated the RNA stability and translation of Anillin (ANLN) and VEGFA, promoting trophoblast proliferation and secretion. Dysregulation of this regulatory axis was observed in placentas from pregnancies with fetal growth restriction (FGR) or preeclampsia, revealing the pathogenic effects of imbalanced m6A modifications. Therefore, our findings provide novel insights into the functions and regulatory mechanisms of m6A modifications in placental development and placental-related gestational diseases., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Objective: This study aimed to analyze the functional roles and molecular mechanism of Wilms' tumor 1-associating protein (WTAP) in the tumorigenesis of nonsmall-cell lung cancer (NSCLC)., Methods: Retrospective analysis was used. Tumor tissues and surrounding nontumor tissues of 150 patients with NSCLS who were surgically resected in the Fourth Hospital of Hebei Medical University from January 2016 to January 2018 were selected. The expression of WTAP in NSCLC tissues was detected by immunohistochemistry. Clinicopathologic parameters were then subjected to univariate and multivariate Cox regression analysis in purpose of uncovering the independent risk factors for overall survival time. MTS (3-[4,5-dimethylthiazol-zyl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazoliuzolium, inner salt) assay, colony formation assay, and transwell assays were performed to estimate cell proliferation, migration, and invasion. Meanwhile, the relationship between WTAP and the cell migration and invasion marker-related proteins were evaluated by Western blot analysis and RT-qPCR. WTAP expression was knocked-down in cell lines by shRNA, and RNA-Seq was performed to investigate the pathways regulated by WTAP., Results: In NSCLC patients, WTAP was highly expressed in tumor tissues and the higher expression was significantly associated with poor overall survival (OS) ( P <0.01). Compared with the control group in vitro, the overexpression of WTAP could significantly promote cell proliferation, migration, and invasion ( P <0.01), while knock-down WTAP significantly reduces the above effects ( P <0.01). In a mouse orthotopic implantation model, higher WTAP abundance could significantly promote tumor enlargement compared with the control group ( P <0.01). Compared with the control group, the knock-down of WTAP significantly inhibit the expression of carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) in cell lines ( P <0.01). Besides, in NSCLC, knocked-down CEACAM5 significantly reduced the impact of WTAP on cell proliferation, migration, and invasion compared with the control group ( P <0.05)., Conclusions: This study suggests that high expression of WTAP was associated with poor clinical outcomes. CEACAM5 may play a synergistic role with WTAP to jointly promote NSCLC progression by enhancing cell proliferation, invasion, and migration., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Sex steroid hormones, primarily synthesized by gonadal somatic cells, are pivotal for sexual development and reproduction. Mice studies have shown that two transcription factors, steroidogenic factor 1 (SF-1) and Wilms' tumor 1 (WT1), are involved in gonadal development. However, their role in human gonadal somatic differentiation remains unclear. We therefore aimed to investigate the roles of SF-1 and WT1 in human gonadal steroidogenic cell differentiation., Methods: Using a transient lentivirus-mediated gene expression system, we assessed the effects of SF-1 and WT1 expression on the steroidogenic potential of human amniotic membrane-derived mesenchymal stem cells (hAmMSCs)., Results: SF-1 and WT1-KTS, a splice variant of WT1, played distinct roles in human steroidogenic differentiation of hAmMSCs. SF-1 induced hAmMSC differentiation into progesterone- and androgen-producing cell lineages, whereas WT1-KTS promoted hAmMSC differentiation into estrogen-producing cell lineages., Conclusion: Our findings revealed that SF-1 and WT1-KTS play important roles in human gonadal steroidogenic cell differentiation, especially during ovarian development. These findings may pave the way for future studies on human ovarian differentiation and development., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Miyazaki, Orisaka, Fujita, Mizutani, Yazawa and Yoshida.)

Background: Dysregulated lipid oxidation occurs in several pathological processes characterized by cell proliferation and migration. Nonetheless, the molecular mechanism of lipid oxidation is not well appreciated in liver fibrosis, which is accompanied by enhanced fibroblast proliferation and migration., Methods: We investigated the causes and consequences of lipid oxidation in liver fibrosis using cultured cells, animal models, and clinical samples., Results: Increased ecto-nucleotide pyrophosphatase/phosphodiesterase (ENPP1) expression caused increased lipid oxidation, resulting in the proliferation and migration of hepatic stellate cells (HSCs) that lead to liver fibrosis, whereas fibroblast-specific ENPP1 knockout reversing these results. Elevated ENPP1 and N 6 -methyladenosine (m 6 A) levels were associated with high expression of Wilms tumor 1 associated protein (WTAP). Mechanistically, WTAP-mediated m 6 A methylation of the 3'UTR of ENPP1 mRNA and induces its translation dependent of YTH domain family proteins 1 (YTHDF1). Additionally, ENPP1 could interact with hypoxia inducible lipid droplet associated (HILPDA) directly; overexpression of ENPP1 further recruits HILPDA-mediated lipid oxidation, thereby promotes HSCs proliferation and migration, while inhibition of ENPP1 expression produced the opposite effect. Clinically, increased expression of WTAP, YTHDF1, ENPP1, and HILPDA, and increased m 6 A mRNA content, enhanced lipid oxidation, and increased collagen deposition in human liver fibrosis tissues., Conclusions: We describe a novel mechanism in which WTAP catalyzes m 6 A methylation of ENPP1 in a YTHDF1-dependent manner to enhance lipid oxidation, promoting HSCs proliferation and migration and liver fibrosis., (© 2024. The Author(s).)

This study aims to explore the possible effect and mechanism of heterogeneous nuclear ribonucleoprotein L (HNRNPL) on the lipid droplet and proliferation ability of clear cell renal cell carcinoma (ccRCC). The mRNA and protein expressions of HNRNPL and WSB1 on ccRCC tissues and cells were detected using qRT-PCR and western blot. The lipid droplet of cells was assessed after Oil Red O staining and BODIPY 493/503 staining. Cell proliferation was detected by CCK-8 assay. The interaction between HNRNPL and WSB1 was verified using RNA immunoprecipitation (RIP) and RNA-pull down assay. WSB1 mRNA stability was measured by Actinomycin D. Elevated expressions of HNRNPL and WSB1 were found in both ccRCC tissues and cells. HNRNPL knockdown can lead to suppressed lipid droplet and cell proliferation ability of ccRCC cells, while expression pattern was found in cells with HNRNPL overexpression. RIP and RNA-pull down assay clarified the binding of HNRNPL with WSB1. HNRNPL can facilitate the stability and expression of WSB1 mRNA. Rescue assay identified the promotive effect of HNRNPL on lipid droplets and cell proliferation of ccRCC cells can be abolished in response to WSB1 knockdown. Collected evidence summarized that HNRNPL can increase the stability of WSB1 mRNA to promote lipid droplet and proliferation ability in ccRCC cells., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The abundance of mRNA is mainly determined by the rates of RNA transcription and decay. Here, we present a method for unbiased estimation of differential mRNA decay rate from RNA-sequencing data by modeling the kinetics of mRNA metabolism. We show that in all primary human tissues tested, and particularly in the central nervous system, many pathways are regulated at the mRNA stability level. We present a parsimonious regulatory model consisting of two RNA-binding proteins and four microRNAs that modulate the mRNA stability landscape of the brain, which suggests a new link between RBFOX proteins and Alzheimer's disease. We show that downregulation of RBFOX1 leads to destabilization of mRNAs encoding for synaptic transmission proteins, which may contribute to the loss of synaptic function in Alzheimer's disease. RBFOX1 downregulation is more likely to occur in older and female individuals, consistent with the association of Alzheimer's disease with age and gender."mRNA abundance is determined by the rates of transcription and decay. Here, the authors propose a method for estimating the rate of differential mRNA decay from RNA-seq data and model mRNA stability in the brain, suggesting a link between mRNA stability and Alzheimer's disease."

Comprehensive multiplatform analysis of 80 uveal melanomas (UM) identifies four molecularly distinct, clinically relevant subtypes: two associated with poor-prognosis monosomy 3 (M3) and two with better-prognosis disomy 3 (D3). We show that BAP1 loss follows M3 occurrence and correlates with a global DNA methylation state that is distinct from D3-UM. Poor-prognosis M3-UM divide into subsets with divergent genomic aberrations, transcriptional features, and clinical outcomes. We report change-of-function SRSF2 mutations. Within D3-UM, EIF1AX- and SRSF2/SF3B1-mutant tumors have distinct somatic copy number alterations and DNA methylation profiles, providing insight into the biology of these low- versus intermediate-risk clinical mutation subtypes.

In this study, we report that TIP39, a parathyroid hormone ligand family member that was recently identified to be expressed in the skin, can induce decorin expression and enhance wound repair. Topical treatment of mice with TIP39 accelerated wound repair, whereas TIP39-deficient mice had delayed repair that was associated with formation of abnormal collagen bundles. To study the potential mechanism responsible for the action of TIP39 in the dermis, fibroblasts were cultured in three-dimensional collagen gels, a process that results in enhanced decorin expression unless activated to differentiate to adipocytes, whereupon these cells reduce expression of several proteoglycans, including decorin. Small interfering RNA-mediated silencing of parathyroid hormone 2 receptor (PTH2R), the receptor for TIP39, suppressed the expression of extracellular matrix-related genes, including decorin, collagens, fibronectin, and matrix metalloproteases. Skin wounds in TIP39-/- mice had decreased decorin expression, and addition of TIP39 to cultured fibroblasts induced decorin and increased phosphorylation and nuclear translocation of CREB. Fibroblasts differentiated to adipocytes and treated with TIP39 also showed increased decorin and production of chondroitin sulfate. Furthermore, the skin of PTH2R-/- mice showed abnormal extracellular matrix structure, decreased decorin expression, and skin hardness. Thus, the TIP39-PTH2R system appears to be a previously unrecognized mechanism for regulation of extracellular matrix formation and wound repair.

Proteins of the Rbfox family act with a complex of proteins called the Large Assembly of Splicing Regulators (LASR). We find that Rbfox interacts with LASR via its C-terminal domain (CTD), and this domain is essential for its splicing activity. In addition to LASR recruitment, a low-complexity (LC) sequence within the CTD contains repeated tyrosines that mediate higher-order assembly of Rbfox/LASR and are required for splicing activation by Rbfox. This sequence spontaneously aggregates in solution to form fibrous structures and hydrogels, suggesting an assembly similar to the insoluble cellular inclusions formed by FUS and other proteins in neurologic disease. Unlike the pathological aggregates, we find that assembly of the Rbfox CTD plays an essential role in its normal splicing function. Rather than simple recruitment of individual regulators to a target exon, alternative splicing choices also depend on the higher-order assembly of these regulators within the nucleus.

Post-translational modifications contribute to the spliceosome dynamics by facilitating the physical rearrangements of the spliceosome. Here, we report USP15, a deubiquitinating enzyme, as a regulator of protein-protein interactions for the spliceosome dynamics. We show that PRP31, a component of U4 snRNP, is modified with K63-linked ubiquitin chains by the PRP19 complex and deubiquitinated by USP15 and its substrate targeting factor SART3. USP15SART3 makes a complex with USP4 and this ternary complex serves as a platform to deubiquitinate PRP31 and PRP3. The ubiquitination and deubiquitination status of PRP31 regulates its interaction with the U5 snRNP component PRP8, which is required for the efficient splicing of chromosome segregation related genes, probably by stabilizing the U4/U6.U5 tri-snRNP complex. Collectively, our data suggest that USP15 plays a key role in the regulation of dynamic protein-protein interactions of the spliceosome.

Recent studies have characterized the extensive somatic alterations that arise during cancer. However, the somatic evolution of a tumor may be significantly affected by inherited polymorphisms carried in the germline. Here, we analyze genomic data for 5,954 tumors to reveal and systematically validate 412 genetic interactions between germline polymorphisms and major somatic events, including tumor formation in specific tissues and alteration of specific cancer genes. Among germline-somatic interactions, we found germline variants in RBFOX1 that increased incidence of SF3B1 somatic mutation by 8-fold via functional alterations in RNA splicing. Similarly, 19p13.3 variants were associated with a 4-fold increased likelihood of somatic mutations in PTEN. In support of this association, we found that PTEN knockdown sensitizes the MTOR pathway to high expression of the 19p13.3 gene GNA11 Finally, we observed that stratifying patients by germline polymorphisms exposed distinct somatic mutation landscapes, implicating new cancer genes. This study creates a validated resource of inherited variants that govern where and how cancer develops, opening avenues for prevention research.Significance: This study systematically identifies germline variants that directly affect tumor evolution, either by dramatically increasing alteration frequency of specific cancer genes or by influencing the site where a tumor develops. Cancer Discovery; 7(4); 410-23. ©2017 AACR.See related commentary by Geeleher and Huang, p. 354This article is highlighted in the In This Issue feature, p. 339.

Skin pigmentation is a complex trait that varies largely among populations. Most genome-wide association studies of this trait have been performed in Europeans and Asians. We aimed to uncover genes influencing skin colour in African-admixed individuals. We performed a genome-wide association study of melanin levels in 285 Hispanic/Latino individuals from Puerto Rico, analyzing 14 million genetic variants. A total of 82 variants with p-value ≤1 × 10-5 were followed up in 373 African Americans. Fourteen single nucleotide polymorphisms were replicated, of which nine were associated with skin colour at genome-wide significance in a meta-analysis across the two studies. These results validated the association of two previously known skin pigmentation genes, SLC24A5 (minimum p = 2.62 × 10-14, rs1426654) and SLC45A2 (minimum p = 9.71 × 10-10, rs16891982), and revealed the intergenic region of BEND7 and PRPF18 as a novel locus associated with this trait (minimum p = 4.58 × 10-9, rs6602666). The most significant variant within this region is common among African-descent populations but not among Europeans or Native Americans. Our findings support the advantages of analyzing African-admixed populations to discover new genes influencing skin pigmentation.

Alternative splicing (AS) is a major post-transcriptional mechanism to enhance the diversity of proteome in response to environmental signals. Among the numerous external signals perceived by plants, light is the most crucial one. Plants utilize complex photoreceptor signaling networks to sense different light conditions and adjust their growth and development accordingly. Although light-mediated gene expression has been widely investigated, little is known regarding the mechanism of light affecting AS to modulate mRNA at the post-transcriptional level. In this minireview, we summarize current progresses on how light affects AS, and how sensory photoreceptors and retrograde signaling pathways may coordinately regulate AS of pre-mRNAs. In addition, we also discuss the possibility that AS of the mRNAs encoding photoreceptors may be involved in feedback control of AS. We hypothesize that light regulation of the expression and activity of splicing factors would be a major mechanism of light-mediated AS. The combination of genetic study and high-throughput analyses of AS and splicing complexes in response to light is likely to further advance our understanding of the molecular mechanisms underlying light control of AS and plant development.

Alternative splicing, a key regulatory process of gene expression, is controlled by trans-acting factors that recognize cis-elements in premature RNA transcripts to affect spliceosome assembly and splice site choices. Extracellular stimuli and signaling cascades can converge on RNA binding splicing regulators to affect alternative splicing. Defects in splicing regulation have been associated with various human diseases, and modification of disease-causing splicing events presents great therapeutic promise. Determining splicing regulators and/or upstream modulators has been difficult with low throughput, low sensitivity, and low specificity. IRAS (identifying regulators of alternative splicing) is a novel cell-based high-throughput screening strategy designed specifically to address these challenges and has achieved high throughput, high sensitivity, and high specificity. Here, we describe the IRAS method in detail with a pair of dual-fluorescence minigene reporters that produces GFP and RFP fluorescent signals to assay the two spliced isoforms exclusively. These two complementary mini-gene reporters alter GFP/RFP output ratios in the opposite direction in response to only a true splicing change. False positives from a signal screen do not stimulate opposite changes in GFP/RFP ratios. The reporter pair in conjunction with robotic liquid handlers and arrayed libraries allows IRAS to screen for both positive and negative splicing regulators with high sensitivity and specificity in a high-throughput manner.

Mutations in SF3B1, which encodes a spliceosome component, are associated with poor outcome in chronic lymphocytic leukemia (CLL), but how these contribute to CLL progression remains poorly understood. We undertook a transcriptomic characterization of primary human CLL cells to identify transcripts and pathways affected by SF3B1 mutation. Splicing alterations, identified in the analysis of bulk cells, were confirmed in single SF3B1-mutated CLL cells and also found in cell lines ectopically expressing mutant SF3B1. SF3B1 mutation was found to dysregulate multiple cellular functions including DNA damage response, telomere maintenance, and Notch signaling (mediated through KLF8 upregulation, increased TERC and TERT expression, or altered splicing of DVL2 transcript, respectively). SF3B1 mutation leads to diverse changes in CLL-related pathways.

Myelodysplastic syndromes (MDS) are heterogeneous myeloid disorders with prevalent mutations in several splicing factors, but the splicing programs linked to specific mutations or MDS in general remain to be systematically defined. We applied RASL-seq, a sensitive and cost-effective platform, to interrogate 5502 annotated splicing events in 169 samples from MDS patients or healthy individuals. We found that splicing signatures associated with normal hematopoietic lineages are largely related to cell signaling and differentiation programs, whereas MDS-linked signatures are primarily involved in cell cycle control and DNA damage responses. Despite the shared roles of affected splicing factors in the 3 splice site definition, mutations in U2AF1, SRSF2, and SF3B1 affect divergent splicing programs, and interestingly, the affected genes fall into converging cancer-related pathways. A risk score derived from 11 splicing events appears to be independently associated with an MDS prognosis and AML transformation, suggesting potential clinical relevance of altered splicing patterns in MDS.

Many RNA binding proteins (RBPs) bind specific RNA sequence motifs, but only a small fraction (∼15%-40%) of RBP motif occurrences are occupied in vivo. To determine which contextual features discriminate between bound and unbound motifs, we performed an in vitro binding assay using 12,000 mouse RNA sequences with the RBPs MBNL1 and RBFOX2. Surprisingly, the strength of binding to motif occurrences in vitro was significantly correlated with in vivo binding, developmental regulation, and evolutionary age of alternative splicing. Multiple lines of evidence indicate that the primary context effect that affects binding in vitro and in vivo is RNA secondary structure. Large-scale combinatorial mutagenesis of unfavorable sequence contexts revealed a consistent pattern whereby mutations that increased motif accessibility improved protein binding and regulatory activity. Our results indicate widespread inhibition of motif binding by local RNA secondary structure and suggest that mutations that alter sequence context commonly affect RBP binding and regulation.

Alternative splicing is prevalent in the mammalian brain. To interrogate the functional role of alternative splicing in neural development, we analyzed purified neural progenitor cells (NPCs) and neurons from developing cerebral cortices, revealing hundreds of differentially spliced exons that preferentially alter key protein domains-especially in cytoskeletal proteins-and can harbor disease-causing mutations. We show that Ptbp1 and Rbfox proteins antagonistically govern the NPC-to-neuron transition by regulating neuron-specific exons. Whereas Ptbp1 maintains apical progenitors partly through suppressing a poison exon of Flna in NPCs, Rbfox proteins promote neuronal differentiation by switching Ninein from a centrosomal splice form in NPCs to a non-centrosomal isoform in neurons. We further uncover an intronic human mutation within a PTBP1-binding site that disrupts normal skipping of the FLNA poison exon in NPCs and causes a brain-specific malformation. Our study indicates that dynamic control of alternative splicing governs cell fate in cerebral cortical development.

The protein SF3B1 is a core component of the spliceosome, the large ribonucleoprotein complex responsible for pre-mRNA splicing. Interest in SF3B1 intensified when tumor exome sequencing revealed frequent specific SF3B1 mutations in a variety of neoplasia and when SF3B1 was identified as the target of three different cancer cell growth inhibitors. A better mechanistic understanding of SF3B1's role in splicing is required to capitalize on these discoveries. Using the inhibitor compounds, we probed SF3B1 function in the spliceosome in an in vitro splicing system. Formerly, the inhibitors were shown to block early steps of spliceosome assembly, consistent with a previously determined role of SF3B1 in intron recognition. We now report that SF3B1 inhibitors also interfere with later events in the spliceosome cycle, including exon ligation. These observations are consistent with a requirement for SF3B1 throughout the splicing process. Additional experiments aimed at understanding how three structurally distinct molecules produce nearly identical effects on splicing revealed that inactive analogs of each compound interchangeably compete with the active inhibitors to restore splicing. The competition indicates that all three types of compounds interact with the same site on SF3B1 and likely interfere with its function by the same mechanism, supporting a shared pharmacophore model. It also suggests that SF3B1 inhibition does not result from binding alone, but is consistent with a model in which the compounds affect a conformational change in the protein. Together, our studies reveal new mechanistic insight into SF3B1 as a principal player in the spliceosome and as a target of inhibitor compounds.

Differentiating erythroblasts execute a dynamic alternative splicing program shown here to include extensive and diverse intron retention (IR) events. Cluster analysis revealed hundreds of developmentally-dynamic introns that exhibit increased IR in mature erythroblasts, and are enriched in functions related to RNA processing such as SF3B1 spliceosomal factor. Distinct, developmentally-stable IR clusters are enriched in metal-ion binding functions and include mitoferrin genes SLC25A37 and SLC25A28 that are critical for iron homeostasis. Some IR transcripts are abundant, e.g. comprising ∼50% of highly-expressed SLC25A37 and SF3B1 transcripts in late erythroblasts, and thereby limiting functional mRNA levels. IR transcripts tested were predominantly nuclear-localized. Splice site strength correlated with IR among stable but not dynamic intron clusters, indicating distinct regulation of dynamically-increased IR in late erythroblasts. Retained introns were preferentially associated with alternative exons with premature termination codons (PTCs). High IR was observed in disease-causing genes including SF3B1 and the RNA binding protein FUS. Comparative studies demonstrated that the intron retention program in erythroblasts shares features with other tissues but ultimately is unique to erythropoiesis. We conclude that IR is a multi-dimensional set of processes that post-transcriptionally regulate diverse gene groups during normal erythropoiesis, misregulation of which could be responsible for human disease.

RNA splicing is a major contributor to total transcriptome complexity; however, the functional role and regulation of splicing in heart failure remain poorly understood. Here, we used a total transcriptome profiling and bioinformatic analysis approach and identified a muscle-specific isoform of an RNA splicing regulator, RBFox1 (also known as A2BP1), as a prominent regulator of alternative RNA splicing during heart failure. Evaluation of developing murine and zebrafish hearts revealed that RBFox1 is induced during postnatal cardiac maturation. However, we found that RBFox1 is markedly diminished in failing human and mouse hearts. In a mouse model, RBFox1 deficiency in the heart promoted pressure overload-induced heart failure. We determined that RBFox1 is a potent regulator of RNA splicing and is required for a conserved splicing process of transcription factor MEF2 family members that yields different MEF2 isoforms with differential effects on cardiac hypertrophic gene expression. Finally, induction of RBFox1 expression in murine pressure overload models substantially attenuated cardiac hypertrophy and pathological manifestations. Together, this study identifies regulation of RNA splicing by RBFox1 as an important player in transcriptome reprogramming during heart failure that influence pathogenesis of the disease.

Human genetic studies have identified the neuronal RNA binding protein, Rbfox1, as a candidate gene for autism spectrum disorders. While Rbfox1 functions as a splicing regulator in the nucleus, it is also alternatively spliced to produce cytoplasmic isoforms. To investigate the function of cytoplasmic Rbfox1, we knocked down Rbfox proteins in mouse neurons and rescued with cytoplasmic or nuclear Rbfox1. Transcriptome profiling showed that nuclear Rbfox1 rescued splicing changes, whereas cytoplasmic Rbfox1 rescued changes in mRNA levels. iCLIP-seq of subcellular fractions revealed that Rbfox1 bound predominantly to introns in nascent RNA, while cytoplasmic Rbox1 bound to 3' UTRs. Cytoplasmic Rbfox1 binding increased target mRNA stability and translation, and Rbfox1 and miRNA binding sites overlapped significantly. Cytoplasmic Rbfox1 target mRNAs were enriched in genes involved in cortical development and autism. Our results uncover a new Rbfox1 regulatory network and highlight the importance of cytoplasmic RNA metabolism to cortical development and disease.

Congenital heart disease (CHD) patients have an increased prevalence of extracardiac congenital anomalies (CAs) and risk of neurodevelopmental disabilities (NDDs). Exome sequencing of 1213 CHD parent-offspring trios identified an excess of protein-damaging de novo mutations, especially in genes highly expressed in the developing heart and brain. These mutations accounted for 20% of patients with CHD, NDD, and CA but only 2% of patients with isolated CHD. Mutations altered genes involved in morphogenesis, chromatin modification, and transcriptional regulation, including multiple mutations in RBFOX2, a regulator of mRNA splicing. Genes mutated in other cohorts examined for NDD were enriched in CHD cases, particularly those with coexisting NDD. These findings reveal shared genetic contributions to CHD, NDD, and CA and provide opportunities for improved prognostic assessment and early therapeutic intervention in CHD patients.

Background: N6-methyladenosine (m6A) is one of the most extensive RNA methylation modifications in eukaryotes and participates in the pathogenesis of numerous diseases including ischemic stroke. Peripheral blood neutrophils are forerunners after ischemic brain injury and exert crucial functions. This study aims to explore the transcriptional profiles of m6A modification in neutrophils of patients with ischemic stroke., Results: We found that the expression levels of m6A regulators FTO and YTHDC1 were notably decreased in the neutrophils following ischemic stroke, and FTO expression was negatively correlated with neutrophil counts and neutrophil-to-lymphocyte ratio (NLR). The m6A mRNA&lncRNA epigenetic transcriptome microarray identified 416 significantly upregulated and 500 significantly downregulated mRNA peaks in neutrophils of ischemic stroke patients. Moreover, 48 mRNAs and 18 lncRNAs were hypermethylated, and 115 mRNAs and 29 lncRNAs were hypomethylated after cerebral ischemia. Gene ontology (GO) analysis identified that these m6A-modified mRNAs were primarily enriched in calcium ion transport, long-term synaptic potentiation, and base-excision repair. The signaling pathways involved were EGFR tyrosine kinase inhibitor resistance, ErbB, and base excision repair signaling pathway. MeRIP-qPCR validation results showed that NRG1 and GDPD1 were significantly hypermethylated, and LIG1, CHRND, lncRNA RP11-442J17.2, and lncRNA RP11-600P1.2 were significantly hypomethylated after cerebral ischemia. Moreover, the expression levels of major m6A regulators Mettl3, Fto, Ythdf1, and Ythdf3 were obviously declined in the brain and leukocytes of post-stroke mouse models., Conclusion: This study explored the RNA m6A methylation pattern in the neutrophils of ischemic stroke patients, indicating that it is an intervention target of epigenetic regulation in ischemic stroke., (Copyright © 2024 IBRO. Published by Elsevier Inc. All rights reserved.)

Esophageal squamous cell carcinoma (ESCC) represents a frequently seen malignancy with high prevalence worldwide. Although current studies have shown that Wilms' tumor 1-associated protein (WTAP), a major part in the methyltransferase complex, is involved in various tumor pathological processes, its specific role in ESCC remains unclear. Therefore, the present work focused on exploring WTAP's function and mechanism in ESCC progression using clinical ESCC specimens, ESCC cells, and mammalian models. Firstly, we proved WTAP was significantly upregulated within ESCC, and WTAP mRNA expression showed a good diagnostic performance for ESCC. Functionally, WTAP positively regulated in-vivo and in-vitro ESCC cells' malignant phenotype through the AKT-mTOR signaling pathway. Meanwhile, WTAP positively regulated the N6-methyladenosine (m 6 A) modification levels in ESCC cells. Protein tyrosine phase type IVA member 1 (PTP4A1) was confirmed to be the m 6 A target of WTAP, and WTAP positively regulated the expression of PTP4A1. Further study revealed that PTP4A1 showed high expression within ESCC. Silencing PTP4A1 inhibited the AKT-mTOR signaling pathway to suppress ESCC cells' proliferation. Rescue experiments showed that silencing PTP4A1 partially reversed the WTAP-promoting effect on ESCC cells' proliferation ability. Mechanistically, WTAP regulated PTP4A1 expression to activate the AKT-mTOR pathway, promoting the proliferation of ESCC cells. Our study demonstrated that WTAP regulates the progression of ESCC through the m 6 A-PTP4A1-AKT-mTOR signaling axis and that WTAP is a potential target for diagnosing and treating ESCC., (© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

The IL-17 receptor adaptor molecule Act1, an RNA-binding protein, plays a critical role in IL-17-mediated cancer progression. Here, we report a novel mechanism of how IL-17/Act1 induces chemoresistance by modulating redox homeostasis through epitranscriptomic regulation of antioxidant RNA metabolism. Transcriptome-wide mapping of direct Act1-RNA interactions revealed that Act1 binds to the 5'UTR of antioxidant mRNAs and Wilms' tumor 1-associating protein (WTAP), a key regulator in m6A methyltransferase complex. Strikingly, Act1's binding sites are located in proximity to m6A modification sites, which allows Act1 to promote the recruitment of elF3G for cap-independent translation. Loss of Act1's RNA binding activity or Wtap knockdown abolished IL-17-induced m6A modification and translation of Wtap and antioxidant mRNAs, indicating a feedforward mechanism of the Act1-WTAP loop. We then developed antisense oligonucleotides (Wtap ASO) that specifically disrupt Act1's binding to Wtap mRNA, abolishing IL-17/Act1-WTAP-mediated antioxidant protein production during chemotherapy. Wtap ASO substantially increased the antitumor efficacy of cisplatin, demonstrating a potential therapeutic strategy for chemoresistance., (© 2024 Hong et al.)

The Prp19 complex (Prp19C), also named NineTeen Complex (NTC), is conserved from yeast to human and functions in many different processes such as genome stability, splicing, and transcription elongation. In the latter, Prp19C ensures TREX occupancy at transcribed genes. TREX, in turn, couples transcription to nuclear mRNA export by recruiting the mRNA exporter to transcribed genes and consequently to nascent mRNAs. Here, we assess the function of the nonessential Prp19C subunit Syf2 and the nonessential Prp19C-associated protein Cwc15 in the interaction of Prp19C and TREX with the transcription machinery, Prp19C and TREX occupancy, and transcription elongation. Whereas both proteins are important for Prp19C-TREX interaction, Syf2 is needed for full Prp19C occupancy, and Cwc15 is important for the interaction of Prp19C with RNA polymerase II and TREX occupancy. These partially overlapping functions are corroborated by a genetic interaction between Δcwc15 and Δsyf2 Finally, Cwc15 also interacts genetically with the transcription elongation factor Dst1 and functions in transcription elongation. In summary, we uncover novel roles of the Prp19C component Syf2 and the Prp19C-associated protein Cwc15 in Prp19C's function in transcription elongation., (© 2024 Henke-Schulz et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Human protein arginine methyltransferase (PRMT) 9 symmetrically dimethylates arginine residues on splicing factor SF3B2 (SAP145) and has been functionally linked to the regulation of alternative splicing of pre-mRNA. Site-directed mutagenesis studies on this enzyme and its substrate had revealed essential unique residues in the double E loop and the importance of the C-terminal duplicated methyltransferase domain. In contrast to what had been observed with other PRMTs and their physiological substrates, a peptide containing the methylatable Arg-508 of SF3B2 was not recognized by PRMT9 in vitro. Although amino acid substitutions of residues surrounding Arg-508 had no great effect on PRMT9 recognition of SF3B2, moving the arginine residue within this sequence abolished methylation. PRMT9 and PRMT5 are the only known mammalian enzymes capable of forming symmetric dimethylarginine (SDMA) residues as type II PRMTs. We demonstrate here that the specificity of these enzymes for their substrates is distinct and not redundant. The loss of PRMT5 activity in mouse embryo fibroblasts results in almost complete loss of SDMA, suggesting that PRMT5 is the primary SDMA-forming enzyme in these cells. PRMT9, with its duplicated methyltransferase domain and conserved sequence in the double E loop, appears to have a unique structure and specificity among PRMTs for methylating SF3B2 and potentially other polypeptides.

RNA splicing plays a fundamental role in human biology. Its relevance in cancer is rapidly emerging as demonstrated by spliceosome mutations that determine the prognosis of patients with hematologic malignancies. We report studies using FD-895 and pladienolide-B in primary leukemia cells derived from patients with chronic lymphocytic leukemia and leukemia-lymphoma cell lines. We found that FD-895 and pladienolide-B induce an early pattern of mRNA intron retention - spliceosome modulation. This process was associated with apoptosis preferentially in cancer cells as compared to normal lymphocytes. The pro-apoptotic activity of these compounds was observed regardless of poor prognostic factors such as Del(17p), TP53 or SF3B1 mutations and was able to overcome the protective effect of culture conditions that resemble the tumor microenvironment. In addition, the activity of these compounds was observed not only in vitro but also in vivo using the A20 lymphoma murine model. Overall, these findings give evidence for the first time that spliceosome modulation is a valid target in chronic lymphocytic leukemia and provide an additional rationale for the development of spliceosome modulators for cancer therapy.

Mutations in the splicing factor SF3B1 are found in several cancer types and have been associated with various splicing defects. Using transcriptome sequencing data from chronic lymphocytic leukemia, breast cancer and uveal melanoma tumor samples, we show that hundreds of cryptic 3' splice sites (3'SSs) are used in cancers with SF3B1 mutations. We define the necessary sequence context for the observed cryptic 3' SSs and propose that cryptic 3'SS selection is a result of SF3B1 mutations causing a shift in the sterically protected region downstream of the branch point. While most cryptic 3'SSs are present at low frequency (

The pairing of 5' and 3' splice sites across an intron is a critical step in spliceosome formation and its regulation. Interactions that bring the two splice sites together during spliceosome assembly must occur with a high degree of specificity and fidelity to allow expression of functional mRNAs and make particular alternative splicing choices. Here, we report a new interaction between stem-loop 4 (SL4) of the U1 snRNA, which recognizes the 5' splice site, and a component of the U2 small nuclear ribonucleoprotein particle (snRNP) complex, which assembles across the intron at the 3' splice site. Using a U1 snRNP complementation assay, we found that SL4 is essential for splicing in vivo. The addition of free U1-SL4 to a splicing reaction in vitro inhibits splicing and blocks complex assembly prior to formation of the prespliceosomal A complex, indicating a requirement for a SL4 contact in spliceosome assembly. To characterize the interactions of this RNA structure, we used a combination of stable isotope labeling by amino acids in cell culture (SILAC), biotin/Neutravidin affinity pull-down, and mass spectrometry. We show that U1-SL4 interacts with the SF3A1 protein of the U2 snRNP. We found that this interaction between the U1 snRNA and SF3A1 occurs within prespliceosomal complexes assembled on the pre-mRNA. Thus, SL4 of the U1 snRNA is important for splicing, and its interaction with SF3A1 mediates contact between the 5' and 3' splice site complexes within the assembling spliceosome.

The Cdc45/Mcm2-7/GINS (CMG) helicase separates DNA strands during replication in eukaryotes. How the CMG is assembled and engages DNA substrates remains unclear. Using electron microscopy, we have determined the structure of the CMG in the presence of ATPγS and a DNA duplex bearing a 3' single-stranded tail. The structure shows that the MCM subunits of the CMG bind preferentially to single-stranded DNA, establishes the polarity by which DNA enters into the Mcm2-7 pore, and explains how Cdc45 helps prevent DNA from dissociating from the helicase. The Mcm2-7 subcomplex forms a cracked-ring, right-handed spiral when DNA and nucleotide are bound, revealing unexpected congruencies between the CMG and both bacterial DnaB helicases and the AAA+ motor of the eukaryotic proteasome. The existence of a subpopulation of dimeric CMGs establishes the subunit register of Mcm2-7 double hexamers and together with the spiral form highlights how Mcm2-7 transitions through different conformational and assembly states as it matures into a functional helicase.

Saccharomyces cerevisiae has been used as a model system to investigate the mechanisms of pre-mRNA splicing but only a few examples of alternative splice site usage have been described in this organism. Using RNA-Seq analysis of nonsense-mediated mRNA decay (NMD) mutant strains, we show that many S. cerevisiae intron-containing genes exhibit usage of alternative splice sites, but many transcripts generated by splicing at these sites are non-functional because they introduce premature termination codons, leading to degradation by NMD. Analysis of splicing mutants combined with NMD inactivation revealed the role of specific splicing factors in governing the use of these alternative splice sites and identified novel functions for Prp17p in enhancing the use of branchpoint-proximal upstream 3' splice sites and for Prp18p in suppressing the usage of a non-canonical AUG 3'-splice site in GCR1. The use of non-productive alternative splice sites can be increased in stress conditions in a promoter-dependent manner, contributing to the down-regulation of genes during stress. These results show that alternative splicing is frequent in S. cerevisiae but masked by RNA degradation and that the use of alternative splice sites in this organism is mostly aimed at controlling transcript levels rather than increasing proteome diversity.

Pladienolide B (PB) is a potent cancer cell growth inhibitor that targets the SF3B1 subunit of the spliceosome. There is considerable interest in the compound as a potential chemotherapeutic, as well as a tool to study SF3B1 function in splicing and cancer development. The molecular structure of PB, a bacterial natural product, contains a 12-member macrolide ring with an extended epoxide-containing side chain. Using a novel concise enantioselective synthesis, we created a series of PB structural analogs and the structurally related compound herboxidiene. We show that two methyl groups in the PB side chain, as well as a feature of the macrolide ring shared with herboxidiene, are required for splicing inhibition in vitro. Unexpectedly, we find that the epoxy group contributes only modestly to PB potency and is not absolutely necessary for activity. The orientations of at least two chiral centers off the macrolide ring have no effect on PB activity. Importantly, the ability of analogs to inhibit splicing in vitro directly correlated with their effects in a series of cellular assays. Those effects likely arise from inhibition of some, but not all, endogenous splicing events in cells, as previously reported for the structurally distinct SF3B1 inhibitor spliceostatin A. Together, our data support the idea that the impact of PB on cells is derived from its ability to impair the function of SF3B1 in splicing and also demonstrate that simplification of the PB scaffold is feasible.

Alternative splicing (AS) enables programmed diversity of gene expression across tissues and development. We show here that binding in distal intronic regions (>500 nucleotides (nt) from any exon) by Rbfox splicing factors important in development is extensive and is an active mode of splicing regulation. Similarly to exon-proximal sites, distal sites contain evolutionarily conserved GCATG sequences and are associated with AS activation and repression upon modulation of Rbfox abundance in human and mouse experimental systems. As a proof of principle, we validated the activity of two specific Rbfox enhancers in KIF21A and ENAH distal introns and showed that a conserved long-range RNA-RNA base-pairing interaction (an RNA bridge) is necessary for Rbfox-mediated exon inclusion in the ENAH gene. Thus we demonstrate a previously unknown RNA-mediated mechanism for AS control by distally bound RNA-binding proteins.

During meiosis in yeast, global splicing efficiency increases and then decreases. Here we provide evidence that splicing improves due to reduced competition for the splicing machinery. The timing of this regulation corresponds to repression and reactivation of ribosomal protein genes (RPGs) during meiosis. In vegetative cells, RPG repression by rapamycin treatment also increases splicing efficiency. Downregulation of the RPG-dedicated transcription factor gene IFH1 genetically suppresses two spliceosome mutations, prp11-1 and prp4-1, and globally restores splicing efficiency in prp4-1 cells. We conclude that the splicing apparatus is limiting and that pre-messenger RNAs compete. Splicing efficiency of a pre-mRNA therefore depends not just on its own concentration and affinity for limiting splicing factor(s), but also on those of competing pre-mRNAs. Competition between RNAs for limiting processing factors appears to be a general condition in eukaryotes for a variety of posttranscriptional control mechanisms including microRNA (miRNA) repression, polyadenylation, and splicing.

Hypomyelinating leukodystrophy (HLD) is an autosomal recessive disorder characterized by defective central nervous system myelination. Exome sequencing of two siblings with severe cognitive and motor impairment and progressive hypomyelination characteristic of HLD revealed homozygosity for a missense single-nucleotide variant (SNV) in EPRS1 (c.4444 C > A; p.Pro1482Thr), encoding glutamyl-prolyl-tRNA synthetase, consistent with HLD15. Patient lymphoblastoid cell lines express markedly reduced EPRS1 protein due to dual defects in nuclear export and cytoplasmic translation of variant EPRS1 mRNA. Variant mRNA exhibits reduced METTL3 methyltransferase-mediated writing of N 6 -methyladenosine (m 6 A) and reduced reading by YTHDC1 and YTHDF1/3 required for efficient mRNA nuclear export and translation, respectively. In contrast to current models, the variant does not alter the sequence of m 6 A target sites, but instead reduces their accessibility for modification. The defect was rescued by antisense morpholinos predicted to expose m 6 A sites on target EPRS1 mRNA, or by m 6 A modification of the mRNA by METTL3-dCas13b, a targeted RNA methylation editor. Our bioinformatic analysis predicts widespread occurrence of SNVs associated with human health and disease that similarly alter accessibility of distal mRNA m 6 A sites. These results reveal a new RNA-dependent etiologic mechanism by which SNVs can influence gene expression and disease, consequently generating opportunities for personalized, RNA-based therapeutics targeting these disorders., (© 2024. The Author(s).)

N6-methyladenosine (m 6 A) is the most abundant dynamic and reversible internal chemical modification of RNA in eukaryotic cells and is essential in multiple pathophysiological processes. However, it has not been reported in atopic dermatitis (AD). We used Arraystar m 6 A-mRNA epitranscriptomic microarray to screen for differentially expressed genes and their m 6 A levels and m 6 A-related enzymes in patients with AD. We confirmed that the m 6 A RNA methyltransferase WTAP and 2 candidate differentially expressed genes (S100A9 and SERPINB3) were significantly upregulated in keratinocytes in public data and epidermal lesions of patients with AD. In vitro cell experiments confirmed that WTAP influenced the expression of the 2 candidate differentially expressed genes and promoted primary human epidermal keratinocyte proliferation while inhibiting human epidermal keratinocyte differentiation. Furthermore, we showed that WTAP, S100A9, and SERPINB3 expression correlated with AD severity. Our findings revealed that WTAP-mediated m 6 A modification promoted the expression of S100A9 and SERPINB3 to aggravate human epidermal keratinocyte proliferation and dysdifferentiation contributing to the pathophysiological development of AD., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

A fast evolution within mitochondria genome(s) often generates discords between nuclear and mitochondria, which is manifested as cytoplasmic male sterility (CMS) and fertility restoration (Rf) system. The maize CMS-C trait is regulated by the chimeric mitochondrial gene, atp6c, and can be recovered by the restorer gene ZmRf5. Through positional cloning in this study, we identified the nuclear restorer gene, ZmRf5, which encodes a P-type pentatricopeptide repeat (PPR) family protein. The over-expression of ZmRf5 brought back the fertility to CMS-C plants, whereas its genomic editing by CRISPR/Cas9 induced abortive pollens in the restorer line. ZmRF5 is sorted to mitochondria, and recruited RS31A, a splicing factor, through MORF8 to form a cleaving/restoring complex, which promoted the cleaving of the CMS-associated transcripts atp6c by shifting the major cleavage site from 480th nt to 344 th nt for fast degradation, and preserved just right amount of atp6c RNA for protein translation, providing adequate ATP6C to assembly complex V, thus restoring male fertility. Interestingly, ATP6C in the sterile line CMo17A, with similar cytology and physiology changes to YU87-1A, was accumulated much less than it in NMo17B, exhibiting a contrary trend in the YU87-1 nuclear genome previously reported, and was restored to normal level in the presence of ZmRF5. Collectively these findings unveil a new molecular mechanism underlying fertility restoration by which ZmRF5 cooperates with MORF8 and RS31A to restore CMS-C fertility in maize, complemented and perfected the sterility mechanism, and enrich the perspectives on communications between nucleus and mitochondria., (© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Background: Bladder cancer (BCa) is the most frequent type of cancer in humans. The association between m6A modification and the anti-tumor effects of natural killer (NK) cells has been described in BCa. This study intended to investigate the implications of m6A regulators in modulating SYTL1 expression in BCa and the association with the anti-tumor effects of NK cells., Methods: The prognostic role of SYTL1 in BCa was investigated using bioinformatics analysis, and the correlation between SYTL1 expression and NK cells was analyzed. The effects of SYTL1 on the anti-tumor response of NK-92 cells were examined by RT-qPCR, cytotoxicity, western blot, and ELISA assays. The relationships among WTAP, YTHDF2, and SYTL1 were investigated by RT-qPCR, RIP-qPCR, ELISA, and actinomycin D treatment. Finally, the effects of WTAP and SYTL1 on BCa tumor growth and the anti-tumor response of NK cells were verified in vivo ., Results: SYTL1 was reduced in BCa tissues and had a prognostic significance, which was related to NK cell-mediated anti-tumor responses. NK-92 cells produced toxicity to BCa cells, which was further enhanced by SYTL1 overexpression in BCa cells through prompting LDH, NKG2D, NKp30, and NKp44 and IFN-γ levels. WTAP enhanced the degradation of the SYTL1 mRNA by YTHDF2. WTAP and YTHDF2 impaired the anti-tumor response of NK cells in BCa. SYTL1 inhibited the BCa progression in mice while enhancing the anti-tumor response of NK cells., Conclusions: WTAP inhibited the anti-tumor response of NK cells to BCa cells by promoting the degradation of SYTL1 mRNA through YTHDF2-mediated m6A methylation., (©The Author(s) 2024. Open Access. This article is licensed under a Creative Commons CC-BY International License.)

Here, we identify RBM41 as a novel unique protein component of the minor spliceosome. RBM41 has no previously recognized cellular function but has been identified as a paralog of U11/U12-65K, a known unique component of the U11/U12 di-snRNP. Both proteins use their highly similar C-terminal RRMs to bind to 3'-terminal stem-loops in U12 and U6atac snRNAs with comparable affinity. Our BioID data indicate that the unique N-terminal domain of RBM41 is necessary for its association with complexes containing DHX8, an RNA helicase, which in the major spliceosome drives the release of mature mRNA from the spliceosome. Consistently, we show that RBM41 associates with excised U12-type intron lariats, is present in the U12 mono-snRNP, and is enriched in Cajal bodies, together suggesting that RBM41 functions in the post-splicing steps of the minor spliceosome assembly/disassembly cycle. This contrasts with U11/U12-65K, which uses its N-terminal region to interact with U11 snRNP during intron recognition. Finally, while RBM41 knockout cells are viable, they show alterations in U12-type 3' splice site usage. Together, our results highlight the role of the 3'-terminal stem-loop of U12 snRNA as a dynamic binding platform for the U11/U12-65K and RBM41 proteins, which function at distinct stages of the assembly/disassembly cycle., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

RNA-binding proteins (RBPs) make vital impacts on tumor progression and are important potential targets for tumor treatment. Previous studies have shown that RBP regulator of differentiation 1 (ROD1), enriched in the nucleus, is abnormally expressed and functions as a splicing factor in tumors; however, the mechanism underlying its involvement in gastric cancer (GC) is unknown. In this study, ROD1 is found to stimulate GC cell proliferation and metastasis and is related to poor patient prognosis. In vitro experiments showed that ROD1 influences GC proliferation and metastasis through modulating the imbalance of the level of the oncogenic gene OIP5 and the tumor suppressor gene GPD1L. Further studies showed that the N6-methyladenosine (m6A) "reader" protein YTHDC1 can interact with ROD1 and regulate the balance of the expression of the downstream molecules OIP5/GPD1L by promoting the nuclear enrichment of ROD1. Therefore, YTHDC1 stimulates GC development and progression through modulating nuclear enrichment of the splicing factor ROD1., (© 2024. The Author(s).)

Background: Protein arginine methyltransferase 5 (PRMT5) methylates multiple substrates dysregulated in cancer, including spliceosome machinery components. PF-06939999 is a selective small-molecule PRMT5 inhibitor., Patients and Methods: This phase I dose-escalation and -expansion trial (NCT03854227) enrolled patients with selected solid tumors. PF-06939999 was administered orally once or twice a day (q.d./b.i.d.) in 28-day cycles. The objectives were to evaluate PF-06939999 safety and tolerability to identify maximum tolerated dose (MTD) and recommended part 2 dose (RP2D), and assess pharmacokinetics (PK), pharmacodynamics [changes in plasma symmetric dimethylarginine (SDMA) levels], and antitumor activities., Results: In part 1 dose escalation, 28 patients received PF-06939999 (0.5 mg q.d. to 6 mg b.i.d.). Four of 24 (17%) patients reported dose-limiting toxicities: thrombocytopenia (n = 2, 6 mg b.i.d.), anemia (n = 1, 8 mg q.d.), and neutropenia (n = 1, 6 mg q.d.). PF-06939999 exposure increased with dose. Steady-state PK was achieved by day 15. Plasma SDMA was reduced at steady state (58%-88%). Modulation of plasma SDMA was dose dependent. No MTD was determined. In part 2 dose expansion, 26 patients received PF-06939999 6 mg q.d. (RP2D). Overall (part 1 + part 2), the most common grade ≥3 treatment-related adverse events included anemia (28%), thrombocytopenia/platelet count decreased (22%), fatigue (6%), and neutropenia (4%). Three patients (6.8%) had confirmed partial response (head and neck squamous cell carcinoma, n = 1; non-small-cell lung cancer, n = 2), and 19 (43.2%) had stable disease. No predictive biomarkers were identified., Conclusions: PF-06939999 demonstrated a tolerable safety profile and objective clinical responses in a subset of patients, suggesting that PRMT5 is an interesting cancer target with clinical validation. However, no predictive biomarker was identified. The role of PRMT5 in cancer biology is complex and requires further preclinical, mechanistic investigation to identify predictive biomarkers for patient selection., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Ferroptosis is involved in various pathophysiological diseases, including triple-negative breast cancer (TNBC). Targeting ferroptosis is considered as a novel anti-TNBC strategy. Nevertheless, the regulatory mechanism of ferroptosis during TNBC progression is unclear. Here, the role of WTAP in ferroptosis during TNBC progression  was investigated. The clinicopathological significance of WTAP, NUPR1 and LCN2 was analyzed by Kaplan-Meier method. Cell viability was assessed using MTT assay. Transwell assay was employed to analyze cell migration and invasion. GSH/GSSG and Fe 2+ levels in TNBC cells were analyzed using kits. m 6 A level was examined using m 6 A dot blot assay. NUPR1 mRNA stability was analyzed using RNA degradation assay. RIP was performed to analyze the interaction between eIF3a and NURP1. Herein, our results revealed that WTAP, NUPR1 and LCN2 expressions were significantly elevated in TNBC. NUPR1 silencing inhibited TNBC cell proliferation, migration and invasion by inducing ferroptosis. NUPR1 positively regulated LCN2 expression in TNBC cells, and LCN2 knockdown induced ferroptosis to suppress TNBC cell malignant behaviors. Our molecular study further revealed that WTAP promoted NUPR1 expression in an m 6 A-EIF3A mediated manner. And, as expected, WTAP knockdown promoted ferroptosis to suppress TNBC cell malignant behaviors, which were abrogated by NUPR1 overexpression. WTAP upregulated LCN2 by regulation of NUPR1 m 6 A modification, thereby suppressing ferroptosis to contribute to accelerate TNBC progression. Our study revealed the cancer-promoting effect of WTAP, NUPR1 and LCN2 in TNBC and clarified the relevant mechanism, providing a theoretical basis for developing novel diagnostic and therapeutic strategies for TNBC., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Progeroid syndromes such as Hutchinson Gilford Progeroid syndrome (HGPS), Werner syndrome (WS) and Cockayne syndrome (CS), result in severely reduced lifespans and premature ageing. Normal senescent cells show splicing factor dysregulation, which has not yet been investigated in syndromic senescent cells. We sought to investigate the senescence characteristics and splicing factor expression profiles of progeroid dermal fibroblasts. Natural cellular senescence can be reversed by application of the senomorphic drug, trametinib, so we also investigated its ability to reverse senescence characteristics in syndromic cells. We found that progeroid cultures had a higher senescence burden, but did not always have differences in levels of proliferation, DNA damage repair and apoptosis. Splicing factor gene expression appeared dysregulated across the three syndromes. 10 µM trametinib reduced senescent cell load and affected other aspects of the senescence phenotype (including splicing factor expression) in HGPS and Cockayne syndromes. Werner syndrome cells did not demonstrate changes in in senescence following treatment. Splicing factor dysregulation in progeroid cells provides further evidence to support this mechanism as a hallmark of cellular ageing and highlights the use of progeroid syndrome cells in the research of ageing and age-related disease. This study suggests that senomorphic drugs such as trametinib could be a useful adjunct to therapy for progeroid diseases., (© 2023. The Author(s).)

Tight regulation of macrophage immune gene expression is required to fight infection without risking harmful inflammation. The contribution of RNA-binding proteins (RBPs) to shaping the macrophage response to pathogens remains poorly understood. Transcriptomic analysis reveals that a member of the serine/arginine-rich (SR) family of mRNA processing factors, SRSF7, is required for optimal expression of a cohort of interferon-stimulated genes in macrophages. Using genetic and biochemical assays, we discover that in addition to its canonical role in regulating alternative splicing, SRSF7 drives transcription of interferon regulatory transcription factor 7 (IRF7) to promote antiviral immunity. At the Irf7 promoter, SRSF7 maximizes STAT1 transcription factor binding and RNA polymerase II elongation via cooperation with the H4K20me1 histone methyltransferase KMT5a (SET8). These studies define a role for an SR protein in activating transcription and reveal an RBP-chromatin network that orchestrates macrophage antiviral gene expression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

RNA modifications have been substantiated to regulate the majority of physiological activities in the organism, including the metabolism of reactive oxygen species (ROS), which plays an important role in cells. As for the effect of RNA modification genes on ROS metabolism in glioblastoma (GBM), it has not been studied yet. Therefore, this study aims to screen the RNA modification genes that are most related to ROS metabolism and explore their effects on the biological behavior of GBM in vitro . Here, an association between WTAP and ROS metabolism was identified by bioinformatics analysis, and WTAP was highly expressed in GBM tissue compared with normal brain tissue, which was confirmed by western blotting and immunohistochemical staining. When using a ROS inducer to stimulate GBM cells in the WTAP overexpression group, the ROS level increased more significantly and the expression levels of superoxide dismutase 1 (SOD1) and catalase (CAT) also increased. Next, colony formation assay, wound healing assay, and transwell assay were performed to investigate the proliferation, migration, and invasion of GBM cells. The results showed that WTAP, as an oncogene, promoted the malignant progression of GBM cells. Functional enrichment analysis predicted that WTAP was involved in the regulation of tumor/immune-related functional pathways. Western blotting was used to identify that WTAP had a regulatory effect on the phosphorylation of PI3K/Akt signaling. Finally, based on functional enrichment analysis, we further performed immune-related analysis on WTAP. In conclusion, this study analyzed WTAP from three aspects, which provided new ideas for the treatment of GBM.